Copper-Induced Stimulation of Nitrification in Biological Rapid Sand Filters for Drinking Water Production by Proliferation of Nitrosomonas spp.

Copper is a cofactor of the ammonia monooxygenase, an essential enzyme for the activity of ammonia oxidizing prokaryotes (AOP). Copper dosing at less than 1 µg/L stimulated ammonium removal in the poorly nitrifying biological filters of three full-scale drinking water treatment plants. Upon copper dosing, the ammonium concentration in the effluent decreased from up to 0.18 to less than 0.01 mg NH4+-N/L. To investigate how copper dosing affected the filter microbial community, we applied amplicon sequencing and qPCR targeting key nitrifying groups, including complete ammonia oxidizing (comammox) Nitrospira. Copper dosing increased the abundance of different nitrifiers. Multiple Nitrosomonas variants (betaproteobacterial ammonia oxidizers), which initially collectively represented 1% or less of the total community, increased almost 10-fold. Comammox Nitrospira were abundant and increased too, but their relative abundance within the AOP decreased because of Nitrosomonas proliferation. No other consistent change in the filter communities was detected, as well as no adverse effect of copper on the filters functionality. Our results show that copper dosing in three independent treatment plants was associated with consistent growth of AOP and that efficient nitrification was achieved through the joint contribution of comammox Nitrospira and an increasing fraction of betaproteobacterial ammonia oxidizers.

Does universal 16S rRNA gene amplicon sequencing of environmental communities provide an accurate description of nitrifying guilds?

Universal (i.e., targeting most bacteria/prokaryotes) 16S rRNA gene based amplicon sequencing is widely used for assessing microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community. However, it is currently unclear if it can yield reliable information on specific microbial guilds, as obtained by using primer sets targeting functional genes or specific16S rRNA gene sequences. Here, we compared the relative abundance, diversity, richness, and composition of selected guilds (nitrifiers), obtained from universal 16S rRNA gene based amplicon sequencing and from guild targeted approaches. The universal amplicon sequencing provided 1) accurate estimates of nitrifier composition, 2) clustering of the samples based on these compositions consistent with sample origin, 3) estimates of the relative abundance of the guilds correlated with those obtained from the targeted approaches and within ~1.2 orders of magnitude of them, but with measurable bias that should be considered when comparing estimates from both approaches. In contrast, the diversity and richness estimations using the universal 16S rRNA based amplicon sequencing were likely limited by the sequencing depth; therefore, we suggest preferring targeted approaches for assessing nitrifiers diversity and richness or using sequencing depth larger than those currently typically practiced. Overvall, we conclude that universal amplicon sequencing provides, in a single analysis, useful information on the abundance and composition of diverse guilds in complex environmental communities.

Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR.

Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantitative PCR methods to enumerate ammonia-oxidizing bacteria (AOB): one targeting the phylogenetic gene 16S rRNA and the other, the functional gene amoA. Cloning-sequencing with both primer sets on DNA from two waterworks revealed contrasting images of AOB diversity. The amoA-based approach preferentially recovered sequences belonging to Nitrosomonas Cluster 7 over Cluster 6A ones, while the 16S rRNA one yielded more diverse sequences belonging to three AOB clusters, but also a few non-AOB sequences, suggesting broader, but partly unspecific, primer coverage. This was confirmed by an in silico coverage analysis against sequences of AOB (both isolates and high-quality environmental sequences). The difference in primer coverage significantly impacted the estimation of AOB abundance at the waterworks with high Cluster 6A prevalence, with estimates up to 50-fold smaller for amoA than for 16S rRNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets.

Widespread green algae Chlorella and Stichococcus exhibit polar-temperate and tropical-temperate biogeography.

Chlorella and Stichococcus are morphologically simple airborne microalgae, omnipresent in terrestrial and aquatic habitats. The minute cell size and resistance against environmental stress facilitate their long-distance dispersal. However, the actual distribution of Chlorella- and Stichococcus-like species has so far been inferred only from ambiguous morphology-based evidence. Here we contribute a phylogenetic analysis of an expanded SSU and ITS2 rDNA sequence dataset representing Chlorella- and Stichococcus-like species from terrestrial habitats of polar, temperate and tropical regions. We aim to uncover biogeographical patterns at low taxonomic levels. We found that psychrotolerant strains of Chlorella and Stichococcus are closely related with strains originating from the temperate zone. Species closely related to Chlorella vulgaris and Muriella terrestris, and recovered from extreme terrestrial environments of polar regions and hot deserts, are particularly widespread. Stichococcus strains from the temperate zone, with their closest relatives in the tropics, differ from strains with the closest relatives being from the polar regions. Our data suggest that terrestrial Chlorella and Stichococcus might be capable of intercontinental dispersal; however, their actual distributions exhibit biogeographical patterns.