Vol. 25, No. 10, pp.1443-1446, Printed in Great Britain. All rights reserved
Soil Bid. Biochem.
0038-0717/93$6.00+ 0.00 Copyright c 1993Pergamon Press Ltd
BACTERIAL RESISTANCE TO HEAVY METALS RELATED TO EXTRACTABLE AND TOTAL METAL CONCENTRATIONS IN SOIL AND MEDIA J. SCOTT ANGLE,‘* ‘Department
CHANEY* and DAVID RHEE’
of Agronomy, University of Maryland, College Park, MD 20742 and 2Environmental Chemistry Laboratory, USDA-ARS, Beltsville, MD 20705, U.S.A. (Accepted I5 March 1993)
Summary-To assess the effects of heavy metal pollution on terrestrial systems, bacteria are frequently isolated from such environments and metal tolerance of the isolates determined by plating on media amended with high concentrations of metal salts. We have found that metal concentrations added to media in traditional assays are orders-of-magnitude higher than water soluble concentrations of Zn, Cd and Ni
in soil, even highly contaminated soil. Further, many soil bacteria are intrinsically resistant to high concentrations of heavy metals, thereby precluding the need to adapt to metal contamination. Most bacteria isolated from soil were resistant to very high concentrations of heavy metals, regardless of whether or not the soils were contaminated with metals. The average tolerance to Zn of bacteria isolated from highly contaminated soil was 75 mg I-’ but the concentration of extractable Zn from the soil was only 0.47 mg kg-’ In a non-contaminated soil, the average bacterial tolerance of Zn and extractable soil concentration of Zn were 26 mg I-’ and 0.04 mg kg-‘, respectively. Defining soil metal concentrations by extractable concentrations as opposed to total concentrations is a more appropriate measure of potential bacterial changes and may elucidate ecosystem changes that might otherwise go undetected.
INTRODUCTION Industrial activities and disposal of waste products have resulted in the contamination of many terrestrial environments with heavy metals. The extent to which these environments are polluted and whether the metals are adversely affecting biological systems is difficult to determine. Simple methods to assess whether heavy metal pollution is adversely affecting the environment have long been sought. One method purported to assess environmental effects of heavy metals is to determine the number of metal-resistant bacteria isolated from an environment affected by heavy metals, as bacteria are capable of rapidly responding to changes in their environment (Allen et al., 1977; Marques et al., 1979; Olson and Thornton, 1982; Duxbury and Bicknell, 1983). Theoretically, if a significant proportion of the bacteria1 population is resistant to a high concentration of the metal contaminant, then the judgement is made that the soil is negatively affected by the presence of the metal (Olson and Thornton, 1981). Previous investigations in our laboratory have failed to detect any significant change in metal tolerance of bacterial populations isolated from soil contaminated with metals, regardless of whether the source of the metals was sewage sludge (Kinkle et al., 1987) or smelter emissions (Angle and Chaney, 1991; El-Aziz et al., 1991). No significant changes were detected in metal tolerance of the bacterial isolates, *Author for correspondence.
