Analysis of in situ manganese(II) oxidation in the Columbia River and offshore plume: linking Aurantimonas and the associated microbial community to an active biogeochemical cycle

Abstract

The Columbia River is a major source of dissolved nutrients and trace metals for the west coast of North America. A large proportion of these nutrients are sourced from the Columbia River Estuary, where coastal and terrestrial waters mix and resuspend particulate matter within the water column. As estuarine water is discharged off the coast, it transports the particulate matter, dissolved nutrients and microorganisms forming nutrient-rich and metabolically dynamic plumes. In this study, bacterial manganese oxidation within the plume and estuary was investigated during spring and neap tides. The microbial community proteome was fractionated and assayed for Mn oxidation activity. Proteins from the outer membrane and the loosely bound outer membrane fractions were separated using size exclusion chromatography and Mn(II)-oxidizing eluates were analysed with tandem mass spectrometry to identify potential Mn oxidase protein targets. Multi-copper oxidase (MCO) and haem-peroxidase enzymes were identified in active fractions. T-RFLP profiles and cluster analysis indicates that organisms and bacterial communities capable of oxidizing Mn(II) can be sourced from the Columbia River estuary and nearshore coastal ocean. These organisms are producing up to 10 fM MnO₂ cell⁻¹ day⁻¹. Evidence for the presence of Mn(II)-oxidizing bacterial isolates from the genera Aurantimonas, Rhodobacter, Bacillus and Shewanella was found in T-RFLP profiles. Specific Q-PCR probes were designed to target potential homologues of the Aurantimonas manganese oxidizing peroxidase (Mop). By comparing total Mop homologues, Aurantimonas SSU rRNA and total bacterial SSU rRNA gene copies, it appears that Aurantimonas can only account for ~1.7% of the peroxidase genes quantified. Under the broad assumption that at least some of the peroxidase homologues quantified are involved in manganese oxidation, it is possible that other organisms oxidize manganese via peroxidases.

Figure 1

Sample locality map of the Columbia River, estuary, Oregon coast and offshore where sampling depths are indicated alongside sample type identifier (E=Estuary, P=Plume, C=Coastal and O=Ocean). Samples indicated in bold and italics font represent 60 L volume samples for both DNA and protein extraction whereas samples represented in regular font were 10 L volume samples for DNA extraction only. Samples O1 and C1 were selected as control samples as they had less influence from the Columbia River. Samples P1, P2 C1, C2 and C3 were taken on June 1^st 2008, 4 days after neap tide whereas samples P3 and P4 were taken on the 3^rd June 2008, during the spring tidal cycle. E1 and E2 where taken on June 2^nd 2008.

Figure 2

Cluster analysis of bacterial communities in relation to practical salinity units (PSU). Panel (A) represents a hindcast of the salinity at the surface on June 1^st 2008 at 5pm. Note the extreme freshening of coastal waters as the Columbia River water discharges. Panel (B) represents a hindcast of salinity at depth during the spring tidal cycle on June 2^nd 2008 at midday. Note the saltwater incursion into the estuary. Panel (C) represents a UPGMA/Pearson product moment correlation cluster analysis of T-RFLP bacterial community fingerprints from all the samples collected. Scale bar is the Pearson product moment correlation r-value. Numbers at the nodes are cophenetic correlation coefficients. The bacterial community represented at sites P1_2m and E1_12m are river derived and indicate a transfer of microorganisms from the river/estuary to offshore whereas microorganisms from the coast (more saline) are influencing the community at E2_11m. The near surface plume environment is heavily influenced by deep coastal microorganisms suggesting mixing of deep and surface waters while organisms from the near surface coastal environment north and south of the river mouth and offshore samples form a distinctive cluster on their own.

Figure 3

In situ whole cell Mn(II) oxidation activity normalized to total bacterial cell number. The total bacterial cell numbers were calculated from Q-PCR data using an average of three SSU rRNA gene copies per cell. The figure representing E1_12m is not accurate due to sediment interference in the LBB assay.

Figure 4

AluI T-RFLP electropherograms of the Bacterial communities from the river and plume samples E1_12m and P1_2m respectively. Arrows indicate the predicted terminal restriction fragment for the genus Aurantimonas and Mn-oxidizing bacteria isolated from the Columbia River.

Figure 5

Percent Aurantimonas SSU rRNA copies in proportion to total Bacterial SSU rRNA gene copies amplified by Q-PCR from the same genomic DNA extracts. The 2007 samples were collected during the summer in the Columbia River Estuary. ETM_In was collected during an estuarine turbidity maximum (ETM) event and ETM_Out was collected after the ETM had passed.

Figure 6

Copies of the Aurantimonas manganese oxidizing peroxidase gene (mopA) and homologs compared to the numbers of Aurantimonas manganoxydans SSU rRNA gene copies amplified by Q-PCR from the same genomic DNA extracts. The 2007 samples were collected during the summer within the Columbia River Estuary. ETM_In was collected during an estuarine turbidity maximum (ETM) event and ETM_Out was collected after the ETM had passed.