MICROBIAL REMEDIATION OF SOILS CO-CONTAMINATED WITH 2,4-DICHLOROPHENOXYACETIC ACID AND CADMIUM
T.M. Roane and I.L. Pepper
Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ 85721, Phone: 520(621)5988; FAX: (520)621-1647
One-third of organically-polluted sites are also contaminated with metals; however, the bioremediation potential of such sites is not clear. While metals are thought to inhibit the abilities of microbial communities to degrade organic pollutants, several microbial-metal resistance mechanisms are known to exist. This study utilizes cadmium-resistant soil microorganisms to enhance the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of cadmium. Cadmium-resistant bacteria were isolated from both a 40-year-old metal-contaminated soil and an uncontaminated soil. During growth experiments, it was found that the uncontaminated soils had a greater number of resistant isolates at low concentrations of cadmium, while cadmium-contaminated soils exhibited higher microbial resistance with increased cadmium concentrations. ERIC PCR fingerprints discriminated among the cadmium-resistant isolates identified by BIOLOG as Bacillus, Corynebacterium, Pseudomonas, and Xanthomonas spp. These isolates were resistant to concentrations ranging from 5 to 275 ppm soluble cadmium. In conventional degradation studies, two resistant isolates, a Bacillus and an unidentified Gram positive rod, supported the degradation of 500 ppm 2,4-D by the cadmium-sensitive 2,4-D degrader Alcaligenes eutrophus JMP134 in the presence of 20 and 40 ppm soluble cadmium, respectively.
Keywords: co-contaminated soil, cadmium-resistance, bacteria, 2,4-D, remediation
While little is known about the degradative capabilities of microorganisms in the presence of a metal stress, it has generally been thought that metal toxicity inhibits degradation of organics (Said and Lewis, 1991). Yet Springael, et al. (1993) and Mergeay, et al. (1985) have shown that 2,4-D degradation can occur in the presence of nickel and zinc, showing it is possible to have organic biodegradation in metal-contaminated soils. The issue to co-contamination, the presence of both an organic and a metallic pollutant, is a serious one. Approximately 30% of all organically-contaminated sites are also contaminated with metals (Kovalich, 1991).
While metals are toxic to most biological systems, attacking nucleic acid and enzymatic pathways, microorganisms have developed a variety of mechanisms for protection against metal toxicity (Roane, et al., 1996). Metal resistance strategies are either to prevent entry of the metal into the cell or to actively pump the metal out of the cell. This can be accomplished by either sequestration, active transport, or chemical transformation through metal oxidation or reduction. Despite microbial metal resistance, not all resistance mechanisms render the metal nontoxic to biological systems. Consequently, unlike organics, metals cannot be degraded and metal remediation strategies have to rely on detoxification and immobilization of the metal to protect biological systems and to retard metal transport.
The use of metal-resistant microorganisms in the bioremediation of co-contaminated soils has been poorly documented. In this study, several cadmium-resistant bacteria, isolated from both metal-contaminated and a pristine soil, were examined for their ability to detoxify cadmium (Cd) in a co-contaminated system in order to facilitate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Alcaligenes eutrophus JMP134 containing the pJP4 plasmid encoding for 2,4-D degradation.
MATERIALS AND METHODS
Field Soil Characterization
In the late 1940s, more than 3.2 million pounds of aluminum-dross was deposited in the area now known as the Olive Grove neighborhood, located just east of Tucson, AZ, bordering the Davis-Monthan Air Force Base. Discarded before 1980 when the Resource Conservation and Recovery Act (RCRA) was established, aluminum-dross, a metallic ash by-product of the meltdown of scrap aluminum, contains potentially toxic levels of cadmium and lead. Containment efforts are currently underway to reduce human exposure. For this study, two dross-impacted soils (OG1 and OG2) were collected from the Olive Grove neighborhood. Collected from the University of Arizona Campbell Avenue Agricultural Station, Tucson, an uncontaminated control soil, Brazito, was used for comparison purposes.