despite the fact that these bacteria were isolated from soil that was extremely polluted with heavy metals (i.e. 20mg total Cd and 2000mg total Zn kg-‘) (Beyer, 1988). The question, therefore, arose as to why changes in metal tolerance that have been reported for bacteria collected from other environments were not observed in our previous studies. To investigate this question the intrinsic level of bacterial tolerance to heavy metals was determined and compared to the use of conventional methods for assessing metal effects on bacteria. MATERIALS AND
Soil [Weikert silt loam (coarse-skeletal, mixed, mesic shallow Typic Dystrochrept)] was collected from four locations in Palmerton, Pa containing different concentrations of heavy metals. Soil samples were all similar (except for concentrations of heavy metals), having all developed from the same parent material and similar cropping practices [i.e. alfalfa (Medicago sativa L.)]. The texture, pH (pH 6.2-6.5) and cation exchange capacity (approx. 6 cmol kg-‘) of all samples were nearly identical. Additional details have been provided by El-Aziz et al. (1991). Samples were collected from an area in close proximity (O-2 km) to a zinc smelter that had been in operation for nearly 100 yr. Metal oxides (primarily Cd and Zn) had been emitted during this time, although emissions have greatly decreased over the last decade with the installation of pollution control scrubbers. Because metal emissions have been emitted
for nearly 100 yr, metal concentrations are generally in equilibrium with the soil solution. This allowed for the examination of extremely contaminated soils in which the free metal ion concentrations were relatively stable. Metal concentration in soil decreased in the prevailing direction (east) of the wind, with distance. A soil was also collected approx. 1 km upwind from the smelter. Metal concentrations in this soil were not affected by the smelter, although all other soil characteristics were similar. Soils were collected from a depth of O-15 cm and analyzed for Zn, Cd, and Ni by the Aqua Regia procedure to estimate total metal concentrations (McGrath and Cuncliffe, 1985). Soil (5 g) was refluxed with concentrated HN03 and Aqua Regia, and the extract was redissolved in 1.0 M HNO, and analyzed by atomic absorption (AA) spectrophotometry with deuterium background correction for analyses of Cd and Zn. Concentrations were corrected for metal contaminants in the extracting solutions. Extractable metals were measured by shaking 10 g (dry wt) moist field soil for 2 h in 20 ml deionized water or 10 mM Ca(NO,),. Samples were filtered and acidified with concentrated HNOj before analysis. The extraction of metals from soil using water or 10 IIIM Ca(NO,), provides reasonable estimates of the free-ion concentration of metals in soil (Haq et al., 1980) and, thus, are closely related to the potential effect on the bacterial community of soil. The tolerance of the soil bacterial community to metals was determined using traditional (direct plating of soil onto metal-salt amended media) as well non-traditional methods. Each soil was serially diluted and plated onto HEPES-MES (HM) [N-2hydroxyethylpiperazine-N-2 ethanesulfonic acid; 2(N-morpholino) ethanesulfonic acid] mineral salts (pH = 6.8) minimal medium (Cole and Elkan, 1973). The same soil solutions were also plated onto the same medium supplemented with CdCI,.ZH,O, NiC1,.6H,O or ZnCl, at concentrations of 500, 100 and 100 mg of the free metal l-‘, respectively. Metal stocks were autoclaved separately and added with filter-sterilized arabinose to molten HM medium. These metal concentrations were selected because they are commonly used in assessing metal tolerance of microorganisms isolated from the environment
(Olson and Thornton, 1981). To assess metal tolerance of the soil community of bacteria, rather than to specifically examine only organisms resistant to metals, it is necessary to examine the metal tolerance of individual bacteria randomly collected from soil. We initially serially diluted and plated the same soils onto HM medium. No metal selection was used during the initial isolation. Fifty randomly selected isolates were collected from each soil and purified by repeated streaking onto HM medium. Each isolate was then streaked onto media containing a range of each metal. Heavy metals were added to the HM medium as before. Metal ion concentrations examined included: Zn, 5-600 mg 1-l; Cd, l&400 mg I-‘; and Ni, 5-600 mgl-I. Specific concentrations added to the medium varied depending on the range of observed maximum resistance levels (MRL); however, at least 15 concentrations within each range were examined for each metal. After inoculation, plates were kept for 7 days at 28°C. The highest metal concentration that permitted growth of each isolate was called the MRL. RESULTS
