BACTERIAL DIVERSITY AT A MIXED WASTE CONTAMINATED SITE

Joynt J.*§, Konopka A.π,  Nakatsu C.*

*Department of Agronomy, π Department of Biology, Purdue University, West Lafayette, Indiana, USA, 47907

§ jjoynt@purdue.edu

 

ABSTRACT

Many scientific investigations have studied the effects of heavy metals and organic contaminants on microbial biomass and activities in soil.  It remains unclear however, whether organic or metal contamination limit the bioremediation potential at a mixed waste site.  We investigated the potentially selective factor limiting remediation by comparing the diversity of microbial populations present along a 21.6 m transect at a mixed waste site contaminated with lead, chromium and various organic compounds

.  Soil microbial DNA was extracted from 24 locations and population patterns for each sample location were compared by denaturant gradient gel electrophoresis (DGGE) of polymerase chain reaction amplified 16S rDNA.  The DGGE patterns demonstrated that sample locations with high concentrations of total xylenes, methylene chloride, toluene, high lead and chromium have a different community composition from the community with low organics.

 

INTRODUCTION

Hazardous waste sites often contain both heavy metal and organic contaminants in the soil.  Many reports have shown an acute effect of heavy metal stress is decreased microbial biomass (Kandeler et al., 1996; Knight et al., 1997), and a chronic effect is decreased microbial metabolic activity (Bååth et al., 1991; Doelman and Haanstra, 1979; Insam et al., 1996). However, these studies have also suggested that over time there is a gradual change in the microbial composition towards a more metal tolerant community.  If a tolerant community is able to develop in soils, than the apparent decrease in biomass and activity that has been reported may not be due to the toxicity of the heavy metal itself, but rather due to an indirect effect of metals such as the lack of available organic carbon in the soil.  Bioremediation at a mixed waste contaminated site may therefore, not be limited by the heavy metals but by the toxicity and availability of the organics that are present. 

 

By looking at the microbial community structure at a long-term mixed waste site, the diversity of the community can be compared between high and low metal and organic contaminant concentrations.  If bacteria become tolerant and are able to adapt to metal stress, then a relatively diverse community would be expected irrespective of the metal concentrations present because the metals are not inhibiting the bacteria. However, if the community cannot adapt to heavy metals than fewer members would be expected to survive and diversity would be lowered as metal concentrations increased.

 

The objective of this study was to investigate how microbial community structure is altered across high to low metal and organic gradients at a mixed waste contaminated site. Bacterial DNA was extracted directly from soil samples, and a fragment of the 16S rDNA gene was PCR amplified and separated using DGGE to provide a picture of the community complexity and structure. 

 

 

MATERIALS AND METHODS

Field site

The sampling site was located at an Indiana Department of Transportation (INDOT) property in Seymour, Indiana. Waste road paint containing lead and chromium, and waste cleaning solvents containing toluene, xylenes, ethylbenzene, methylene chloride and trichloroethylene were dumped at the site and covered with backfill material since the late 1960's.  In July, 1999 soil was sampled along a 21.6 m long and 0.5 m wide trench at the site. A total of twenty four samples were collected along the transect taken at 0.9 m intervals alternating along the north/south sides of the trench  at a 0.6 to 0.9 m depth. 

 

Chemical analysis

Soil was sent to Brookside Laboratories Inc. in New Knoxville, Ohio for chemical and physical analysis.  The following parameters were measured: pH, total organic carbon, total lead, total chromium, nitrogen, phosphorous, potassium, volatile organic carbon, texture, and cation exchange capacity.

 

Soil DNA extraction/ Polymerase chain reaction (PCR) / Denaturing gradient gel electrophoresis (DGGE)

Soil DNA was extracted in triplicate using the FastDNA Spin Kitâ (Bio101) soil DNA extraction kit following manufacturers instructions. DNA amplification was carried out for subsequent use in DGGE using universal bacterial primers that flank the V3 region of the 16S rDNA gene.  The primers (synthesized by Integrated DNA Technologies Inc.) are: 5’ AC TCC TAC GGG AGG CAG CAG 3’ corresponding to position 338-358 in Escherichia coli, with a GC clamp (5’-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G-3’) attached to the 5’ end, and reverse primer 5’-ATT ACC GCG GCT GCT GG-3’ corresponding to position 534-518 in E. coli (Konopka et al., 1999).  The GC clamp is present in order to stabilize the DNA in the DGGE gel. 

