4pplt('d (;eoche)mstrx. Vol I. pp 10q-1tl9.

lt~Se, 088L-2927/Nb S3 (K) * ( ~

Printed in (}teat Brilain ~ lONe, P e r g a m o n Pros, 1 It]

Microbiological exploration for mineral deposits: a new technique

GLENDA B. MICHAELS
Western State College, Gunnison, CO 81231), U.S.A.

and

WALTER C, RIESE
ARCO Exploration Company, P.O. Box 1346, Houston, TX 77251. U.S.A.

Abstract-~The measurement of metal resistance in microorganisms is a cost-effective technique that

should be used to supplement the presently employed inorganic geochemical methods of exploration. The
method is based on the measurement of metal tolerances in the total microbial population by means of the
use of culture techniques. It is strongly recommended that the culture technique demonstrated here bc
integrated into reconnaissance exploration programs that require efficient focusing to be successful. Thc
technique was developed and successfully tested in the Tomichi Mining District, Colorado, U.S.A., where
the environment is subalpine to tundra and the metals sought include Cu, Pb, Zn, Ag, and Au. It is easily
adapted to most environments and metals, and draws additional advantage from being able to detect
mineralization at greater distance and with lower sample density than standard inorganic sampling
techniques.

INTRODUCTION al., 1978; SJOGREN and PORT, 1981; TIMONEY et al.,

TRADITIONAt, geochemical exploration for mineral
deposits involves the sampling of various inorganic
media and their subsequent analysis for the element
being sought or its pathfinders. The presence of
mineral deposits also influences the biotic community
and, therefore, some exploration programs include
the sampling of biological materials, most notably
vegetation. Microorganisms also represent a poten-
tial tool for exploration.
Like other organisms, bacteria are sensitive to the
toxic effects of heavy metals. Genetic mechanisms
which render organisms tolerant to the effects of
metals in their environment are widespread, how-
ever, and many bacteria are thus able to live in the
presence of relatively high metal concentrations. This
genetic information is most commonly present on
elements called plasmids, pieces of D N A separate
from the bacterial chromosome. Plasmid-borne resis-
tance has been demonstrated for Cd (DEVANASet al.,
1980; NAKAHARAet al., 1977; PEARL, 1980; SUMMERS,
1984), Hg (SUMMERSet al., 1978), Ag (HAEFEIAet al.,
1984; McHuGH et al., 1975). As (CHEN et al., 1985;
NOVlCK and ROTH, 1968; SUMMERSet al., 1978), Pb
and Sb (SUMMERS, 1984), Te (TAYLORand SUMMERS,
1979), Cr, Ni and Zn (SUMMERSet al., 1978).
Because the toxicity of heavy metals represents a
selection pressure influencing the microbial commu-
nity, more metal tolerant organisms should be found
where metals are present than in locations unaffected
by metals, The occurrence of such tolerant forms in
ecosystems polluted by heavy metals and in clinical
settings where silver nitrate is used as an antiseptic is
well documented (DEVANASet al., 1980; HOUBAand
REMACLE, 1980; McHUGH et al., 1975; NAKAHARAet
1978; TRAXLERand WOOD, 1981 ; VARMAet al., 1976).
Little attention has been given to the distribution of
metal tolerant bacteria in the vicinity of ore deposits,
however.
Preliminary studies in the Tomichi Mining District
of Colorado demonstrated higher than expected
levels of antibiotic and heavy metal resistant bacteria
in Tomichi Creek near known mineral deposits. The
presence of both antibiotic and heavy metal resis-
tance is to be expected because genes for both types
of resistances are frequently located on the same
plasmid (CALOMIRISet al,, ! 984; HARNEqq"and GYLES,
1984; McHuGH et al., 1975; NAKAHARAet al., 1977:
SJOGREN and PORT, 1981). These preliminary data
suggested the use of antibiotic or heavy metal resis-
tant bacteria as a potential exploration tool.
Resistance to an antibiotic can be readily
demonstrated by incorporation of the antibiotic into
a suitable growth medium for bacteria. Therc arc,
however, two negative aspects to the use of antibiotic
resistance for exploration. There is no definite corre-
lation between specific antibiotics and the presence
of a particular metal or metals, This is due primarily
to the unstable nature of bacterial plasmids which
frequently undergo rearrangements of thc genes
which they contain (CAMPBELl., 1981; CHAU et al.,
1982; RUBENSet al., 1979). Antibiotics are also unsta-
ble and require refrigeration; this makes them incon-
venient for field work.
Heavy metals can be incorporated into growth
media to demonstrate the presence of resistant strains
if the necessary concentrations are determined.
Metals can also be used in othcr methods such as
luminometry and isotope incorporation because the
effects of metals do not require actively growing cells.

