0038.0717/79/1001-0487P02.

00/0

EFFECTS OF LEAD ON THE SOIL BACTERIAL

MICROFLORA

P. DOELMAN and L. HAANSTRA

Research Institute for Nature Management, Kemperbergerweg 67, 6816 KM Arnheim,
The Netherlands

(Accepted 30 May 1979)

Summary-The sensitivity of soil bacteria towards Pb was investigated. Soil suspensions from fourteen
different soil types with a high or low Pb content were plated out on soil extract agar containing
various concentrations of PbC12.
In agar with a high Pb content, higher bacterial counts were found with suspensions from Pb-contain-
ing soils than with those of soils with a low Pb content. In the Pb-containing soils, proportionally
more gram-negative rods were present while coryneform bacteria decreased. In an additional experi-
ment, in which Pb was added to a sandy soil, more Pb-tolerant bacterial strains were found 3 years
later than in the same soil without Pb.
When pure cultures of the bacteria isolated from the soils were tested in liquid media for Pb tolerance,
a higher proportion of tolerant strains was found in Pb-containing soils. Among strains of gram-negative
bacteria isolated from these soils a higher proportion of tolerant strains was found than in corneform
bacteria.
It was concluded that as a consequence of Pb pollution of soil a selection of Pb-tolerant bacteria may
take place

INTRODUCTION have been made on bacteria. Jordan and Lechevalier

We (Doelman and Haanstra, 1979a,b) have shown
that when Pb (PbCl*) is added to the soil its respir-
ation may be retarded. A relationship was found
between the cation-exchange capacity of the soil and
the inhibition of respiration. Decomposition of
various organic compounds, such as glucose, starch,
cellulose, amino acids and millepede excrements was
also delayed in Pb-amended soils. The effects lasted
for a long time but they gradually decreased after
several years. It was concluded, therefore, that adap-
tation of respiration and decomposition to Pb pol-
lution may be due to either selection of Pb-resistant
microorganisms, to the adaptation and mutation of
the original microflora or to a combination of both.
There are hardly any indications in the literature as
to which of these mechanisms prevails in the “adap-
tation” of the soil microflora to Pb. However, studies
on the effects of pesticides on microbial activities in
the soil have shown that fungi may adapt to some
other heavy metals. For instance, Phytophthora infes-
tans became resistant to Cu, either by adaptation or
mutation (Horsfall, 1956). Strains of Aspergillus niger
resistant to Zn were isolated by Elorza (1969). The use
of mercurial fungicides has induced a higher resist-
ance to Hg in Pyrenophora auenae (Greenaway and
Cowan, 1970). However, it has been found that Chry-
sosporium panrwrum also builds up a resistance but a
Mortierella species and a Trichoderma species
remained sensitive (Pugh et al., 1975). From the above
data it may be concluded that the use of heavy metals
in soil may give rise to a selection process as well as
to the adaptation of a species, i.e. the above mechan-
isms may also occur in the case of Pb.
In contrast to the work on fungi, few similar studies
487
(1975) studied the effects of Zn smelter emissions on
forest-soil microflora, and suggested that in Zn pol-
luted soils an initial reduction of bacteria, actinomy-
cetes and fungi was followed by a selection of Zn-
tolerant strains. In the case of Pb, mainly its effects on
pure cultures have been studied. Micrococcus luteus
and an Azotobacter species were both found to be
resistant to Pb (Tornabene and Edwards, 1972). It
should be noted, however, that these studies were per-
formed using rich culture media, in which the micro-
organisms were protected against the action of Pb by
organic compounds and phosphates (Ramamoorthy
and Kushner, 1975). In contrast to the above observa-
tions made by Tornabene and Edwards (1972). Foully
(1976) found some species of Enterobacteriaceae to be
susceptible to Pb but, at the same concentrations,
Bacillus species were resistant. Recently, Williams et
al. (1976) provided some evidence for increased Pb
tolerance in microbes from mine waste.
We have studied the effects of Pb on the bacteria
microflora in soil. The resistance to Pb of the soil
bacteria present in soils polluted for a long time, has
been compared with that of the bacteria present in
a number of non-polluted soils. In addition, the
occurrence of changes in composition of mixed flora
under the influence of Pb was investigated by deter-
mining the proportions of gram-negative rods and
coryneform bacteria, respectively.
MATERIALS AND METHODS
The following soils were used to study the effects
of Pb on their bacterial flora. As reference soils, in
which no Pb accumulation occurred, a sandy soil
“Eng” and a clay soil “Nude” were used. Both are
488 P. DOELMAN and L. HAANSTHA