 

Denaturing gradient gel electrophoresis was carried out as described previously (Konopka et al., 1999).  Briefly, PCR products were resolved on an 8% (wt/vol) polyacrylamide gel (37.5:1 acrylamide:bisacrylamide) in 0.5X TAE (20 mM Tris-HCl, 10 mM acetate, 0.5 mM EDTA) plus denaturant (100% denaturant contains 7M urea, 40% formamide in 0.5X TAE). Electrophoresis was performed on a D-Gene apparatus (BioRad) at a constant voltage of 20V for 15 minutes, followed by 200V for 4.5 hours.  All gels were initially run using a 30-55% denaturant gradient. Additionally, 30-70% denaturant gradients were used to show bands with high G+C contents that migrated off the 30-55% gels.

 

RESULTS

Chemical analysis

Figure 1 shows the lead and chromium concentrations across the site as well as total organic carbon for the north side of the transect.  Lead (Pb) and chromium (Cr) concentrations in the soil ranged from 4 ppm to 17 000 ppm and 4 ppm to 3200 ppm respectively, and volatile organic concentrations ranged from 23 000 ppm to <1 ppm in soil. Soil was mainly fill material, consisting largely of sand (62-88.6%) with some pockets of high clay (37.6%). Total soil concentrations of Pb and Cr were higher in samples that also contained high organic contaminants, and had generally lower clay content.  

 

DGGE analysis

Bands in the DGGE profile correspond to 16S rDNA fragments that differ in their nucleotide sequences, and as a result migrate to different positions in the gel. DGGE analysis of soils along the transect is shown in Figure 2.  Comparison of the bacterial communities from each of the samples shows distinctly different profiles from left to right across the gel.  Samples HN1, HN2 and HN3 appear to have similar banding profiles, whereas samples HN6 to HN12 also have a similar profile. These samples correspond to samples with high and low metals respectively.  Sample HN4 and HN5 have bands common to both sets of communities and appear to be transition samples.  Several populations with high G+C contents were found only in samples HN1 to HN3, which have high volatile organic concentrations.  Not all of these populations can be seen in Figure 2, but are discernable when the denaturant is extended to 70% (data not shown). Two bands were present in all samples irrespective of metal or organic concentrations.  There were more unique bands however, than common bands across the site. 

 

DISCUSSION

In this study the primary interest was investigating the bacterial community structure and diversity at a metal and organic contaminated site using DGGE.  The DGGE profile shown in Figure 3 demonstrates that the community across the site has several different populations of bacteria present in both high and low Pb and Cr soil concentrations as indicated by the number of bands present in each sample.  This supports the inference that a bacterial community is able to adapt to a heavy metal environment.  The separation of the profile patterns into two distinct groups (HN1to HN3 and HN6 to HN12) coincides with the presence of high levels of organic contamination in one set (HN1 to HN3) and the absence of organic contamination in the other (HN6 to HN12).  Both sets of profiles have high levels of Pb and Cr.  However, in the profiles for HN6 to 12, sample HN10 has a Pb concentration of 351 ppm, and sample HN11 has a Pb concentration of 3.73 ppm, yet the profiles remain consistent irrespective of the metal concentration. These results suggest the organics rather than metals at the site may predominantly influence the community composition in the present study.  Recently, Konopka et al., 1999 investigated microbial diversity by DGGE at a lead contaminated site, and found that lead soils in that study were more comparable to an agricultural soil with a smear of bands, than the organic contaminated soil in which the diversity was severely reduced.  The soils in the present study showed a diverse community, although they did not produce a smear of bands as reported in Konopka et al., 1999, which may be attributable to the organic contaminant influence. However, if the community composition was solely influenced by the organics, than soils with low organic contamination should have a greater diversity than soils with high organic contamination. This was not the case.  Two reasons may explain this.  First, soil chemical data at the site show trace levels of organics contaminants present in the HN6-HN12 samples which indicates organics were present in these locations at some point.  These samples may therefore have reduced diversity as a result of these previous contaminants.  Second, soil properties such as texture and CEC differ between the two profile sets, which may be influencing the community structure.

 

In conclusion, it can be seen that lead chromium and organic contamination are influencing the microbial community structure present at the site, although the extent of their influence is not known.  More work must be done to determine the dominant selective pressure on the community.  Future work entails nucleotide sequence determination of the dominant bands present in distinct sample profiles, and monitoring of community shifts with metal and organic and both added to the soil.

 

Acknowledgements: Funding was provided by the DOE-NABIR Program, grant #DE-FG02-98ER62681

 

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Figure 1: Total lead, chromium and total organic carbon concentrations along the north side of the transect.

 

 

Figure 2: Samples HN1 to HN12 across the site transect.  Samples were extracted from soil and a section of the V3  region of 16S rDNA gene was PCR amplified, then run on a  30-55% DGGE gel.  Bands at different positions in the gel represent different bacterial populations. Samples in figure start with a marker lane, followed by samples HN1 to HN12 consecutively.