# Presentation: 487704

## Novel, Fe(III)-, Sulfate-, and Nitrate-Reducing Bacterial Enrichments from FRC Sediments

**Novel, Fe(III)-, Sulfate-, and Nitrate-Reducing Bacterial Enrichments from FRC Sediments**
-  M.W. Fields1, S.L. Carroll1, Q. Ye2, Y. Roh1, K.A. Bergman1, Z. Yang1, and J. Zhou1
-  1Oak Ridge National Laboratory, Oak Ridge, TN, 2University of Missouri, Columbia, MO

**Abstract**.  The NABIR-Field Research Center (FRC) is designated as an experimental site for biogeochemistry and bioremediation studies (_[http://www.](http://www.esd.ornl.gov/nabirfrc/)__[esd](http://www.esd.ornl.gov/nabirfrc/)__[.](http://www.esd.ornl.gov/nabirfrc/)__[ornl](http://www.esd.ornl.gov/nabirfrc/)__[.](http://www.esd.ornl.gov/nabirfrc/)__[gov](http://www.esd.ornl.gov/nabirfrc/)__[/](http://www.esd.ornl.gov/nabirfrc/)__[nabirfrc](http://www.esd.ornl.gov/nabirfrc/)__[/](http://www.esd.ornl.gov/nabirfrc/)_).  Subsurface sediments (~15 m) were enriched for iron-reducing bacteria with acetate or lactate as the electron donors, or for sulfate- and nitrate-reducing bacteria with lactate and ethanol.  Results indicated that iron-reducing bacteria were present at 50-120 cells ml-1 in the sediment samples from FW-100 and that nitrate- or sulfate-reducing bacteria were present at 5000 cells ml -1 and 1000 cells ml -1, respectively, for FW-109 sediments. Clonal libraries of the SSU 16S rRNA gene were constructed from the different enrichments after being transferred four times.  The iron enrichments could reduce Fe3+-citrate with either acetate or lactate as the electron donor, but only the lactate enrichment in GM could reduce amorphous iron oxides.  The nitrate enrichment was predominated by microorganisms closely related to *Pseudomonas* NZHA7, *Citrobacter*, and *Azoarcus*, could reduce nitrate to dinitrogen gas, and uranium could not be reduced by this culture.  The enrichments provided with iron sulfate could produce iron sulfides with lactate and ethanol as electron donors, and the enrichment culture could reduce U(VI).  The GM+lactate enrichment could also reduce U(VI), and mineralogical analyses indicated that U(IV) could be co-precipitated with Fe(II).  The results indicated that the enrichments were dominated by 4 to 10 species bacteria.  Gram-negative bacteria dominated the nitrate enrichment, and Gram-positive microorganisms dominanted the iron and sulfate enrichments.  The majority of the clones had less than 95% similarity with previously observed sequences, and some had less than 90% sequence similarity with previously cultivated microorganisms.  The GM+lactate enrichment was dominated by *Anaerovibrio* species, and also contained *Sporomusa*, *Clostridium*, and *Anaeromusa*.  The sulfate-reducing enrichment also contained *Clostridium* spp., but the predominant microorganisms appeared to be *Desulfosporosinus*.  The results indicated the following:  1.) iron-, sulfate-, nitrate-, and uranium-reducing communities are present in Area 3 at the FRC, 2.) differences in community composition resulted in different community phenotypes for enrichment cultures, 3.) the enrichment cultures represented microorganisms that had been previously uncultivated, and 4.) some of the cultures were dominated by novel, Gram-positive bacteria that had the capacity for U(VI) reduction when cultured in the presence of other indigenous microorganisms.

