Microbial Activity in Arsenic Contaminated Soil
Samantha Wolf, Mark Emerson, Derek Laskowsky.
Abstract:
Hypothesis: Will soil previously contaminated with CCA contain microbes that show more resilience to prolonged exposure to CCA wood than microbes in an uncontaminated soil? What is the prevalence of microbes in each soil? Our method was to grow the cultures in flasks, perform a dilution series and plate the samples on agar, incubate them for 24 hours and then perform a colony count to determine the prevalence of microbial growth in each sample. We found that the sample with the highest colony count was the CCA soil with the CCA wood chip additive, while the control sample with no additives had the lowest colony count. We concluded that there must be a variety of microbes present in the CCA soil that are tolerant of or perhaps prefer the presence of arsenic, and were effectively selected for growth in the presence of the CCA wood. We did not have the time or material resources to perform a genetic analysis on these microbial cultures.
Introduction:
The purpose of CCA is to prevent rot and damage to lumber from termites, effectively acting as a pesticide, in addition to strengthening the lumber. The EPA has deemed that CCA is too toxic for use on lumber intended for homes and play grounds. CCA PT wood came into use in the 1940s, and was widely used in the 1970s for residence purposes. By Dec. 2003, manufacturers were prohibited from applying CCA to lumber intended for homes or playgrounds
During the 1940’s the lumber industry began its large-scale treatment of lumber with chromated copper arsenate (CCA) for preservation. This treated lumber had a wide spread application ranging from playground equipment and residential homes, along with a variety of commercial uses. Although chromated copper arsenate was intended as a wood preservative and to prevent insect infestation, this chemical mixture itself brought forth wide spread soil microbe degradation.
The complications brought on by the CCA treated lumber is the leaching of chromated copper arsenate into the surrounding soil, the leaching prevents any microbial activity and therefore incapacitating the microbes and thus preventing any breakdown of organic pollutants. Even as soil composition itself has wide spread effects on microbe activity and availability, the presence of CCA has been proven in recent studies to have an adverse effect on microbial communities.
The focus of this study is to determine if CCA exposed microbes are resilient to the CCA, compared to non-CCA exposed microbes. To evaluate the population of microbe colonies within CCA contaminated soil in comparison to soil free of CCA contamination. The results of this study heighten our awareness to microbe activity and the deep affects that CCA treated lumber has on microbe activity (Turpeinen, Kairesalo, Haggblom.,2006).
The site selected is a rental residence once belonging to a local handyman. Within the door yard was a small pile of Pressure Treated lumber, the pile consist of scrap pieces of Chromated Copper Arsenate and Alkaline Copper Quaternary treated lumber. The lumber of main interest is that treated with Arsenic.
There are no records indicating when the previous owner began stock piling this debris, but has been there since September of 2009. The pile is located adjacent to the driveway and shares the same grade as the driveway. The pile is scattered amongst a patch of what appears to be berry bushes, the bushes themselves don’t appear to be producing fruit.
Our research focuses on a) the amount of copper and arsenic found in soil that has been exposed to Chromated Copper Arsenate (CCA) pressure treated wood, and b) the presence and activity of microbes in four soil samples: one control soil, one CCA soil, and one control sample and one CCA sample that have been inoculated with PT wood chips containing CCA.
Methods:
Soil Sampling and analysis:
The initial soil samples were extracted during winter, from predetermined depths of 3, 6, and 9 inches, speculating that the arsenic metal wouldn’t be much deeper. During the sampling process, it was discovered that the frost in that area wasn’t any deeper than three inches, partly due to the fact that the contaminated site was mostly buried under a snow bank. We used a power auger to break through the initial frost line and then hand dug with small spade to remaining depths. 
GPS reading for the selected site was N 44, 39.738 and W 71, 56.199 and an elevation of 1533 feet above sea level.
Microbial enrichment cultures:
On February 17th, we began incubating soil samples of sand, loam, and gravel in a brain-heart nutrient broth at 60 degrees F in a shaker-incubator for one week. The wood chip inoculant, however, was not added in time, so we began the process again. The second enrichment culture, using the same nutrient broth, consisted of one control flask with uncontaminated top soil, one control flask with CCA contaminated soil, one flask with uncontaminated top soil inoculated with a 2.2 gram piece of CCA wood, and one flask of CCA contaminated soil inoculated with a 2.2 gram piece of CCA wood. Incubation of these specimens began on March 1st, under the same conditions for 1 week. At the end of 1 week, undiluted samples were plated for a count of colony forming units. We found that this concentration was far too high to count, so we then performed a 4 x 10 dilution series at a concentration of .5 ml of sample and plated the samples again, incubating at 30°C for 24 hours. Results were ideal to count after 24 hours.