The soil samples obtained represent the top 10 cm of the soil surface horizon. The following soil analyses were performed by the University of Arizona Soil, Water and Plant Analysis Laboratory: pH (Page, et al., 1982), percent organic carbon (Artiola, 1990), and total cadmium and lead (U.S. EPA Method 6010).
The total microbial numbers in each study soil were based on direct acridine orange staining of diluted soil (between 10-100 cells per field) slurries (1 g-soil dry weight with 9.5 ml sterile sodium pyrophosphate, Na4P207(H20)10. Twenty fields per slide were examined using brightfield microscopy (Hobbie, et al., 1977).
Culturable soil bacterial numbers were determined using conventional plating techniques, from the soil slurries described above, onto the minimal-nutrient medium for heterotrophic organisms, R2A (Difco, Baltimore, MD): 0.5 g yeast extract; 0.5 g proteose peptone; 0.5 g casamino acids; 0.5 g dextrose, C6H1206; 0.5 g soluble starch, (C6H1005)n; 0.3 g sodium pyruvate, NaC3H303; 0.3 g potassium phosphate, K2HP04; 0.05 g magnesium sulfate, MgS04; and 15 g agar, pH 7.2. Plates were incubated for one week at 25oC before counting.
Community Cadmium Resistance
The degree of cadmium toxicity on a community level was assessed by exposing two different soil microbial communities, from the metal-contaminated OG1 and the uncontaminated Brazito, to various cadmium-as CdCl2-concentrations in a defined mineral medium: 0.5 g sodium citrate, C6H5Na307; 0.1 g magnesium sulfate, MgS04; 1.0 g ammonium sulfate, (NH4)2S04; 1.0 g glucose, C6H1206, and 0.1 g sodium pyrophosphate, Na4P207(H20)10, buffered to pH 6.0 with potassium phthalate, KHC8H404.
Soluble cadmium concentrations ranged from 0-40 ppm. One-ml of a 1:10 soil slurry (1 g soil-dry weight with 9.5 ml sterile sodium pyrophosphate, vortexed 2 min.) was used to inoculate 25 ml of the defined mineral medium amended with cadmium.
Bacterial growth was monitored every 24 hr for nine days when stationary phase growth was reached. To determine the actual numbers of cadmium-resistant bacteria, the above cultures were plated onto the previously defined mineral medium amended with the same level of cadmium as the corresponding liquid culture flask.
Soluble Cadmium Determination
Soluble cadmium concentrations were determined using a flame atomic absorption spectrophotometer by measuring the amount of cadmium left in solution following a 0.2 µm filtration.
Characterization of Cadmium-Resistant Isolates
Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC PCR) was used to genetically distinguish the isolates from each other (Versalovic, et al., 1994). ERIC PCR uses primers representing conserved DNA sequences found in all bacteria. The product of the random insertion and amplification of differently sized DNA segments, the resulting genetic fingerprints differentiated among the isolates. PCR products were electrophoresed in a 2% agarose gel at 100 V cm-1.
The BIOLOG bacterial identification system (Biolog, Inc., Hayward, CA) uses a 96-well carbon utilization assay to metabolically fingerprint and identify individual isolates. In this study, the BIOLOG system was used to identify, to the genus level, six randomly chosen cadmium-resistant isolates from both metal-contaminated and uncontaminated soils.
Cadmium Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) of cadmium, which is defined as the level at which toxicity is first observed as a decrease in viable numbers, was determined for each isolate. The MIC of cadmium reflected the degree of cadmium-resistance. Of two isolates, A and B, for example, with MICs of 10 and 20 ppm cadmium, respectively, isolate B is more resistant because it takes a greater amount of cadmium to decrease the number of organisms.
