Using metatranscriptomics to better understand the role of microbial nitrogen cycling in coastal sediment benthic flux denitrification efficiency.

Spatial and temporal variability in benthic flux denitrification efficiency occurs across Port Phillip Bay, Australia. Here, we assess the capacity for untargeted metatranscriptomics to resolve spatiotemporal differences in the microbial contribution to benthic nitrogen cycling. The most abundant sediment transcripts assembled were associated with the archaeal nitrifier Nitrosopumilus. In sediments close to external inputs of organic nitrogen, the dominant transcripts were associated with Nitrosopumilus nitric oxide nitrite reduction (nirK). The environmental conditions close to organic nitrogen inputs that select for increased transcription in Nitrosopumilus (amoCAB, nirK, nirS, nmo, hcp) additionally selected for increased transcription of bacterial nitrite reduction (nxrB) and transcripts associated with anammox (hzo) but not denitrification (bacterial nirS/nirk). In sediments that are more isolated from external inputs of organic nitrogen dominant transcripts were associated with nitrous oxide reduction (nosZ) and changes in nosZ transcript abundance were uncoupled from transcriptional profiles associated with archaeal nitrification. Coordinated transcription of coupled community-level nitrification-denitrification was not well supported by metatranscriptomics. In comparison, the abundance of archaeal nirK transcripts were site- and season-specific. This study indicates that the transcription of archaeal nirK in response to changing environmental conditions may be an important and overlooked feature of coastal sediment nitrogen cycling.

Temporal profiling resolves the drivers of microbial nitrogen cycling variability in coastal sediments.

Here we describe the potential for sediment microbial nitrogen-cycling gene (DNA) and activity (RNA) abundances to spatially resolve coastal areas impacted by seasonal variability in external nutrient inputs. Three sites were chosen within a nitrogen-limited embayment, Port Phillip Bay (PPB), Australia that reflect variability in both proximity to external nutrient inputs and the dominant form of available nitrogen. At three sediment depths (0-1; 1-5; 5-10 cm) across a 2 year study key genes involved in nitrification (archaeal amoA and bacterial ß-amoA), nitrite reduction (clade I nirS and cluster I nirK, archaeal nirK-a), anaerobic oxidation of ammonium (anammox 16S rRNA phylogenetic marker) and nitrogen fixation (nifH) were quantified. Sediments impacted by a dominance of organic nitrogen inputs were characterised at all time-points and to sediment depths of 10 cm by the highest transcript abundances of archaeal amoA and archaeal nirk-a. Proximity to a dominance of external nitrate inputs was associated with the highest transcript abundances of nirS which temporally co-varied with seasonal changes in sediment nitrate. Sediments isolated from external inputs displayed the greatest depth-specific decrease in quantifiable transcript abundances. In these isolated sediments bacterial ß-amoA transcripts were temporally associated with increased sediment ammonium levels. Across this nitrogen limited system variability in the abundance of bacterial ß-amoA, archaeal amoA, archaeal nirk-a or nirS transcripts from the sediment surface (0-1 and 5 cm) demonstrated a capacity to improve our ability to monitor coastal zones impacted by anthropogenic nitrogen inputs. Specifically, the spatial detection sensitivity of bacterial ß-amoA transcripts could be developed as a metric to determine spatiotemporal impacts of large external loading events. This temporal study demonstrates a capacity for microbial activity metrics to facilitate coastal management strategies through greater spatial resolution of areas impacted by external nutrient inputs.

Primer selection influences abundance estimates of ammonia oxidizing archaea in coastal marine sediments.

Quantification of the α-subunit of ammonia monooxygenase (amoA) through PCR is an established technique for estimating the abundance of ammonia oxidizing archaea (AOA) in environmental samples. This study quantified AOA with two established primer sets in 1 cm increments from the sediment surface (0-1 cm) to a depth of 10 cm at two locations within Port Phillip Bay (PPB), Australia. Primer choice had a significant effect on within sample estimates of AOA with copy numbers ranging from 102 to 104 copies per ng DNA. Variation in AOA abundance patterns with increasing sediment depth were site and primer specific. Sequence mismatches between the primer binding region of the isolated amoA sequences from PPB and Nitrosopumilus maritimus SCM1 were identified and may explain the high variation identified between primer estimates. Our results highlight the need for testing multiple primer pairs that target different regions of the AOA amoA sequence prior to large-scale marine sediment environmental studies.