arable soils from the region around Wageningen. A
fortified peat soil “Trio” and peat soils W, and W,
from the “Weerribben”, a nature reserve in the north
of the Netherlands, were also included in the investi-
gations as reference soils. The soil samples L,, L,
and L6 originated from refuse-matter soils of old lead
mines at la Calamine and Plombieres (Belgium). Soils
La and Ls were covered by a varied vegetation ; on
soil Lb, an old silt pond, there was no vegetation.
The soil samples IB, IO, IIB, 110, IIIB and III10 were
from Pb~contaminated grassland soils around a
metallurgical plant in Hoboken (BeIg~um). The
B-samples were from the O-IOcm layer, the O-sam-
ples, which also contained considerable quantities of
Pb, from the lO-20cm layer. All the other soils were
sampled to a depth of approximately 10 cm. For each
soil sample, IO-15 cores were taken at random and
homogenized by sieving just before analyzing the
samples. For the IB, IO, IIB, 110, IIIB and 1110
samples only 3 cores were taken. Some properties of
the soils are given in Table 1.
Viable bacteria were counted by plating dilutions of
soil on an agar medium which contained yeast extract
0.1 g, agar 15 g, soil extract 1 I (Doelman and Haan-
stra, 1979a), pH 6.ti.8. Lead (PbCI,) was added to
the agar medium at concentrations of 0, 1, 2.4, 8, 16,
32, 64 and 128 pg Pb.ml- ‘, respectively. After incu-
bation at 25°C for 10-14 days, the number of bac-
terial colonies were counted. The bacterial counts
were made as follows: the final dilution was prepared
so that 100-300 colonies developed on each plate
1 g, agar 15 g, soil extract 1 I., pH 6.66.8. The strains
on both media were examined microscopically after 1
day and after 8 days to classify them into morphologi-
cal groups such as gram-negative rods, coryneform
bacteria and spore-forming bacteria.
The Pb-sensitivity of the isolates classified in this
way, was estimated by cultivation in a medium con-
taining yeast extract 0.1 g glucose I g, soil extract
2OOm1, demineralized water 800 ml, pH 6.6-6.8, to
which PbCl, was added in concentrations of 0, 2,
4,8, IO, 15, 20, 2.5 and 3Oc(g PbZ+ .ml-‘, respectively.
Bacterial suspensions in tubes containing IO ml of the
above medium were grown by shaking for 72 h at
25°C after inoculation with 0.5 ml of a fresh suspen-
sion of the bacteria which were pre-cultivated on the
same medium. Growth was determined by measuring
the turbidity. Bacterial strains were considered to be
Pb-sensitive if no growth could be detected in the
above me~um at 4~~8 Pbeml-‘. They were con-
sidered to be Pb-tolerant if growth occurred at a con-
centration of 30 1.18Pb.ml - ‘. To establish the rate
of “adaptation” to Pb in the soil, 1500 pg Pb.g- ’
were added to sandy soil “Eng”, and then stored out-
doors. Samples of this soil were taken annually from
the same stock and the sensitivity of their bacterial
flora was determined.
RESlJLTS AND DISCUSSION
The occurrence of Pb-tolrrunt bacteria in Pb-polluted
Soil.9