Since degradation is often inhibited in the presence of metal(s), the ability of the cadmium-resistant isolates to support 2,4-D degradation by A. eutrophus JMP134, carrying the pJP4 plasmid for 2,4-D oxidation in the presence of toxic concentrations of cadmium, was examined. Under normal conditions, A. eutrophus JMP134 with the pJP4 plasmid can degrade 2,4-D; however, even with low cadmium levels present (<5 ppm), degradation was inhibited. Of interest was the potential of the selected cadmium-resistant isolates to detoxify cadmium such that A. eutrophus JMP134 could degrade 2,4-D. The cadmium-resistant isolates alone were unable to degrade 2,4-D in the presence of cadmium.
Five-ml of defined mineral medium amended with 500 ppm 2,4-D and either 20 or 40 ppm soluble cadmium, depending on the MIC of the resistant isolate, was inoculated with 106 cells ml-1 metal-resistant isolate. Following a 48 hr incubation at 25oC to allow for growth of the metal-resistant organism, the culture was then inoculated with 106 cells ml-1 A. eutrophus JMP134. Upon addition of the 2,4-D degrader, 2,4-D concentrations were monitored spectrophotometrically at 230 nm every 24 hr. Similarly, each cadmium-resistant isolate was also tested for its ability to degrade 2,4-D in the presence of cadmium. The 2,4-D readings were conducted on culture extracts (1 ml culture-microcentrifuged at 14,000 rpm for 2 min.).
Once established in pure culture, the ability of successful isolates to protect A. eutrophus JMP134 from cadmium-toxicity was examined in artificially metal-contaminated soil. Fifty-grams (dry weight) of uncontaminated Brazito soil was amended with 1% (w/w) glucose, and 500 ppm 2,4-D and 100 ppm cadmium. Glucose was used as a readily metabolizable carbon source for the cadmium-resistant populations. Soil moisture was kept at 16% throughout the experiment. Each soil microcosm (in 500 ml wide-mouth polypropylene jars) containing 50 g amended soil was inoculated with 104 cells g-1 cadmium-resistant organism. Following a 48 hr incubation at 25oC, each microcosm was inoculated with 104 cells g-1 A. eutrophus JMP134. The concentrations of 2,4-D in each microcosm were monitored periodically throughout a seven-week period. Control microcosms consisted of Brazito soil amended with glucose; 2,4-D and cadmium without either inocula; and in addition, Brazito amended with glucose, 2,4-D and cadmium with only the A. eutrophus inoculum.
Soil Physical and Chemical Characteristics
Physiochemical parameters known to influence soil microorganisms and the bioavailability of cadmium were measured (Table 1). Both metal-contaminated soils (OG1 and OG2) and the non-metal-contaminated soil (Brazito) were similar in terms of soil pH (approximately 8), and soil texture (all were sandy loams), and ranged from 0.2-0.5% organic carbon. Soil OG2 had substantially higher cadmium and lead levels than soil OG1 (5 and 55 ppm, and 75 and 1660 ppm, respectively) and the uncontaminated Brazito soil. The similarities in soil parameters, other than metal levels, provided the basis for the microbiological comparisons of these soils.
Total bacterial numbers were similar in all three soils, ranging from 7.2x107 to 2.7x1010 cells g-1 soil (Table 1). As expected, total numbers were higher in all soils when compared to viable numbers with the uncontaminated soil exhibiting the greatest culturable recovery (3.2x107 CFU g-1 soil). Likewise, both metal-contaminated soils showed decreased cultural counts (9.9 and 5.7x105 CFU g-1 soil, respectively). The culturable cells recovered from the metal-contaminated soils were four to five orders of magnitude less than the total cells. For Brazito, a far greater percentage of total cells were culturable.
Community Growth Response to Cadmium
After a 24-hour incubation, as the cadmium concentration increased, the number of cells in all soils decreased. However, both metal-contaminated soils, OG1 and OG2, exhibited one to two orders of magnitude greater populations than the uncontaminated Brazito soil. Figure 1 represents the growth response with respect to increasing cadmium concentrations for the microbial communities in these soils. Each of the communities exhibited cadmium toxicity upon initial exposure to 5 ppm cadmium with soil OG1 least affected, while both Brazito and OG2 declined sharply.