103
1()4 G.B. Michaels and W. C. Riese
METHOD DEVELOPMENT
Culture media a n d metal concentrations
A medium with a low nutrient content is required
for use in evaluation of oligotrophic (low nutrient)
systems. An excess of nutrients will inhibit the
organisms which are characteristic of waters with low
nutrient c o n t e n t (FLETCHER and MARSHALL. 1982).
previous work in our laboratory has shown the
Casein-Peptone-Starch (CPS) agar of COLUNS and
Wn.LOUGHBV(1962) tO be effective for these systems
as is also documented in the literature (JONES, 1970;
SrAr'LES and FRV, 1973). This medium is not commer-
cially available in dehydrated form, however, and
must be made up from the component chemicals. The
Environmental Protection Agency has recently
developed a medium designated R2A, a low nutrient
medium designed for testing drinking water
(REASONER and GELDREICH, 1985). This medium is as
effective as CPS for evaluating oligotrophic surface
waters (MICHAELS,unpublished data) and is commer-
cially available in dehydrated form suitable for pre-
paring both broth (liquid) and agar (solid) media.
Metal concentrations to be utilized for determining
the presence of tolerant organisms will vary depend-
ing upon the type of system being evaluated and the
medium used. The higher the nutrient content of the
medium, the higher the metal concentrations that
will be required because media components, includ-
ing agar, bind metals (BABICH and STOTZKV, 1980).
The nature of the system being tested will also affect
the required concentration of metals because a
number of ions and organic compounds provide pro-
tective effects, and the toxicity of many metals is
affected by the pH of the system (BABICH and
STOTZKY, 198(};GADDand GRIFFITHS.1978: LAEGREID
et al., 1983). Metal concentrations effective in the
oligotrophic waters of the Gunnison watershed and
which should be effective for use in other oligotrophic
systems are shown in Table 1.
Table 1. Metal concentrations to determine tolerance
Metal (or ion) Concentration (~tg/ml)*
Arsenate 2000, 4000
(as sodium arsenate)
Cadmium 10, 25, 50, I(X)
(as cadmium chloride)
Copper 5[I. 100, 150, 2(X)
(as copper sulfate)
Lead 500, 750, 1000
(as lead acetate or lead nitrate)
Mercury 1,5. 1{I
(as mercuric chloride)
Silver 5, 111
(as silver nitrate)
Tellurite 150, 2511,5011
(as potassium tellurite)
Zinc 2IX)[),4000
(as zinc chloride)
* Concentration ol melal ion.
A range of metal concentrations should be used for
each metal ion being evaluated. Attempts to use a
single concentration may lead to deceptive results: if
the concentration is too low, everything will appear
resistant: and if concentrations are too high, either no
organisms will grow or there will not be enough
growth to give statistically valid data. Use of a range
of concentrations will also be helpful in assessing data
variations caused by seasonal changes in aquatic
p o p u l a t i o n s (BELL et al., 1980; GOULDER, 1980;
HAACK and McFETERS, 1982: NELSON and VISSER,
1978). Attempts to use mixtures of metals provide
inconclusive results, although bacteria resistant to
multiple metals can be isolated (HARNEa-rand GYLES,
1984; TRAXLERand WOOD, 1981: MICHAELS, unpub-
lished data).
The incubation time required for adequate
development of visible colonies on agar medium or
membrane filters is 5 to 7 days at a temperature
approximating that of the water sampled. Visible
colonies may appear earlier, but the numbers will be
low and the organisms represent only the fastest
growing segment of the population. A significant
increase in population occurs beween 3 and 5 days.
Cultures must not be incubated at temperatures
above 20°C if the water does not exceed that tempera-
ture (BABICHand STOTZKY,1980; MOSSERet al., 1976:
MICHAELS, unpublished data) or selection again
occurs for only a fraction of the population and these
may not be the metal-tolerant forms. Some data
suggest that while the quantitative numbers of
colonies growing on the plates may be the same at 5
and 20°C there is a definite qualitative difference in
the population (NELSON and VmSER, 1978). Because
culture methods are already selected for that fraction
of the population that will grow on the chosen
medium, any factors which may further limit the
types of organisms present should be minimized.
Little problem is encountered with overgrowth of
the plates by fungi when using either agar or mem-
brane filters with broth-soaked pads. Te and Pb are
the most likely metals to show fungal colonies, but
even this is limited. If a problem with fungi does
occur, it can be eliminated by the addition of cyclo-
heximide to the medium.
S a m p l e types
Three types of samples can be obtained from aqua-
tic systems: grab samples, which represent the water
column; sediment samples, which represent the
populations of the stream bottom: and samples of the
sessile populations which are present on surfaces
within the streams. Of these samples, the grab sample
is the easiest to obtain and is the most representative
of the drainage area. It has the fewest organisms,
however, so particular care must be taken during
sampling and experimental procedures (EL-
SHAARAWI et al., 1981; JONES and SIMON, 1980;
KOSmSKI et al., 1979). Grab samples should always
 New microbiological technique for mineral exploration
be taken in an area of rapid flow and as far from the
stream bank as possible.