f4cm dia with 0 fig Pb.ml-‘. The average number of
colonies derived from 4 replicate piates was con-
sidered to be a sample of a Poisson distribution. The
standard error of the mean value of a set of counting
experiments did not exceed 10%.
About 600 bacterial strains were isolated at random
from the 0 fig Pb .rni- ’ plates for ail the soils and also
from the Pb-containing plates for “Eng” and “Nude”
soils (controls) and the soils IIB, La, L, and L6 (Pb
soils). The bacteria were cultivated on two agar media
of the following composition. The first medium con-
tained: yeast extract 7 g, glucose log, agar 15 g, soil
extract 250 ml, demineralized water 750m1, pH
6.6-6.8; and the second one contained: yeast extract
Table 2 and Fig. 1 give the relative number of col-
onies found when suspensions of the various soil
types were plated on a Pb-containing agar medium.
A medium poor in nutrients was used in this experi-
ment because available Pb is easily diminished by
compounds such as amino acids and phosphates. The
results (Table 2) demonstrate that in Pb-cont~n~ng
soils, a higher proportion of Pb-tolerant bacteria
occurs than in normal soils with low concentrations
of Pb.
With a Wilcoxon Ranksum test, used as a permu-
tation test, on the percentage of bacteria surviving
in the individual 14 soils, the grouping Pb-polluted
and unpolluted soils (Fig. 1) has a rather extreme

Table 1. Properties of Pb-containing and non Pb-containing soils used in the present investigation

Number of
microorganisms
Organic Mineral &;tion (%) (mgP& ’ (106-g-r dry
Soil &, matter (%) Clay Sand dry soil) soil)

IB 4.7 3.7 6 10 84 600 55

IO 4.7 2.3 5 9 86 340 38
IIB 5.2 4.4 7 13 80 522 330
II0 5.9 3.0 6 10 84 295 280
IIIB 5.3 12.7 5 10 85 737 120
III0 5.4 2.9 8 13 79 236 21
L, 6.0 20.f 13 8 79 184 2.9
L3 6.7 25.3 19 10 71 200 51
L6 7.0 3.7 76 23 1 1177 3.1
‘“Eng” 4.1 2.8 12 81 7 12 22
“Nude” 7.0 3.2 99 0 I 12 105
“Trio” 5.6 45.7 6 22 72 41 480
W, 4.9 31 43 38 19 1.5 375
Ws 5.0 28.5 27 60 13 15 310
 Pb and bacterial composition 489

Table 2. The influence of Pb. incorporated in the agar medium used in the plate counts, on the
relative bacterial numbers in soil suspensions of various soils (the number of colonies on the Opg
Pb.ml-’ plates = 100%). The soils are arranged in decreasing order of Pb. The numbers are averages
of 4 replicates and the standard error of the mean value of a set of experiments did not exceed
10%

Pb concentration pg Pb’ml- ’ in agar plates

Soil type in soil (pg.g-‘) I 2 4 8 16 32 64 128

L, 1177 97 103 103 103 92 64 42 3

IIIB 737 110 II2 104 95 94 109 96 38
IB 600 103 100 78 73 74 85 75 48
IIB 522 92 78 87 71 80 87 66 15
IO 340 105 107 I 07 108 84 85 II6 101
II0 295 95 ND 77 63 67 85 87 33
1110 236 94 82 74 68 70 96 73 51
L5 200 100 II4 01 85 100 100 56 32
LS 184 100 95 84 89 83 91 83 65
“Trio” 41 99 82 61 32 15 3 0 0
W, I5 83 80 56 48 30 7 0 0
W3 15 91 76 58 39 22 II 0 0
“Eng” 12 103 86 72 34 30 44 10 0
“Nude” 12 79 63 44 35 31 62 48 8

ND = not determined.

