# Presentation: 504945

## Polyphasic characterization of microbial communities under the stressful conditions of nitrate, heavy metals, radionuclides, and acidic pH in contaminated groundwater

- M.W. Fields1, T. Yan2, X. Liu2, C.E. Bagwell3, S.L. Carroll2, P.M. Jaridne2, C.S.Criddle4, T.C. Hazen5, and J. Zhou1
- Miami University1, Oak Ridge National Laboratory2, Savannah River Technology Center3, Stanford University4, and Lawarence Berkeley National Laboratory5

**Abstract**
- Clonal libraries of multiple genes (SSU rRNA gene, *nir*K, *nir*S, *amo*A, *pmo*A, and *dsr*AB) were constructed from groundwater samples (n=6) that varied in degrees of contamination.  The sites were in proximity to a concentration plume and the following geochemical parameters were measured:  pH, nitrate, uranium, nickel, sulfate, and TOC (total organic carbon).  Principle components analysis (PCA) was used to compare the relationships between the sites with respect to the unique operational taxonomic unit (OTU) distributions within the different clonal libraries.  When geochemical characteristics were analyzed, the data suggested that the samples varied significantly with respect to nitrate and sulfate.  The sites were grouped differently when compared upon the basis of the SSU rDNA genes or each of the functional genes.  The diversity of amoA genes was much lower compared to the other functional genes and the one sample with high nitrate and circum neutral pH appeared to be different from the other sites.  When all gene OTUs were used in the analyses, the sites were more similar than in any other comparison, and the background site was grouped with the acidic, contaminated sites.  The results indicated that a combination of the contaminants (i.e., nitrate, uranium, nickel) and pH levels have impacted the bacterial communities at the respective sites in different and dynamic ways.  These data suggested that even though the background site was phylogenetically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site.

-  The pH, nitrate, uranium, and nickel levels in the groundwater samples from four FRC groundwater samples.  FW-300 represents the background area.  Values were determined by the FRC management team at Oak Ridge National Laboratory.
- Well	          		pH		nitratea	uraniumb	nickelc	aluminumc
- 					(mM)		(μM)		(μM)		(mM)
- FW-300         	 6.1		0.02		ND		0.85		0.01
- FW-005         	 3.9		6.27 		27.0 		84.3 		1.74
- FW-010          	3.5		713   		0.71  		322		41.5
- FW-015          	3.4		173   		32.4		147		22.9
- TPB-16		6.3		0.48		4.62		ND		0.01
- FW-003		6.0		17.1		0.04		0.26		0.02
- a nitrate was determined via ion chromatography
- b uranium was determined via ICP-mass spectroscopy
- C nickel and aluminum were determined via ICP

**The estimated species number and diversity based upon cloned SSU rRNA genes**.   The number of clones screened and the estimated species number based on less than or equal to 98% nucleotide identity for the same species (unique OTU).  The Shannon-Weiner and reciprocal Simpson’s index are diversity indices, and a higher number represents more diversity.  The evenness index estimates the homogeneity of the clone distribution, and a community has an increased number of different clones with a similar distribution as E approaches 1.  The site TPB-16 appeared to be more diverse than background, and the acidic wells, FW-005, -010, and –015 had significantly decreased diversity.  FW-003 also had decreased diversity, and this site also had significantly increased nitrate levels compared to background.  The high nitrate sites also displayed a more dominated community structure compared to FW-300 and TPB-16.

**Principal components analyses**.  The clonal sequences for the SSU rRNA genes, *nir*K, *nir*S, *amo*A/*pmo*A, and *dsr* were compared via RFLP and/or direct sequence determination, and the occurrence and distribution of phylogenetically distinct OTUs was determined.  The data was reduced with  factor analysis using principal components.
**Geochemistry**.  When the geochemical parameters of pH, nitrate, TOC, uranium, nickel, sulfate and aluminum were used, 85% of the variance could be explained by two components.  The background site and TPB-16 were distinct from the other sites (least contaminated), and FW-010 and FW-015 were very similar.  The background and TPB-16 had circum neutral pH and the lowest nitrate, nickel, and aluminum levels.
**SSU rRNA sequences**.  When the SSU rRNA gene sequences were compared the background site was grouped with FW-003, TPB-16 was distinct from all other sites, and the three acidic sites were grouped.  The two components accounted for only 58% of the variance in SSU rRNA sequences between the sites.
***amo*****A/*****pmo*****A**.  The FW-003 site was distinct from the others when the *amo*A and *pmo*A sequences were compared and this site had circum neutral pH and increased nitrate levels.
***nir*****K and *****nir*****S**.  When the *nir*S sequence were compared, the two contaminated sites with circum neutral pH were distinct from the other sites as well as from one another.  The background site and acidic sites were grouped.  The distribution of *nir*K sequences were even more similar, however FW-015 was distinct from the other sites that were all closely grouped.
***dsr*****AB**.  When the distribution of unique *dsr*AB sequences were compared, site TPB-16 and FW-015 were distinct from the other sites which were closely grouped.  The background site, FW-003, FW-010, and FW-005 were affected by the loading factors in a similar fashion.
**All gene sequences**.  When the distribution of all unique gene sequences were compared, overall the sites were more closely grouped.  Two components could explain 85% of the variance of gene distribution among the sites, and this was similar to the explained variance by the geochemical characteristics.  However, the background site did group more closely with the acidic sites, and the circum neutral sites, FW-003 and TPB-16 were distinct.
- The PC1 from the geochemical characteristics explained 65% of the observed variance.  When the geochemical PC1 value was compared to the diversity index (1/D) estimated from the SSU rRNA gene sequences, a power function correlation was observed (r=0.85).  When the all gene PC1 value (74%) was compared to the 1/D estimation, a significant correlation was not observed.

|  | Screened Clones | Unique OTUs |
| --- | --- | --- |
| SSU rRNA genea | 1630 | 445 |
| nirKb | 958 | 48 |
| nirSb | 1162 | 143 |
| amoAc | 539 | 63 |
| pmoAc | 26 | 14 |
| dsrABd | 1812 | 900 |

- a Fields et al. (in review); b Yan et al., 2003;
- c Yan et al., (in preparation); d Bagwell et al., (in review)

**Number of Screened and Unique Clones for each Gene Set**

- http://www.esd.ornl.gov/nabirfrc/

**Conclusions**.  The measured groundwater chemistry differed markedly between sampling wells within the contaminant plume and the background site.  PCA analyses was sufficient for simple separation of sites by groundwater chemistry, but none of the measured chemical analytes appeared primarily or solely responsible for the overall geochemical variability.  High variability in geochemistry between sampling locations was evident.  Therefore, different combinations of chemical variables may be necessary to explain spatial differences in the microbiology within and along the contaminant plume.  In addition, correlations between groundwater chemistry and the recovery and diversity of different functional gene sequences gave very different results.  Sites with drastically different geochemistry could have similar OTU distributions and sites with different sequences had similar geochemical parameters.  Clear linear relationships between the chemistry and microbiology seems unlikely at the system level.  The data reduction with  PCA did consider phylogenetically distinct OTUs, but the analysis did not consider the relatedness between the distinct OTUs.  Future work is underway to compare distance matrices of all sequences between sites in order to capture the degree of similarity within the genomics sequences of the sampled community.