An aquifer in Northport, Maine has naturally occurring elevated levels of arsenic. Microorganisms may release arsenic in groundwater by reducing bedrock surface coatings or by altering arsenic speciation, thus affecting the sorption of arsenic. Currently there is competing evidence whether microbially mediated iron or arsenic reduction is more important for arsenic release. This work will determine the relative importance of iron and arsenic reduction within the Northport aquifer. Clone libraries for two sampling dates are being constructed to examine changes in the composition of the microbial communities between low arsenic recharge wells and high arsenic discharge wells to assess the impact microorganisms may have on the groundwater chemistry of the aquifer. Since little is known about the long term stability of microbial community structure in a groundwater environment this work will examine the temporal shifts in the microbial communities within the Northport aquifer. The importance of microbial arsenate reduction and arsenite oxidation is will be examined culturing techniques.
Hundreds of millions of people around the world have been exposed to arsenic through contaminated groundwater drinking sources. In Maine, 5-6% of the state’s residents may use groundwater sources with arsenic concentrations that exceed the US EPA’s Maximum Contaminant Level (MCL) of 10µg/L (Loiselle et al., 2002).
Adsorption of arsenic to iron oxyhydroxides and arsenic speciation can each affect arsenic mobility in groundwater. The relative importance of these two activities needs to be studied further.
In a fractured bedrock aquifer located in Northport, Maine there are naturally occurring elevated levels of arsenic (Lipfert et al, 2006). An arsenate respiring microorganism, Sulfurospirillum species NP4, was isolated from a well in the Northport aquifer and the proportion of it in groundwater samples correlated well with the arsenite concentration in the groundwater (Weldon and MacRae, 2006). Another probe which was designed to detect a genus of iron reducing bacteria, Geobacter, showed a correlation between Geobacter and the total arsenic concentration in groundwater samples (Weldon and MacRae, 2006).
Preliminary results from the clone library study show there is an increase in the proportion of microbial clones identified as ? proteobacteria when comparing the low arsenic recharge well R to the high arsenic discharge wells D1 and D2 (Fig. 1). This shift points to the reduction of iron as an important activity in the aquifer. Within ? proteobacteria there are many organisms that are capable of iron reduction, for example Geobacter sulfurreducens (Caccavo et al., 1994). Microbial reduction of iron has been linked to an increased retention of arsenate in the solid phase which, indicates that organisms capable of reducing arsenate may play a key role in arsenic mobilization while iron reduction is occurring within an aquifer (Kocar et al., 2006).
Figure 1: Complete microbial community analysis for groundwater wells in Northport, ME.
The shift of the microbial population in the discharge wells toward ? and ? proteobacteria could be relevant for arsenic cycling and release within the aquifer system (Fig. 1). Many ? proteobacteria species such as Hydrogenophaga species CL3 and Azoarcus species DAO1 are capable of oxidizing arsenite to arsenate, either aerobically or anaerobically (Rhine et al., 2005). Conversely ? proteobacterial species such as Enterobacter sp GS4, Citrobacter species TSA-1, and strain MLMS-1 are capable of dissimilatory arsenate reduction (Rhine et al., 2005). Based on the dominance of microorganisms from both ? and ? proteobacteria in the high arsenic discharge wells of the Northport aquifer, there could be microbially mediated arsenic speciation and cycling in this system.
Additional sequence data was combined with the preliminary results to and similar shifts in the microbial populations were observed. Minor changes in the community composition of less than 5% were observed in some of the wells but the predominant trends remained the same. While the overall shifts in the microbial populations were similar there was an improvement in the statistical significance of the combined results over the preliminary analysis. These results point to the potential importance of iron reduction and possibly arsenic reduction within the Northport aquifer.
Figure 2: Microbial community composition for each well for samples from May & November of 2007.
In addition to an increased number of sequence data from 1 sampling date sequence data was obtained for a second set of samples to observe temporal shifts in the microbial population. The overall shifts in the microbial population composition observed between the wells remained. Also, the microbial community appears to be relatively stable with only minor shifts in the populations within individual wells in a six month period.
A generalized approach that does not target specific microbes, the construction of clone libraries, was used to examine the composition of the microbial community. The libraries were constructed from samples from 4 observation wells installed in the aquifer. Two of the wells have low arsenic levels: one was in the overburden materials, well O, and the other well, R, is in the recharge region of the aquifer. The other two observation wells are located in the high arsenic discharge region of the aquifer, wells D1 and D2.
Previously a limited number of clones were sequenced. Prior to sequencing restriction fragment length polymorphism, RFLP, patterns which were frequently observed in individual libraries were reanalyzed using a second pair of enzymes to ensure the proper classification of the most frequent clone types. The additional data was used to improve the statistical significance of the results from the original clone library. To test the temporal stability of the community structure, cloned PCR products from a second time point were purified and sequenced. The sequence data obtained will be aligned using Clustal W and classified using phylogenetic trees constructed using Mega®. The relevance of observed differences between microbial populations will be statistically tested using mothur and Arlequin software packages.
Arsenite oxidizing bacteria were isolated using groundwater enrichments with added arsenite and nitrate. Colonies from solid media enrichments have been picked and transferred back into liquid media.
Jennifer Weldon (student investigator)
University of Maine
5711 Boardman Hall
Orono, ME 04469
Jean D. MacRae
University of Maine
5711 Boardman Hall
Orono, ME 04469
Image Description: Complete microbial community analysis for groundwater wells in Northport, ME
Image Description: Microbial community composition for each well for samples from May & November of 2007
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