Comparing DNA isolation and sequencing strategies for 16S rRNA gene amplicon analysis in biofilm containing environments.

Bacteria are primarily responsible for biological water treatment processes in constructed wetland systems. Gravel in constructed wetlands serves as an essential substrate onto which complex bacterial biofilms may successfully grow and evolve. To fully understand the bacterial community in these systems it is crucial to properly isolate biofilms and process DNA from such substrates. This study looked at how best to isolate bacterial biofilms from gravel substrates in terms of bacterial richness. It considered factors including the duration of agitation during extraction, extraction temperature, and enzyme usage. Further, the 16S taxonomy data subsequently produced from Illumina MiSeq reads (using the SILVA 132 ribosomal RNA (rRNA) database on the DADA2 pipeline) were compared with the 16S data produced from Oxford Nanopore Technologies (ONT) MinION reads (using the NCBI 16S database on the EPI2ME pipeline). Finally, performance was tested by comparing the taxonomy data generated from the Illumina MiSeq and ONT MinION reads using the same (SILVA 132) database. We found no significant differences in the effective number of species observed when using different bacterial biofilm detachment techniques. However, enzyme treatment enhanced the total concentration of DNA. In terms of wetland community profiles, relative abundance differences within each sample type were clearer at the genus level. For genus-level taxonomic classification, MinION sequencing with the EPI2ME pipeline (NCBI database) produced bacterial abundance information that was poorly correlated with that from the Illumina MiSeq and DADA2 pipelines (SILVA132 database). When using the same database for each sequencing technology (SILVA132), the correlation between relative abundances at genus-level improved from negligible to moderate. This study provides detailed information of value to researchers working on constructed wetlands regarding efficient biofilm detachment techniques for DNA isolation and 16 s metabarcoding platforms for sequencing and data analysis.

Enhanced phosphate removal and potential recovery from wastewater by thermo-chemically calcinated shell adsorbents.

Shell from the seafood processing industry is an under-utilised waste resource worldwide. Calcite, the major component of shell is commonly used in wastewater treatment for the removal of phosphorus (P). Here, mussel and oyster shell-based adsorbents (MSB and OSB) were used for removal of P as phosphate (PO43-) from aqueous solution and secondary wastewater, following preparation through chemical calcination at 700 °C. Batch adsorption experiments were carried out to identify the effects of various operating parameters (e.g., pH, dosage, contact time, initial concentration of P ions, co-existing ions), while a desorption study helped to understand the availability of the bonded P. The optimal contact time for PO43- removal was 120 min using both adsorbents with the dose at 200 mg. Characterisation of the adsorbent was performed using SEM-EDX, pHpzc, BET, FTIR and XRD. The XRD analysis showed that both calcite and lime were present on the surface of the shell particles. P was adsorbed effectively through inner-sphere complexation and surface microprecipitation mechanisms, while an enhanced maximum P adsorption capacity of 12.44 mg/g for MSB and 8.25 mg/g for OSB was reached. The Redlich-Peterson isotherm model fitted well with the equilibrium isotherm data (R2 ≥ 0.97) which also suggested a heterogenic surface. The desorption study (on the saturated adsorbent) found that ~97% of bonded P could be plant available in soil. These results suggest that a shell-based adsorbent can serve as a promising material for P removal from real wastewater effluent and subsequently could be used as a soil conditioner.

Restoration of afforested peatland: Immediate effects on aquatic carbon loss.

Peatland restoration is undertaken to bring back key peatland ecosystem services, including carbon storage. In the case of drained, afforested blanket peatlands, restoration through drain blocking and tree removal may impact upon aquatic carbon concentrations and export, which needs to be accounted for when considering the carbon benefits of restoration. This study investigated concentrations and export of aquatic carbon from a drained, afforested blanket bog catchment, where 12% of the catchment underwent drain blocking and conifer removal (termed 'forest-to-bog' restoration), and from two control catchments: one in open bog and one that remained afforested. Using a before-after-control-impact (BACI) design, we found no significant increases in concentrations or export of aquatic carbon (DOC, POC or DIC) in the first year following forest-to-bog restoration (i.e. across the whole post-restoration period). However, increased DOC concentrations were observed in the first summer (2015) post-restoration, and seasonally increased DOC export was noted during storm events in the autumn of the same year. The lack of significant effects of forest-to-bog restoration on aquatic carbon export may be a consequence of the small proportion of the catchment (12%) undergoing management. In terms of management, the removal of more of the forestry residues (i.e., brash) may help to mitigate effects on aquatic carbon, by removing a potential DOC and POC source. Restoring small areas at a time (≤12%) should result in minimal aquatic carbon export issues, in contexts similar to the current study.

Assessing hospital impact on pharmaceutical levels in a rural 'source-to-sink' water system.