Following a 72-hour incubation, it was noticed that the OG1 population was two to three orders of magnitude greater at 40 ppm than 20 ppm bioavailable cadmium. This phenomenon was repeatedly observed in subsequent trials (Fig. 2). Again, as expected, numbers declined upon cadmium exposure; however, the numbers seemed to recover upon further exposure, indicating cadmium toxicity followed by the appearance of a resistant population. This phenomenon was not evident in the uncontaminated Brazito soil and was seen to a lesser extent in the other metal-contaminated soil OG2.
Isolate Identification and Cadmium Resistance
Six culturable cadmium-resistant organisms were selected and characterized as summarized in Table 2. While isolate I1a could not be identified, BIOLOG gave the closest match for the other isolates. The genera observed, Bacillus, Corynebacterium, Pseudomonas and Xanthomonas, represented common soil organisms. ERIC PCR confirmed that each isolate was genetically unique and could be distinguished from each of the others based on their DNA fingerprints (Fig. 3).
The MIC of cadmium for these organisms was represented by the highest concentration of cadmium tolerated before a significant decline in microbial numbers was observed. For these six isolates, the MIC of bioavailable cadmium varied from <5 ppm to 275 ppm.
Pure culture: In order for the cadmium-sensitive 2,4-D degrading A. eutrophus JMP134 to survive and metabolize 2,4-D in the presence of cadmium, bioavailable cadmium concentrations must be rendered nontoxic. The ability of four of the most cadmium-resistant isolates, D9, H1, H9 and I1a, to detoxify cadmium such that A. eutrophus JMP134 could degrade 500 ppm 2,4-D, was determined (Fig. 4). Earlier experiments showed that A. eutrophus alone in the presence of cadmium could not degrade 2,4-D, presumably because of cadmium toxicity. Likewise, in the presence of cadmium, none of the cadmium-resistant organisms tested could degrade 2,4-D. However, in the mineral salts-medium amended with 2,4-D, cadmium and 106 cells ml-1 of cadmium-resistant isolates I1a or H9, 20 and 40 ppm soluble cadmium, respectively, were detoxified. This allowed an initial inoculum of 106 cells ml-1 A. eutrophus to degrade 500 ppm 2,4-D to 37 and 52 ppm. Isolates D9 and H1 were unable to detoxify cadmium.
Soil microcosms: Artificially-contaminated Brazito soil with both 2,4-D and cadmium was partially remediated through the addition of cadmium-resistant isolates to facilitate cadmium detoxification and subsequent 2,4-D degradation by A. eutrophus JMP134. Each of the isolates examined, within 50 days, allowed for the complete degradation of 500 ppm 2,4-D (Table 3). Interestingly, neither the indigenous microbial flora nor the cadmium-resistant isolates alone could degrade 2,4-D in the presence of cadmium. Additionally, A. eutrophus JMP134, without the assistance of one of the resistant isolates, could not degrade 2,4-D in the presence of cadmium, indicative of cadmium toxicity.
A limitation in metal-microbial studies is that the total metal concentration does not necessarily reflect the amount of metal that is biologically toxic or bioavailable. Recently, investigators have been trying to elucidate the ecological implications of bioavailable metal. Primarily toxic in their free form, metals are thought to be more toxic when soluble versus sorbed to humic or clay colloids (McLean and Beveridge, 1990). Consequently, in this study, an effort was made to report the influences of bioavailable soluble cadmium only on the remediation of co-contaminated soils.
The primary difference among the soils examined in this study was metal concentration which was reflected in both the total and culturable numbers of microorganisms present in these soils. Several studies have shown that chronic metal stress results in decreased bacterial diversity, biomass, and activity (Baath, 1989). In the present study, while the uncontaminated control soil had a typical number of culturable cells present (~107 cells g-1 soil), both metal-contaminated soils had lower culturable organisms but perhaps more striking was that the culturable numbers were four to five order of magnitude less than the total number of organisms in the soils. This indicated a high number of viable but nonculturable organisms, that is, microorganisms which are unable to grow on conventional laboratory growth media. The high number of viable but nonculturable organisms was another manifestation of the metal stress in these soils. In comparison, the uncontaminated control soil had similar total and culturable numbers, a reflection of ecosystem health.