Sediment samples represent populations of longer
residence time and are thus more characteristic of the
immediate area, although upstream organisms may
also attach to the sediments. In rapidly flowing
streams, bacteria are usually free in the water column
(GEEsEY and Cos'rERTON, 1979) and are less likely to
attach to sediments. In standing water or slow-flow-
ing streams, a significant proportion of the bacteria in
the water column are attached to suspended particles
which more readily precipitate (BELL and ALBmC3HT,
1982). Sediment samples are often more difficult to
obtain and in cases where stream beds are extremely
rocky may not be available. Because more bacteria
are present per unit volume of sediment than are
present in the water column, dilution of the sample is
necessary which may pose a problem under field
conditions. Sediments also provide adsorption sites
for metal ions, particularly if the clay mineral content
is high, and this can cause problems with effective
metal concentrations (BAB1CHand STOTZKY, 1980"
GADD and GRIFm'HS, 1978).
Samples of the sessile populations in the glycocalyx
covering rocks and other surfaces in an aquatic system
represent organisms of longer residence time but
which are influenced by upstream conditions.
Organisms growing in the glycocalyx release "swarm
cells" which colonize new areas; these cells are pre-
sent in the water column (CoSTERTONand IRVIN,
1981). Glycocalyx samples are not always easy to
obtain and are difficult to quantify, making site-to-
site comparison more difficult. The protective effect
provided by metal adsorption to the glycocalyx
polysaccharides may also cause problems with effec-
tive metal concentrations.
In our experience, grab samples are the best for
evaluation of aquatic systems. Sample volume
requirements are quite low, often less than 100 ml,
and media can be inoculated in the field. Prepared
media are easy to transport and field procedures are
simple. Glassware for spreading the control plates
can be conveniently sterilized by dipping into a con-
tainer of alcohol and firing with a lighter. Membrane
filters of the size normally used for cultivating bac-
teria are commercially available. The filter holders
used to create a vacuum for pulling the water sample
through the filters are available as sterile disposable
units or as resterilizable units; hand vacuum pumps
are readily available. Our data indicate that it is not
necessary to use a separate sterile filter holder for
each site: rinsing the holder with water from the site
being tested is sufficient to remove the previous
sample.
It is desirable to process water samples as soon as
possible after they are obtained in order to avoid the
problems caused by continued metabolic activity
within the samples. Organisms will continue to grow
and metabolize while the samples are being trans-
ported, and because not all bacteria grow at the same
105
rate, there may be qualitative changes in the popula-
tion. These changes may lead to misinterpretation of
the data. Additionally, the ultraviolet component of
sunlight is damaging to many organisms (FtmoKA and
NARIKAWA, 1982). Samples should therefore be pro-
tected from direct sunight until they are processed.
Comparison of culture methods
Spread plating, the use of an agar-based medium
on which the water sample is spread with a bent glass
rod, is the most advantageous method for determin-
ing the background or control counts. Membrane
filtration using broth-soaked pads as a source of
nutrients is the most advantageous culture method
for testing metal tolerance. The metals can be added
to the broth after it has cooled, and there is no
problem with solidification during handling as there
is with agar-based media. Moreover, eliminating the
agar also eliminates one source of potential metal
binding. Suitable water samples can be filtered
through commercially available membrane filters
designed for the cultivation of microorganisms, and
the filters placed directly on the media-soaked pad in
disposable dishes of the correct size. Broth-soaked
pads can also be used for controls, but this requires
dilution of the sample.
DESCRIPTION OF THE STUDY AREA
General geology
The area to which we applied this exploration
technique is in the Garfield Quadrangle, Chaffee and
Gunnison counties, in west-central Colorado,
U.S.A. It lies between longitude 1(16°15' and
106°30'W, and latitude 38030 ' and 38°45'N.
The rocks of the area range in age from Pre-
cambrian to Quaternary. Precambrian rocks consist
of metamorphosed sedimentary and intrusive igne-
ous bodies; Paleozoic and Mesozoic rocks are
sedimentary; and the Tertiary rocks are intrusive,
volcanic and volcaniclastic. All are mantled and loc-
ally obscured by Quaternary moraines, glaciofluvial
deposits, landslides, talus, and alluvium which
together with extensive vegetation make exploration
extremely difficult. Structure is complex and domi-
nated in style by Laramide thrusting, folding, and
normal faulting which has dropped Paleozoic carbo-
nates into the Precambrian crystalline rocks where
they, and the ore deposits they host, have been
preserved (DINGS and ROBINSON, 1957: CRAWFORI),
1913).
Description of the ore deposits
Our work centered in the Tomichi Mining District,
in the extreme south-central portion of the quad-
rangle. The deposits here are replacement types and
1116 G. B. Michaels and W. C. Riese