Total and free metal ion concentrations
Nickel concentrations in soil were not affected by smelter emissions since nickel oxides were not a primary emission from the Zn smelter (Table 1). Soil A is highly contaminated with Zn and Cd, while soil Z is relatively free from metal contamination. Soils C and D represented intermediate concentrations of Zn and Cd. These soils were used to examine the progression of metal effects on the soil microbial community. The majority of metals deposited on and mixed into soil were adsorbed or precipitated (in or on the solid phase), resulting in low water or dilute salt extractable concentrations, even though total concentrations were very high. Higher extractable concentrations were found with 10 mM Ca(N03), than when water was used as the extracting solution as the result of competition for metal-binding sites on the soil. Populations of metal-resistant bacteria
The soils were examined for the number of metalresistant bacteria using traditional methods. Total
Table 1. Soil pH and total (Aqua R&-AR) and extractable [IO rn~ Ca(NO,),--CN and water-Hz0 heavy metal concentrations in soil. Soils (top 15 cm)] were collected from alfalfa tields at varying distance from the Zn smelter. All values represent the mean of three replicate analyses ZIl Soil
A C D Z LSD,,0,
6.15 6.34 6.38 5.96 0.21
1540 650 728 156 75
0.47 0.25 0.11 0.04 0.19
1.17 0.10 0.08 0.05 0.24
32.4 8.63 11.6 1.45 15.0
ND = Not determined
CN (mg kg-‘) 0.036 0.026 0.017 0.005 0.009
0.022 0.001 0.004 0.001 0.007
25.4 14.7 24.4 26.1 2.9
0.26 0.028 0.011 0.42 0.13
ND ND ND ND ND
Metal resistance of bacteria bacterial numbers were lO-lOO-fold lower on HM medium than when soil is plated onto richer media (Table 2). HM medium does not support the growth of all soil bacteria, a problem inherent to any biological medium. HM medium was used, however, because Angle and Chaney (1989) examined free metal ion concentrations in HM medium and determined the extent of metal-binding in this medium supplemented with metal salts. We also found that this medium does not select for a fraction of the total bacterial community that is any more or less metalresistant than bacteria cultured on media such as soil extract agar (unpublished data). By dividing the number of bacteria growing on the metal supplemented medium by the total population for each soil, the proportion of the culturable bacterial community resistant to the noted metal concentrations was calculated. There was a general correlation between the extractable metal concentration of Zn and Cd in soil and the percentage of bacteria resistant to the metal. For example, the percentage of bacteria resistant to Zn increased 120 fold between the least (Z) and most (A) contaminated soils (Table 2). The metal pressure imposed upon this community will result in a shift to relative metal resistance. Hence, the traditional approach to assess metal effects on the bacterial community, when the soils are highly contaminated, appears to provide accurate relative comparisons. The difference in biological effects between total and extractable concentrations of metals in soil is best exemplified by comparing extractable Zn concentrations in soil C and the concentration of Zn added to the media to enumerate the proportion of the total bacterial population resistant to Zn. Water and 10 mM Ca(NO,), extractable Zn soil concentrations were 0.01 and 0.25 mg kg-‘, respectively. The arbitrary medium concentration to establish resistance, however, is 500 mg Zn 1-l. Hence, using traditional methods, for an organism to be considered tolerant to Zn, it must be 2000-5000 times more tolerant to Zn than concentrations of the extractable form found in contaminated soil. The use of excessively high total metal additions to biological media may therefore
Table 2. Total culturable soil bacterial populations and percentage of the population resistant to routinely used metal additions incorporated into HM medium. The total population was determined by plating serial dilutions of soil onto HM medium not amended with metal salts. The percentage of the total population resistant to 5OOmgZnl~‘,lOOmgCdI-’ and IOOmg Nil-‘wasdetermined by plating the same serial dilutions onto HM medium supplemented with individual metal salts. All values are means of analyses conducted in triplicate Total population
Soil A C D Z LSD,,,
Total cultarable population (104 g-1) 182 176 228 322 86
(?) 4.8 1.5 0.81 0.04
4.1 3.7 5.2 0.46
0.43 3.1 I.5 0.05
Table 3. Geometric mean (K) of the maximum resistance level (MRL), standard deviation (a) and range (R) of 50 randomly collected soil bacteria. Each purified isolate was plated onto HM medium supplemented with varying concentrations of Zn, Cd or Ni Maximum
75 73 43 26
~ I80 25-500 I35 20-500 26 20-120 86 5-250
65 60 50 24
(mg I-') 84 73 96 20