**Oak Ridge National Laboratory**

**Material and Methods**
_**Enrichments**_**.  **Sediment cores were transported to the laboratory in Mason jars flushed with argon.  Dilutions were performed in an anaerobic chamber, and the enrichments were incubated in the dark at 18С using anaerobic technique.  The GS medium has been described previously (Kostka and Nealson, 1998), and GM medium was based upon groundwater chemistry.  The biochemical and molecular analyses were done after four transfers (~3 m).  The electron acceptors were ferric citrate (15 mM), ferrous sulfate, or potassium nitrate and electron donors were acetate, lactate, or lactate and ethanol.
_**DNA extraction and amplification**_**. **Groundwater samples (1-2 l) were collected, and transported to the laboratory in glass, amber bottles.  Bacteria were harvested by centrifugation (10,000 x g force, 4С for 30 min), and the pellets were stored at -80С until used for DNA extraction.  The cell pellet was resuspended in a lysis buffer and the cells disrupted with a previously described grinding method (Zhou and Tiedje, 1996).  DNA was extracted as previously described (Zhou et al., 1997), and the precipitated DNA was purified by gel electrophoresis plus mini-column preparation (Wizard DNA Clean-Up system, Promega).** **The PCR reactions (20 *u*l) contained 2ul 10×buffer (500 mM KCl, 100mM Tris HCl pH 9.0 and 1% Triton X-100), 1.5ul 25 mM/L MgCl2, 0.2 ul 400ng/ul Bovine serum albumin (Boehringer Mannheim), 0.2 ul 25 mM 4×dNTPs (USB Chemicals), 10 pmol each primer, 2.5 U Taq polymerase, 1ul purified DNA (5-10 ng).  To minimize PCR-induced artifacts, the optimal number of cycles was determined and five PCR reactions were combined prior to cloning as described previously (Qiu et al., 2001). The combined PCR products (326 bp and 774 bp, respectively) were separated by electrophoresis in a low-melting point agarose gel (0.8%), the appropriate band excised, and the DNA extracted with a Wizard Prep Kit (Promega, Madison, WI) according to manufacture instructions.  Recovered DNA was resuspended in 6 ul ddH2O, 2 ul ligated with pCR2.1 vector from a TA-cloning kit, and competent *Escherichia* *coli* cells transformed according to provided protocol (Invitrogen, San Diego, CA).  The SSU 16S rDNA genes were amplified with the FD1 and 1540R primers, and PCR products purified with the ArrayItTM PCR Purification Kit (TeleChem International, Inc.) or treated with ExoSAP-ITTM (US Biochemical Corporation) according to manufacturer instructions.
_**Sequence determination and phylogenetic analysis**_.  DNA sequences were determined with a BigDye Terminator kit (Applied Biosystem) using a 3700 DNA analyzer (Perkin-Elmer) according to the manufacture instructions with the SSU 16S rDNA-specific primer 529R, and compared with sequences from GeneBank or RDB. The sequences were aligned with ClustalW (Thompson et al., 1994).  Phylogenetic analyses of partial SSU 16S rRNA gene sequences were conducted using MEGA version 2.1 (Kumar et al., 2001).  Neighbor-joining phylogenies were constructed from dissimilatory distances and pairwise deletion of gaps and missing data.  Maximum parsimony phylogenies did not differ significantly (data not shown).
_**Mineralology**_.  Cultures were grown anaerobically, and 5 ml of culture medium was used for EDX and SEM as described previously (Roh *et al*., 2002). Fe(II) levels were monitored with a spectrophotometer and the ferrozine method of HCl-extractable iron as described previously (Lovley and Phillips, 1988).  The reduction of uranium was determined by measuring the loss of U(VI) from solution with a kinetic phosphorescence analyzer (Chemcheck, Ins.) as described previously (Lovely and Phillips, 1992). The morphology, mineralogy, and chemistry of precipitated phases by Fe(III)-reducing bacteria were determined with a Phillips 30 FEG  scanning electron microscope (SEM) with energy dispersive X-ray analysis (EDX). XRD analysis was used to monitor mineralogical characteristics and was performed with a Scintag XDS 2000 diffractometer.

- Research was funded by the Natural and Accelerated Bioremediation Research (NABIR) program and Genomes to Life (GTL), U.S. Department of Energy

**Community analysis of GS-acetate-Fe(III) enrichment**.  The GS-acetate enrichment could reduce ferric citrate, but amorphous iron oxide was not reduced.  The predominant population comprised over half of the clonal library, and had 98% sequence similarity *Clostridium sphenoides*.  The other main *Clostridia* group had 98% sequence similarity with *Clostridium* *bifermentans*.  *C*. *bifermentans* was isolated from soil contaminated with chlorinated aliphatic compounds.  Two other predominant sequences constituted approximately 8% of the library each, and the closest, cultivated microorganisms were similar to *Sporomusa* and *Anaerovibrio*.  However, the sequences only had 89% and 80% sequence similarity with *Anaerovibrio glycerini, *respectively*. *

**Community analysis of GS-lactate-Fe(III) enrichment**.  The GS- lactate enrichment could reduce ferric citrate, and amorphous iron oxide was also reduced.  The predominant population comprised over half of the clonal library, and had the highest sequence similarity (90%) with an uncultivated clone from a trichlorobenzene-transforming microbial consortium and 89% sequence similarity with *Anaerovibrio* *glycerini*.  The other main *Anaerovibrio* group was also distantly related to previously cultivated microorganisms, and had 88% sequence similarity with *Selenomonas* *lacticifex*.  The *Clostridium* group constituted a decreased proportion of the clonal library (~20%) compared to the acetate enrichment, and the sub-population had 98 to 99% sequence similarity with *C*. *sphenoides*.* *