Table 2. Levels of heavy metals inhibitory to bacterial populations

Ion Concentration Source

Hg 1 ppm ( 1 /~g/ml) freshwater aquatic systems

1 x 10 4M (20 gg/ml) factory sedimentation pond
5-10 ktg/ml this report
Cd 1 ppm (1 gg/ml) freshwater aquatic system
500 ppm (500 #g/ml) marine sediment
1.8 x 10-4M (20011 p.g/ml) factory sedimentation pond
50-100 gg/ml this report
Ag 1 ppm (l /xg/ml) freshwater aquatic system
1 x 10-3M (1117 /xg/ml) silvermine
10/zg/ml this report
Cu 1 ppm (1 /.Lg/ml) freshwater aquatic system
200/zg/ml this report
Pb 1 ppm (1 gg/ml) freshwater aquatic system
250 ppm (2511/xg/ml) freshwater aquatic system
500--100(1/zg/ml this report
Zn I × l0 2M (6538/xg/ml) factory sedimentation pond
20110-4000 #g/ml this report

occur in a syncline of Paleozoic carbonates which has
been normally faulted into the underlying Precamb-
rian crystalline rocks. These replacements assume
two distinct forms: replacements along fault zones,
the most significant, and replacements along bed-
ding, which resemble the deposits of the Leadville
District, Colorado. The ore mineralogy is a granular
aggregate of sphalerite and galena with variable
amounts of pyrite and chalcopyrite. The galena is
c o m m o n l y argentiferous and the pyrite auriferous.
All occur in a gangue of marbelized limestone or
dolomite with variable amounts of quartz or silicified
limestone. Much of the near-surface ore is strongly
oxidized, but there is little or no oxidation below
depths of about 30 m (DINGS and ROBINSON, 1957).
This area was selected because we believed the
deposits and their attendant mining wastes would
provide a strong metal influx to area streams, which
because of their remote location should be free of any
antibiotic contamination. In this area the microbial
response to metals could be examined in a natural
setting with no influences from the use of antibiotics.
This area also provided drainages with comparable
geology in the same ecozone which have no known
deposits in them; this assured that we would be able
to estabish background responses for the same
species that we were sampling in the mineralized
portions of the district. Mining activity stopped in the
Tomichi Mining District around 1953, so any other
influences on the microbial population have, hope-
fully, also been mitigated.
sedimentation pond and silver mine show higher
resistance levels (HOUBA and REMACLE, 1980:
HAEFEffl et al., 1984). Most of the streams in the
watershed are affected by mineral deposits by the
time they reach the valley floor, hence the elevated
levels of metal resistance present. The numbers of
aquatic bacteria resistant to antibiotics are also high
when c o m p a r e d to other unpolluted streams which
have been studied (BURTON et al.. 1982: FEARYet at.,
1972; KELCH and LEE, 1978: KOBORI et al., 1984).
When specific streams originating above metallo-
genic zones and flowing through them are examined,
elevated levels of resistant bacteria are found below.
This can be shown for Tomichi Creek in the Tomichi
District as well as for nearby Gold Creek in the Gold
Brick District, where Au is the primary metal re-
covered.
Seasonal fluctuations which we have observed in
the data suggest that soil bacteria washed into the
stream by snowmelt and run-off represent a major
source of resistant organisms. This is shown for the
antibiotic chloramphenicol in Table 3. Peaks
observed in early season sampling tend to flatten out
in late s u m m e r when less run-off occurs. Addition-
ally, there are often increases in the native aquatic
population about this time (HAAf'K and MCFETERS,
1982).
Table 3. Effect of snow melt on incidence of chlor-
amphenicol resistance in stream populations