It is widely recognised that inadequate removal of pharmaceuticals in wastewater may lead to their presence in surface waters. Hospitals are key point-sources for pharmaceuticals entering municipal waterways, and rural hospitals are of concern as receiving wastewater treatment plants (WWTPs) may be smaller, less advanced and thus less efficient. While most research has focused on urban settings, here we present results from a rural ''source-to-sink'' study around a hospital. The aim was to determine the contribution of pharmaceuticals discharged to a municipal wastewater system, and, to assess pharmaceutical removal efficiency in the WWTP. Samples were collected daily for one month to assess water quality and pharmaceuticals in the broader water cycle: (i) raw water supply; (ii) treated hospital tap water; (iii) hospital wastewater discharge; (iv) combined WWTP influent; and (v) final WWTP effluent. Target compounds included analgesics/antiinflammatories, antibiotics, psychiatric drugs, and a synthetic estrogen hormone. Concentrations ranged from: 3 ng/L (carbamazepine) to 105,910 ng/L (paracetamol) in hospital discharge; 5 ng/L (ibuprofen) to 105,780 ng/L (paracetamol) in WWTP influent; and 60 ng/L (clarithromycin) to 36,201 ng/L (paracetamol) in WWTP effluent. WWTP removal ranged from 87% (paracetamol) to <0% (carbamazepine and clarithromycin), and significant correlations with water quality characteristics and WWTP flow data were observed for some compounds. Results suggested that the hospital is an important source of certain pharmaceuticals entering municipal wastewater, and associated water quality parameters are impacted. Pharmaceutical persistence in the WWTP effluent highlighted the direct pathway these compounds have into receiving surface water, where their impact remains uncharacterised. Rural regions may face future challenges mitigating environmental risk as WWTP infrastructure ages, populations grow and pharmaceutical use and diversity continue to increase.

Measuring restoration progress using pore- and surface-water chemistry across a chronosequence of formerly afforested blanket bogs.

During the restoration of degraded bogs and other peatlands, both habitat and functional recovery can be closely linked with nutrient cycling, which is reflected in pore- and surface-water chemistry. Several peatland restoration studies have shown that the time required for recovery of target conditions is slow (>10 years); for heavily-impacted, drained and afforested peatlands of northern Scotland, recovery time is unknown. We monitored pore- and surface-water chemistry across a chronosequence of formerly drained, afforested bog restoration sites spanning 0-17 years, using a space-for-time substitution, and compared them with open blanket bog control sites. Our aims were to measure rate of recovery towards bog conditions and to identify the best suite of water chemistry variables to indicate recovery. Our results show progress in recovery towards bog conditions over a 0-17 year period post-restoration. Elements scavenged by trees (Mg, Na, S) completely recovered within that period. Many water chemistry variables were affected by the restoration process itself, but recovered within 11 years, except ammonium (NH4+), Zn and dissolved organic carbon (DOC) which remained elevated (when compared to control bogs) 17 years post restoration. Other variables did not completely recover (water table depth (WTD), pH), exhibiting what we term "legacy" effects of drainage and afforestation. Excess N and a lowered WTD are likely to slow the recovery of bog vegetation including key bog plants such as Sphagnum mosses. Over 17 years, we measured near-complete recovery in the chemistry of surface-water and deep pore-water but limited progress in shallow pore-water. Our results suggest that at least >17 years are required for complete recovery of water chemistry to bog conditions. However, we expect that newer restoration methods including conifer harvesting (stem plus brash) and the blocking of plough furrows (to increase the WTD) are likely to accelerate the restoration process (albeit at greater cost); this should be evaluated in future studies. We conclude that monitoring pore- and surface-water chemistry is useful in terms of indicating recovery towards bog conditions and we recommend monitoring WTD, pH, conductivity, Ca, NH4+, phosphate (PO43-), K, DOC, Al and Zn as key variables.

Ecological and environmental transition across the forested-to-open bog ecotone in a west Siberian peatland.

Climate change may cause increasing tree cover in boreal peatlands, and the impacts of this encroachment will be noted first at forested-to-open bog ecotones. We investigate key metrics of ecosystem function in five such ecotones at a peatland complex in Western Siberia. Stratigraphic analysis of three cores from one of these transects shows that the ecotone has been dynamic over time with evidence for recent expansion of forested peatland. We observed that the two alternative states for northern boreal peatlands (forested/open) clearly support distinct plant and microbial communities. These in turn drive and respond to a number of feedback mechanisms. This has led to steep ecological gradients across the ecotones. Tree cover was associated with lower water tables and pH, along with higher bulk density, aquatic carbon concentrations, and electrical conductivity. We propose that the conditions found in the forested peatland of Western Siberia make the carbon sink more vulnerable to warmer and drier conditions.