value of the test statistic W in relation to all other two curves at concentrations of 2, 4, 8, 10 and 15 pg
possible groupings of the soil. Only 05% of all poss- Pb’ml-‘). When for a control soil (“Nude”) and a
ible groupings has an equal or greater value of W Pb-polluted soil (IIB) strains of the Pb-containing
at concentrations of 4, 8. 16 and 32pg Pb’ml-’ in agar plates were tested in the liquid medium, a pro-
the agar medium. gressively higher proportion of strains isolated from
The occurrence of a higher proportion of tolerant Pb-containing agar plates were Pb-tolerant (Table 3).
bacteria in Pb-containing soils was checked using a This was especially so with strains isolated from the
different method. About 300 strains were isolated Pb-polluted soil (P < 0.05;Wilcoxon signed Rank
from the agar plates without Pb on which suspensions test).
of Pb-containing and control soils were plated out. Not all the strains are equally Pb-resistant in Pb-
The strains were then tested in the liquid media con- containing soils, some of them being quite sensitive.
taining different quantities of Pb. Using this method The presence of Pb-sensitive bacteria may be the
the same results were obtained (Fig. 2), i.e. a higher result of an uneven distribution of Pb in the soil. It is
proportion of tolerant strains was derived from Pb- conceivable that the inside of soil aggregates contains
containing soils than from the control soils (using the less Pb-ions than the outside. Nishio and Furusaka
Wilcoxon Ranksum statistic W, a statistically-signifi- (1970), investigating the bacteriocidal effect of various
cant difference P < 0.028 was obtained between the concentrations of HgClz on numbers of bacteria in
the outer and in the inner part of soil aggregates,
reported that the bacteria in the outer parts of soil
aggregates were more sensitive to HgCl, than the

Fig. 1. The influence of Pb incorporated in ihe agar

medium used in plate counts on the average number of Fig. 2. The inauence of Pb in the liquid medium, on the
bacteria from nine Pb-polluted soils (+O) and from average growth of 600 bacteria isolated from four Pb-pol-
five reference soils (+o). The results are expressed as luted soils (M) and from two reference soils (S-O).
percentages of the numbers growing on the medium with- The results are expressed as percentages of the numbers
out Pb. growing in the medium without Pb.
490 P. DOELMANand L. HAANSTRA

Table 3. The sensitivity in liquid media of bacterial strains isolated from agar plates which contained
various concentrations of Pb. The results are expressed as percentage of the number of strains growing
in liquid media without lead

PbCI, (pg Pb’ml-‘) pg Pb.ml- I in liquid media

Soil type in agar plates N I 2 4 8 IO I5 20 25 30

“Nude” 0 42 93 67 69 67 67 50 43 21 I2
(control 2 44 100 80 80 88 88 84 64 43 7
soil) 8 61 98 66 67 85 85 75 59 25 3
32 51 98 74 72 87 X7 80 67 33 5
IIB 0 55 100 96 91 96 95 96 95 X0 69
(Pb- 2 62 100 97 94 98 97 98 97 89 73
polluted 8 64 100 100 100 98 98 98 9X 98 91
soil) 32 58 100 100 loo 100 100 I 00 IO0 100 86

N = number of purified strains tested.