Percent of heterotrophic population

RESULTS AND DISCUSSIONS resislanl
May June July
The data presented in Table 2 indicate that bacteria
from streams in the Tomichi Mining District are able All sites 14.62 18.2 8.45
Control sites* 19.97 211.45 13.43
to grow in the presence of higher concentrations of
Human/animal 13.111 17.57 8.(11
heavy metals than has generally been reported in the
literature. Only the organisms from a factory * Not influenced by human or domestic animal activity.
 New microbiological technique for mineral exploration
Large animal populations in an area may also
influence the incidence of heavy metal and antibiotic
resistant bacteria. Bacteria from feces of over 200
native animals were obtained and tested. Sixty-five
per cent of these animals host bacteria with some
antibiotic resistance: most show resistance to at least
one metal. These bacteria may be carried into
streams, thereby influencing the number of resistant
organisms present. This further suggests that fecal
samples may be collected for analysis of bacterial
resistance in an area. We observed, for example, that
deer and elk ranging in the vicinity of Homestake's
Pitch Project, a U mine, host high levels of Pb-resis-
tant bacteria. As was the case with the stream data,
the levels of metal and antibiotic resistant bacteria in
fecal samples are higher than previously reported in
the literature (HUBERet al., 1971 : MARE,1968).
Animal and aquatic bacteria may interact via their
ability to transfer plasmids from one bacterium to
another. This process is called conjugation and has
been demonstrated in limited experiments with
strains isolated from the area. The transfer of metal
resistance was not specifically tested in this study but
can be expected due to the genc distribution on
plasmids noted earlier and is well documented in the
literature (URETER,1984: SUMMERSet al., 1978). The
presence of metals in the environment represents a
selection pressure for resistant genes which thus tend
to increase, whether the bacteria are in the water or
in the intestinal tracts of animals which drink the
water and eat the plants affected by the mineral
deposits.
Due to the relationship between metal and anti-
biotic resistance, data of the type presented here
must be interpreted carefully if large numbers of
domestic animals are present in the area of explora-
tion. This is especially true if antibiotic-containing
feeds are being used, because this practice selects for
antibiotic resistant intestinal bacteria.
Resistance to ultraviolet (UV) radiation has been
linked to plasmids with metal and antibiotic resis-
tance (BREEZEand OBASEIKI-EBOR, 1983; DRABBLE
and STOCKER, 1968). UV radiation resistance at high
altitudes has not been studied, although UV resis-
tance is well documented in laboratory experiments.
This'may represent a selection pressure in streams
which originate above timberline and are subject to
intense radiation from sunlight. Preliminary data
indicate elevated levels of UV resistance among bac-
teria from the headwaters of Tomichi Creek which
originates at about 365(I m. This does not appear to
have influenced the outcome or results of this study,
however.
Reports in the literature indicate that starvation
effects in oligotrophic systems may cause the bacteria
present to be extremely small (FLETCHER and
MARSHALL, 1982: FRY and ZIA, 1982: KJELLEBERGel
al., 1983). This caused concern that the standard 0.45
p~ membrane filter might be too large in pore size.
Our data showed, however, that most cultivable
11)7
bacteria are large enough to be trapped on these
filters. In samples collected in late season, a sub-
stantial portion of the cells did pass through a (I.45
filter. This may be related to seasonal population
changes. The major problem which this poses is one
of obtaining a cell-free water sample to use as a
control when other non-cultural techniques are used
for analysis; this study was therefore not affected.