25-300 25-300 15-300 l&50
’ 68 83 182 174 51 53 25 47
l&300 25-500 IS250 5-175
result in the failure to detect metal-induced population changes when metal pressure in soil is low to moderate. It is therefore suggested that lower standard metal additions to media be used to assess metal effects on the soil microbial population. Metal tolerance of individual bacterial isolates
Most isolates were resistant to metal concentrations much higher than extractable metal concentrations found in any of the soils, even the most polluted soil (Table 3). Soil bacteria originally isolated on HM medium possess an intrinsic degree of metal tolerance that generally precludes their need to adapt to the introduction of metal stress. Even the most metal-sensitive isolates were intrinsically tolerant to much higher concentrations of metals than extractable concentrations found in soil. Mean MRLs were much lower (except for soil D, Ni) than concentrations added to the medium in traditional assays (Table 3). Only a very small percentage of the total bacterial community (as is evident in Table 2) was capable of surviving on media amended with 500, 100, and 100 mg 1-l Zn, Cd, and Ni, respectively. The ranges of the MRLs shows that metal concentrations added to media are generally excessively high and may thus fail to detect changes that are occurring at lower metal concentrations in soil. DISCUSSION
It is well established that the free, hydrated metal ion is the toxic form of metals present in the environment. DeKock and Mitchell (1957) reported that chelated heavy metals are not toxic to plants and Halvorson and Lindsay (1977) showed that chelated metals are not available for uptake by plants. Similar results have been reported for shrimp, further demonstrating the importance of free metal ions in solution (Sunda et al., 1978). Numerous extracting agents have been used to estimate metal ion concentrations, or bioavailability, of metals in the soil solution (Lindsay and Norvell, 1978; Logan and Chaney, 1983). Such studies have attempted to relate metal concentrations extracted with dilute salts, dilute acids, water or chelating agents to metal uptake by plants. Each of these extracting solutions has been shown to provide a reasonable estimate of plant-available metals, although no single method is
appropriate for all soils. Theoretically, plant roots and microbes are exposed to the same soil solution and concentrations around a root or bacterial cell should be identical. Extractable metals are not an exact measure of the free ionic concentration of metals in soil, but their determination is a very significant improvement over the use of total metal concentrations. The myriad of inorganic factors in soil, pH, CEC, etc. often are the controlling factors in determining metal availability (Babich and Stotzky, 1980). Extractable metals are a very small fraction of the total concentration in soil. For example, whereas soil A contained 1540 mg kg- ’ total Zn, only 0.47 mg kg-’ was in the extractable form (Table 1). It should be noted that most media, including HM, support only a very small fraction of the total microbial population. It is impossible, however, to examine the metal tolerance of bacteria that cannot be cultured. We, therefore, emphasize that our results and conclusions apply only to the culturable population of soil bacteria. The same reactions that affect the free metal ion concentrations in soil also operate in media and control the extent to which the microbial community is affected by the metal. Ramamoorthy and Kushner (1975) and Collins and Stotzky (1989) demonstrated that complex microbial growth media bind significant quantities of heavy metals. The extent of binding of metals in HM medium, however, is relatively low compared to media containing undefined nutritional components (yeast extract, tryptone, etc.). Therefore, although we believe the use of defined minimal media to be a significant improvement over other, more traditional media to examine metal effects on soil bacteria, problems remain with the interpretation of the observed results. In conclusion, two important observations regarding heavy metal effects on soil bacterial populations and the use of these populations as indices of metal pollution have been made. First, metal concentrations typically added to media to enumerate that proportion of the population which is resistant to metals are excessively high. The use of such high standard metal additions to media may result in the failure to detect important changes in the bacterial population inhabitating contaminated soil. We have also shown that bacterial population changes in metal contaminated soil should be minimal, as most soil bacteria are intrinsically resistant to high concentrations of metals. Acknowledgements-This
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