As would be expected, upon exposure to toxic levels of cadmium, the number of culturable organisms decreased in both the metal-contaminated and the uncontaminated soils. The microbial communities from both metal-contaminated soils were more resistant (in terms of numbers of surviving organisms) than the uncontaminated community to the levels of cadmium examined (5, 20 and 40 ppm soluble cadmium). However, upon closer examination of the cadmium-resistant populations from the metal-contaminated soil OG1, the resistant population became more resistant as the cadmium levels increased. This community was more resistant at 40 ppm versus 20 ppm bioavailable cadmium. Intuitively, one would expect the number of surviving organisms to continue to decrease as cadmium levels became increasingly toxic.
This observation was interesting because it raised the issue of whether or not some metal-resistant microorganisms have and can use multiple mechanisms of resistance to the same metal. There are several known pathways for metal resistance in microorganisms. Examples include the production of a secreted polysaccharide layer which surrounds the cell and can ionically sequester metals preventing their entry into the cell. Interestingly, the production of these polymeric layers often occurs without exposure to metal and is known to be involved in adhesion, nutrient storage, and protection against desiccation and other environmental assaults. Another mechanism may involve the production of specific metal-binding proteins which actively bind metals inside the cell to retard the metal's interaction with essential cellular processes. Some bacteria actively pump the metal back out of the cell once it has crossed the cell membrane with the use of energy-dependent efflux pumps. To date, most metal-resistance research is based on the presence of only one resistance mechanism in a particular organism. However, might such an organism use a "gratuitous" mechanism of resistance ( a mechanism that is not necessarily in response to the metal itself), such as polysaccharide production, to protect itself under less toxic metal exposure, only to switch to a more aggressive, metal-directed resistance when metal levels are more threatening? Two other studies by Roane and Kellogg (1996) in a study of lead-resistance in soil communities from lead-contaminated soils, and by Chech and Miller (personal communication, University of Arizona) in a study of the cadmium-resistance of a naphthalene degrading bacterium, have also observed increasing resistance with increasing metal concentrations. These observations merit further investigation.
Six of the cadmium-resistant populations from the above growth study were identified as either Bacillus, Pseudomonas, or Xanthomonas spp. One population could not be identified using the BIOLOG identification system. While each isolate was also shown to be genetically distinct from each of the others using ERIC PCR, each isolate also differed in its resistance to cadmium. Two of the most resistant isolates were a Bacillus and a Pseudomonas, resistant up to 225 ppm and 275 ppm soluble cadmium, respectively. Yet, two other bacilli were least resistant, only up to 5 ppm cadmium. These six isolates demonstrated the diversity of organisms and variable degrees of cadmium tolerance to be found in response to metal exposure.
It is generally believed that organic degradation is inhibited in the presence of metal, presumably due to metal toxicity; however, few studies have actually addressed this issue. The ultimate goal of this project was to find cadmium-resistant bacteria that could either resist cadmium and degrade 2,4-D (used as a model for organic degradation); or could detoxify cadmium to allow for another, metal-sensitive organism to carry out the degradation. To examine this potential, four of the six characterized populations, resistant to greater than 20 ppm soluble cadmium, were used in 2,4-D degradation studies. Alcaligenes eutrophus JMP134 was used as a metal-sensitive 2,4-D degrader. In pure culture experiments, two of the isolates, resistant to 20 ppm and 275 ppm cadmium, were able to detoxify cadmium to such an extent that complete 2,4-D degradation from an initial concentration of 500 ppm occurred in the presence of the toxic levels of cadmium.