Metal resistance studies must also consider the
effects of pH on the system. In general, an acidic pH
increases the toxic effects of metals by increasing
solubility and changing ionic form, while alkaline pH
tends to decrease toxicity except for Cd (BAB1C}tand
STOTZKY,1980: GADD and GRIFFITHS, 1978). Stream
sites most affected by mineral deposits will show less
significant pH effects than sites which are not so
affected.
The presence of certain divalent cations, especially
Ca and Mg, can also affect the toxicity of metals to
bacteria (BABICH and STOTZKV, 1980: GADD and
GRIFF1THS, 1978). Aquatic systems which show ele-
vated levels of these ions may also show elevated
levels of apparent metal resistance due to the protec-
tive eff~:ct of these elements. When microbiological
data are being used for exploration, Ca and Mg levels
must be considered: areas with extensive outcrops of
dolomite, for example, may frequently show high
concentration of Ca and Mg in stream waters.
Metal ion interactions are also known to occur. Cd
may influence apparent Zn resistance levels: inhibi-
tion by Hg may be affected by Mn, Cu, Co, Ni, Pb,
Fe, or Cr (BABICHand S'rOTZKV, 1981)). Fe affects the
toxicity of Cu, and sulfide as H~S may precipitate
toxic metal ions as insoluble sulfides (GADD and
GRIFEIIHS, 1978). Metal interactions of this type will
not prevent detection of metal resistant bacteria but
the concentrations required in the cultures will be
affected.
Clay minerals and organic compounds such as
humic and fulvic acids have a marked influence on
metal toxicity. These materials adsorb metals and in
so doing reduce the amount available for biotic use
(BABICH and STOTZKY. 1980; GADD and GRIFFITHS,
1978; LAEGREID et al., 1983). Very little organic
material or clays are typically present in systems such
as the one we studied and were not deemed an
influence in this study.
SUMMARY
The data presented in this study indicate that
microorganisms represent a potential exploration
tool and that cultural methods are an effective and
convenient method of measuring the presence of
metal resistant bacteria. Additional background level
data from nonmineralized areas will be helpful in
future investigations because the values reported to
date are almost exlusively concerned with metal
pollution.
The use of several metals is recommended.
1118 G . B . Michaels and W. C. Riese
B e c a u s e t h e r e are p a t h f i n d e r s for m o s t m e t a l s o f
e c o n o m i c c o n s e q u e n c e this s h o u l d n o t be a p r o b l e m .
A d d i t i o n a l l y , r e s i s t a n c e s h o u l d b e m e a s u r e d at
s e v e r a l c o n c e n t r a t i o n s a c r o s s a b r o a d r a n g e . Tripli-
c a t e s are r e c o m m e n d e d f o r e a c h c o n c e n t r a t i o n at
e a c h site in o r d e r to a s s u r e statistical viability o f t h e
resulting data.
A l t h o u g h this s y s t e m was d e v e l o p e d a n d t e s t e d in
an o l i g o t r o p h i c s y s t e m at high e l e v a t i o n s , it can b e
a d a p t e d to o t h e r a r e a s if t h e f a c t o r s r e v i e w e d are
c o n s i d e r e d . W e d o n o t s u g g e s t that a m i c r o b i o l o g i c a l
e x p l o r a t i o n t e c h n i q u e such as this s h o u l d r e p l a c e t h e
presently employed inorganic techniques of
g e o c h e m i c a l e x p l o r a t i o n . If u s e d in c o n j u n c t i o n with
t h e m , h o w e v e r , its use can result in m a r k e d s a v i n g s o f
t i m e a n d m o n e y b e c a u s e it is a very r a p i d e v a l u a t i o n
t e c h n i q u e a n d is itself e x t r e m e l y cost e f f e c t i v e .
Acknowledgements'--We gratefully acknowledge the assist-
ance provided by the Anaconda Minerals Company in
funding this research and for permission to publish the
results. We also thank Jeffrey J. Hill and David J. Schneck
for their assistance in the field and laboratory.
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