**Community analysis of GM-lactate-Fe(III) enrichment**.  The GM medium was a basal salts composition with trace minerals and NH4Cl, and was similar to the general groundwater chemistry.  The predominant population had 91% sequence similarity with *Anaerovibrio* *glycerini*, and was similar to the predominant sequences of the GS-lactate enrichment.  It should also be noted that the predominant sequence had the highest sequence similarity (96%) with a bacterial isolate from the FRC.  The *Clostridium* sub-population accounted for almost 20% of the sampled community, and had 99% sequence similarity with *Clostridium* *algidixylanolyticum*.  *C*. *algidixylanolyticum* were observed in the GS-acetate enrichment, but too a lesser extent.  The C. sphenoides sub-population observed in the GS-acetate and GS-lactate were not detected in the GM enrichment.  Other less predominant populations similar to *Anaerovibrio* and/or *Sporomusa* were observed in the GM-lactate enrichment.  These sequences had between 86% and 88% sequence similarity with *A*. *glycerini*.

- SEM with EDX analysis suggested that uranium could be reduced and co-adsorbed/co-precipitated with iron and phosphorous on bacterial cell surface (F).  X-ray diffraction analysis of the crystalline minerals showed that all peaks were attributable to vivianite [Fe3(PO4)2•8H2O] (G).  U(IV) was produced by the GM-lactate enrichment under growth conditions.

**  ****C**

**  ****E**

**C**

**Conclusions**:
- • iron-, sulfate- and uranium-reducing microbial communities are present in Area 3 at the FRC
- • Microbial enrichment cultures were dominated by low G+C, Gram-positive bacteria
- • Different electron donors resulted in different bacterial communities
- • The GS-acetate enrichment was predominated by sequences with 98% similarity to *C*. *sphenoides* but contained sequences distantly related to *Anaerovibrio* and *Sporomusa*
- • The GS-lactate enrichment was predominated by microorganisms with 89% sequence similarity with *A*. *glycerini* and *S*. *lacticifex*
- • The GM-lactate enrichment was also predominated by sequences with ~90% sequence similarity with *A*. *glycerini*
- • The GM-sulfate enrichment was predominated by *Desulfosporosinus *species, and also contained sequences closely related to *C*. *chromoreductans*
- • The GM-lactate culture could reduce amorphous iron oxides, and could co-precipitate U(VI) with Fe(III) and form U(IV)-Fe(II)-phosphates
- • Novel and unique iron-, sulfate-, and U-reducing microorganisms are present at the FRC

**A**

**B**

**D**

**F**

**G**

**Community analysis of GM-nitrate enrichment from FW-109**.  The predominant population had 99% sequence identity with an uncultivated clone from acid-tolerant epiphytic communities, and was very similar  to sequences cloned directly from FRC groundwater in the same area.  These FRC clones constituted approximately 7 to 10% of the sampled diversity.  Two microorganisms previously isolated from FRC groundwater clustered with these clones.   A second predominant population comprised over 30% of the sampled diversity, and had 97% sequence identity with *Citrobacter* *sedlakii*.  *Azoarcus*-like sequences accounted for 3% of the library, and the representative clone had 96% sequence identity with *Azoarcus* *eutropha*.  One clonal sequence clustered with two FRC isolates that appeared to be -*Proteobacteria*.  The clone had 96% sequence identity with an FRC isolate from Area 2 groundwater, and 93% sequence identity with Ochrobactrum *intermedium*.

**Community analysis of GM-sulfate enrichment from FW-109**.  The predominant population comprised approximately 25% of the sampled diversity and had 88% sequence identity with *Desulfosporosinus* Blif.  Sub-populations that had 95% to 97% sequence identity with *Desulfosporosinus* *orientis* constituted for an additional 37% of the library.  The clone E04-023 constituted just over 10% of the library, and had 98% sequence identity with *Clostridium* *chromoreductans*.  Two other clones that comprised an additional 10% of the library also clustered with *C*. *chromoreductans*.  *C*. *chromoreductans* was recently isolated from soil, and has the capacity to reduce Cr(VI).  A small fraction of the enrichment community (5%) appeared to have only 87% sequence identity with previously uncultivated clones that originated from a chlorobenzene-degrading community.