Results and Data:
Arsenic was found in the 3 inch depth sample at a concentration of 63.5 ppm. In average soil, arsenic is present at 6.5 ppm (excluding sites near arsenic rich mineral deposits) Other metals at high concentrations at 3 inches were cadmium at 2.2 ppm, chromium at 31 ppm, and copper at 44 ppm, all components in CCA wood treatment (See figure 3). Average concentration in topsoil for cadmium is 0.1 to 1.0 ppm; chromium is 70 ppm: copper is 20 ppm (http://pubs.usgs.gov/pp/1270/pdf/PP1270_508.pdf Element Concentrations in Soils and Other Surficia1 Materials of the Conterminous United States, US Geological Professional Paper 1270 published 1984)
|
|
|
|
|
|
Total Metals in Soil |
|
|
|
|
|
|
|||||||||
|
|
|
|
|
|
|
|||||||||||||||
|
Methods |
Microwave-assisted nitric acid digest(SW846-3501) with ICP-OES analysis |
|||||||||||||||||||
|
Lab ID: |
C12-10-1 |
C12-10-2 |
C12-10-3 |
|||||||||||||||||
|
Sample ID: |
CCA 1-3 |
CCA 3-6 |
CCA 6-9 |
|||||||||||||||||
|
Metal |
Concentration in soil, ppm (mg/kg, dry wt. basis) |
|||||||||||||||||||
|
Arsenic (As) |
63.5 |
23.2 |
<1 |
|||||||||||||||||
|
Cadmium (Cd) |
2.2 |
<1 |
<1 |
|||||||||||||||||
|
Chromium (Cr. Total) |
31.0 |
23.4 |
16.1 |
|||||||||||||||||
|
Copper (Cu) |
44.0 |
22.4 |
8.8 |
|||||||||||||||||
|
Lead (Pb) |
10.2 |
8.4 |
9.1 |
|||||||||||||||||
|
Nickel (Ni) |
18.3 |
16.5 |
12.9 |
|||||||||||||||||
|
Zinc (Zn) |
45.4 |
40.3 |
38.5 |
|||||||||||||||||
|
|
Figure 3 |
|
|
|
|
|
||||||||||||||
figure 2
Conclusions:
The CFU count was highest in the CCA soil sample inoculated with the CCA wood chip. The CFU count for this sample was 36 at 4x10 dilution, extrapolated to 360,000 count. The control soil CFU was 50,000. The uninoculated CCA soil had the second highest count, at 240,000. This indicates that the soil has cultured arsenic tolerant bacteria naturally. It may even be that the bacteria prefer the presence of arsenic. Once CCA wood was added as a food source to the CCA soil, colony numbers increased. CCA wood added to the control soil did not produce high numbers of colonies, so microbes present in the control soil are not tolerant of the CCA chemicals.
In summary, what we discovered were the microbes that were in our enrichment cultures were drawn towards carbon and organic matter. Due to time constraints and funds we were unable to further pursue our experiment much further than that. Our research did lead us to the conclusion that the microbes were more than likely dormant from the winter environment. With the microbes being dormant that would account for the high soil reading, the microbes were unable to dissolve the Arsenic, providing these specific microbe feed on Arsenic. During our research period, we did discover that there are a wide range of microbe species and within these species lays a variety of microbes that feed on heavy metals such as arsenic. Not suggesting that all microbes feed on heavy metals, most likely there some that don’t. If more funding was available, the next step would be to collect some more soil samples and within these samples plant Chinese Break Ferns. Previous advancements have shown that the break fern would extract the arsenic from the ground and the plant would store it within. From there the plant could be harvest and then properly disposed of. The soil samples that were collect revealed what we suspected and that was the presence of arsenic, we believed that the pile of pressure treated lumber had been leaching into the surrounding soil. The further down we sampled, the arsenic concentration decreased. This finding put our minds at ease on the severity of the contamination and the possibility of the contaminants reaching the water table.
As an interesting side note, a new bacteria was discovered within the last year. Bacteria were discovered that are able to use arsenic in place of phosphorus in their DNA. The bacteria were discovered by Felisa Wolfe-Simon through a study done on Mono Lake in California. The lake has extremely high levels of arsenic and not many organisms are able to live in the lake. This is a small breakthrough in the science world. Prior to this discovery our rules for the basic needs of life forms remained the same for many years. All life forms need elements like carbon, oxygen, nitrogen and phosphorus. The phosphorus is used in the DNA of the living being. It acts as the structure or the back bone of the DNA. Prior to this discovery we believed that all living things required phosphorus for their DNA. We now know that there are living things, bacteria in this case that are able to use arsenic instead of phosphorus in their DNA. This is possible because arsenic is similar to phosphorus on a molecular level. They can be mistaken for each other in some chemical processes; this is why arsenic is toxic to most living things. Due to the fact that it is similar and not identical when the swap does take place it leads to problems in the living being. In light of this, the fact that something is able to survive off of arsenic is quite extraordinary.
References:
Riina Turpeinen, Timo Kairesalo, Max Haggblom., 5 Jan 2006. Microbial Community Structure and Activity in Arsenic-, Chromium- and Copper- Contaminated Soils.
http://scienceblogs.com/pharyngula/2010/12/its_not_an_arsenic-based_life.php
http://www.nature.com/news/2010/101202/full/news.2010.645.html
The microbe, named strain GFAJ-1, is a salt-loving member of the Halomonadaceae family of proteobacteria that came from the sediments of toxic Mono Lake in eastern California. This old alkaline lake is known for its being hyper-salty and naturally high in arsenic http://www.livescience.com/9046-microbe-eats-arsenic.html