ones in the inner parts. Hattori and Hattori (1976) bacteria or actinomycetes. The overall tolerance of
reported that the number of gram-negative bacteria to isolates from the mine site was only significantly
total bacteria, at field conditions, was usually smaller greater than that of unpolluted soil isolates at Pb
than 10% in the outer part. While on the contrary this concentrations of 125 and 150 ~1 Pb.ml-’ in the
ratio was much higher and frequently exceeded 30% tested medium; but at 200 pg Pb’ml-’ differences
in the inner part. Moreover it is also known that were not significant. Jensen (1977) examining the col-
coryneform bacteria (such as Arthrohacter spp) are onies on dilution plates did not indicate any gross
rather drought resistant and are likely to occur in the change in the composition of the bacterial flora due
outer part of the soil aggregate. Therefore, a shift in to the presence of Pb. In the case of fungi, however,
the composition of the microflora in Pb-containing he observed an increased occurrence of dominant
soils does not necessarily have to be ascribed to the species, such as Paeciliomyces lilacium and Fusarium.
selection of Pb-tolerant bacteria but may also be of It should be emphasized that only after isolation and
an indirect nature. examining the strains under a microscope we could
In the control soils, which contained only low observe the change in composition of the bacterial
quantities of Pb, a considerable number of bacterial flora. It should be noted that 2 yr after the addition of
species were Pb-tolerant. This suggests that tolerance Pb the dissimilation rate of organic compounds, such
is related to certain innate properties shared by quite as glutamic acid, was still somewhat lower than in the
a number of species. control soil (Doelman and Haanstra, 1979b), while
The selection of Pb-tolerant strains in the soil 3 yr after the addition of Pb the soil respiration was
occurs within a few years. When 15OO~g Pb.g-’ was still 30% lower than in the control soil (Doelman and
added to a sandy soil after 1 yr no difference with the Haanstra, 1979a).
control soil in Pb-sensitivity of the bacteria was found
but after 2 yr, and particularly after 3 yr, a selection of Lead sensitivity of gram-negative rods and coryneform
tolerant strains had taken place (Table 4). While Jor- bacteria, isolated from the various soil types
dan and Lechevalier (1975) reported increased Zn In the previous section it was shown that in Pb-
tolerance in isolates from forest soil near a smelter, containing soils a higher proportion of strains
increased Pb tolerance was suggested by Williams et occurred that were tolerant towards this heavy metal.
a/. (1977). Comparing the microflora from Pb-pol- The question arose whether the change in compo-
luted mine waste with unpolluted pasture soil. the sition of the microflora is the result of a selection. To
latter reported that fungi were more Pb-tolerant than investigate this, the proportion of gram-negative rods

Table 4. The sensitivity to Pb of a number of representative bacterial strains, which were isolated from
a sandy soil. amended with and without 1500 pg Pb,g- ‘, after 1, 2. 3 and 4 yr. respectively. The results
are expressed as percentages of the number of strains growing in the medium without Pb

PbCll Number of
Years pg Pb,g-’ bacterial PbCI, (pg Pb’ml-‘)
Of in the strains in the liquid medium
amendment soil tested I 2 4 8 10 I5 20 25 30

I 0 43 ND 98 ND ND 71 ND ND ND 30
I500 41 ND 93 ND ND 78 ND ND ND 27
2 0 96 81 81 81 83 28 I4 I6 I6 II
I500 96 95 95 93 93 69 41 28 26 21
3 0 96 85 52 50 65 43 30 17 II II
1500 96 97 97 95 92 87 66 53 34 32
4 0 62 79 40 77 84 73 39 21 I3 I3
1500 51 82 73 94 92 86 65 29 22 20

ND = not determined.
 Pb and bacterial composition 491

Table 5. The influence of Pb, incorporated in soil extract agar, on the proportion of
gram-negative rods and coryneform bacteria in different soils

Gram-negative rods Coryneform bacteria

% of total count
PbCI, (pg Pb. ml- ‘) in the agar medium
Soil type 0 2 8 32 64 0 2 8 32 64

“Eng” 34 45 48 ND 70 42 31 31 ND
“Nude” 61 65 72 ND 75 31 24 21 ND 2;
IIB 63 63 68 73 ND I8 20 I6 IO ND
L3 64 68 62 ND 88 29 25 35 ND I7
L5 69 ND ND 76 ND 20 ND ND 20 ND
L.5 81 ND 85 ND ND I4 ND 7 ND ND

ND = not determined.

and coryneforms occurring on the various plates with portion of both Pb-sensitive and tolerant gram-nega-
different Pb-concentrations was established (Table 5). tive rods may occur in Pb-containing soils if they are
The following picture emerges from the results. With moist. Similarly, the drought-resistant coryneforms,
increasing concentration in the agar medium, the pro- which are numerous in the outer layer of soil aggre-
portion of gram-negative rods increased and that of gates, may be suppressed.
the coryneform decreased. This was particularly true
of the control soils, particularly when the number of
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