The results of this experiment imply that these two populations actually detoxify cadmium through some sort of sequestration or precipitation mechanism since the toxic or soluble concentration of cadmium had to be reduced to allow for A. eutrophus survival and metabolism. This experiment also demonstrated the potential use of these isolates in microcosm studies involving artificially metal-contaminated soils.
In soil microcosms with artificially-contaminated Brazito soil with 500 ppm 2,4-D and 100 ppm cadmium, all four isolates examined successfully detoxified cadmium, thereby protecting A. eutrophus JMP134 from toxicity. While 2,4-D levels were undetectable after 50 days, implying complete degradation, the rate of degradation would be expected to vary in response to the efficiency of cadmium detoxification as seen in the pure culture work. The Brazito used in these microcosms was not sterile; yet the indigenous community was unable to degrade 2,4-D in the presence of 100 ppm cadmium. Neither could the individual cadmium-resistant isolates degrade the organic with cadmium present. Finally, while A. eutrophus JMP134 could degrade 500 ppm 2,4-D in Brazito without cadmium within 72 hr, with cadmium present in the soil, A. eutrophus was unable to degrade the 2,4-D after 50 days.
This study has demonstrated the potential use of metal-resistant microorganisms in the remediation of co-contaminated soils. The primary mode of action being metal detoxification such that organic degradation is no longer inhibited. This study has also raised the interesting question of whether metal-resistant organisms can select for different mechanisms of detoxification depending on the level of metal stress, having important implications in the remediation of any metal-impacted ecosystem. This idea alone can drastically influence our understanding of how microorganisms respond to their environment.
This work was supported in part by Grant number 5 P42 ESO4940-07 from NIEHS, Superfund Program. We thank the Arizona Department of Environmental Quality for access to the Olive Grove contaminated soils.
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Table 1. Characterization of two metal-contaminated soils, OG1 and OG2, and the uncontaminated control soil Brazito.
Table 2. Isolate identification and cadmium-resistance.
|Isolate||Bacterial identification||BIOLOG Scorea||MICb of Cd (ppm)|
aBIOLOG identified the isolates to varying degrees and in several instances could
only give the closest match.
bMIC was based on soluble cadmium concentrations.
cBIOLOG could not identify this isolate.
Table 3. 2,4-D degradationin Brazito soil amended with 500 ppm 2,4-D and 100 ppm cadmium in the presence of cadmium-resistant isolates (I1a, D9, H9, and H1).
|Soil Amendment||2,4-D Concentration (ppm)|
|Day 0||Day 5||Day 10||Day 40||Day 50|
|100 ppm Cd||500||500||500||500||500|
|A. eutrophus JMP134||500||0||0||0||0|
|100 ppm + A. eutrophus JMP134||500||500||500||500||500|
|100 ppm Cd + Cd-resistant isolatea||500||500||500||500||500|
|100 ppm Cd + Cd-resistant isolate
+ A. eutrophus JMP134
aEach amendment included one of the four cadmium-resistant isolates: I1a, D9, H9, H1.
Fig. 1. Total microbial community response to cadmium
concentration from different soils measured as culturable cell concentrations following a 24 hr exposure.
Both OG1 and OG2 soils were metal-contaminated, while Brazito represented an uncontaminated control.
Fig. 3. Agarose gel of ERIC PCR fingerprints for each
cadmium-resistant isolate. Fingerprints for all isolates were unique. Lanes: 1, 123-bp DNA ladder as a
size standard; 2, Isolate L1; 3, Isolate I1a; 4, Isolate H1; 5, Isolate D9; 6, Isolate H9; 7, negative control
Fig. 2. Increased resistance with 72 hr exposure to a
higher cadmium concentration of cadmium-resistant populations from the metal-contaminated soil OG1
measured as culturable cell concentrations. Each line represented four replicate trials.
Fig. 4. 2,4-D degradation by metal-sensitive A. eutrophus JMP134 in the presence of cadmium and cadmium-resistant isolates I1a, D9, H9 and H1. Within 72 hr, both isolates I1a and H9 allowed for complete degradation of 500 ppm