Identity and ecophysiology of filamentous bacteria in activated sludge

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology.     

Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization.

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [(14)C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H(2)-utilizing and (14)CO(2)-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H(2)-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H(2) utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

Flow-through stable isotope probing (Flow-SIP) minimizes cross-feeding in complex microbial communities

Stable isotope probing (SIP) is a key tool for identifying the microorganisms catalyzing the turnover of specific substrates in the environment and to quantify their relative contributions to biogeochemical processes. However, SIP-based studies are subject to the uncertainties posed by cross-feeding, where microorganisms release isotopically labeled products, which are then used by other microorganisms, instead of incorporating the added tracer directly. Here, we introduce a SIP approach that has the potential to strongly reduce cross-feeding in complex microbial communities. In this approach, the microbial cells are exposed on a membrane filter to a continuous flow of medium containing isotopically labeled substrate. Thereby, metabolites and degradation products are constantly removed, preventing consumption of these secondary substrates. A nanoSIMS-based proof-of-concept experiment using nitrifiers in activated sludge and ¹³C-bicarbonate as an activity tracer showed that Flow-SIP significantly reduces cross-feeding and thus allows distinguishing primary consumers from other members of microbial food webs.Subject terms: Microbiology, Biological techniques, Metabolism, Biogeochemistry, Microbial ecology

Stable isotope probing (SIP) is widely applied to link specific microbial populations to metabolic processes in the environment and has greatly advanced our understanding of the role of microorganisms in biogeochemical cycling. SIP relies on tracing the incorporation of specific isotopically labeled substrates (e.g., ¹³C, ¹⁵N, ¹⁸O, ²H) into cellular biomarkers or bulk cellular biomass [e.g., 1–4]. SIP is considered a robust technique to identify microbial populations that assimilate a labeled substrate of interest in complex environmental communities. However, cross-feeding can occur when isotopically labeled metabolites are released from a primary consumer and then used by other microorganisms, which subsequently also become isotopically labeled. Likewise, when ¹³C-bicarbonate and unlabeled substrate are supplied to assess the activity of specific chemolithoautotrophs [e.g., 5–7], undesired ¹³C-incorporation can occur due to cross-feeding between chemolithoautotrophs whose activity depends on each other, for example in nitrifiers, where ammonia oxidizers provide nitrite oxidizers with their substrate, nitrite. The uncertainties associated with cross-feeding in SIP studies increase as the incubation time of microbial communities increases. While this phenomenon can be used to study microbial interactions and trophic networks [8–11], cross-feeding can lead to erroneous identification of organisms that are not directly responsible for the process of interest, but are rather connected to primary consumers via a microbial food web [2, 10, 12, 13].We developed an approach that significantly reduces the effect of cross-feeding in SIP studies. For this purpose, a thin layer of microbial cells is placed on a membrane filter, and isotopically labeled substrate is supplied at a fixed concentration by continuous flow, which constantly removes released metabolites and degradation products of primary substrate consumers. While previous SIP studies have employed a continuous flow of medium or substrate [e.g., 6, 14–16], in these studies, cross-feeding still occurred, as large amounts of biomass were placed in a 3D space, which allowed for the exchange of metabolites. Here, we present a proof-of-concept experiment with a nitrifying activated sludge microbial community, which converts ammonia to nitrite by the activity of ammonia-oxidizing bacteria (AOB), and subsequently oxidizes nitrite to nitrate by nitrite-oxidizing bacteria (NOB). In our experiments, the carbon source for both groups of autotrophic nitrifiers (the sludge contained no comammox bacteria [17, 18]) was isotopically labeled inorganic carbon (¹³C–NaHCO₃) and, as the sole electron donor, unlabeled ammonium was provided.In the flow-through approach, AOB, but not NOB, should be ¹³C-labeled because the substrate for NOB (nitrite), produced by AOB is continuously removed and thus the NOB should remain metabolically inactive (Fig. 1). In addition to a regular batch incubation, we included a control incubation, where the flow-through was recirculated to determine the impact of the experimental setup (continuous medium flow and retainment of biomass on a membrane filter) in Flow-SIP on the activity of the bacterial cells (in particular on the NOB as their autotrophic activity is used as a read out for cross-feeding in our experiments) in comparison to the batch experiment. Cross-feeding is expected to occur in both recirculated and batch control incubations, where nitrite is not removed and thus both AOB and NOB conserve energy to fix ¹³C–CO₂. After the experiments, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to identify AOB and NOB and combined with nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify ¹³C-assimilation at the single-cell level for all setups.Open in a separate windowFig. 1Schematic representation of the experimental setup: (left) batch, (center) recirculated, and (right) flow-through incubation.In all incubations, the carbon source for both autotrophic nitrifiers (AOB, yellow and NOB, magenta) was isotopically labeled (i.e., CO₂ as ¹³C–NaHCO₃) and ammonia was provided as the only external energy source (as NH₄Cl). Cross-feeding is expected to occur in the batch and recirculated approaches, where NOB consume nitrite produced via ammonia oxidation by AOB and thus both AOB and NOB incorporate ¹³C–CO₂. In the flow-through approach, only AOB are expected to be ¹³C-labeled, as cross-feeding should be eliminated by the continuous removal of nitrite. Other, non-nitrifier cells are indicated in gray.For these experiments, activated sludge from a Danish municipal wastewater treatment plant was initially treated by sonication to disrupt large flocs. Cells were then either placed on a membrane filter for flow-through incubation and the recirculated control experiment, or incubated in a conventional batch experiment. All experiments were set up using the same amount of biomass, and the ratio of biomass to medium volume was the same in batch and recirculated control experiments. Incubations were done using mineral medium containing 250 µM NH₄Cl and 2 mM ¹³C–NaHCO₃ for 24 h. Medium flow was maintained at a rate of 26 ml h⁻¹. We did not select a higher flow rate in order to avoid excessive stress by the medium flow on the microbial cells and to minimize the required amounts of media containing isotopically labeled bicarbonate. Furthermore, modeling nitrite advection and diffusion at different flow rates showed that, for example, a tenfold higher flow rate would only marginally reduce nitrite concentrations surrounding the AOB colonies (Fig. S1). In contrast, in a purely diffusive system without continuous flow, our model showed that nitrite would accumulate to significantly higher concentrations around single AOB colonies (Fig. S2). For example, after 24 h, ~23 µM nitrite would accumulate at a distance of 0–100 µm (with no significant decrease over distance) around an AOB colony of 50 cells, which is 230- to 9200-fold higher (depending on the distance to the colony) than modeled nitrite concentrations at the flow rate used in our experiments. Most inorganic metabolites that can directly be taken up into cells behave similar as nitrite in a diffusive system, i.e., they have a similar diffusion coefficient. Larger molecules tend to have an even smaller diffusion coefficient, which would lead to slower diffusion, and thus even more efficient metabolite removal when a medium flow is applied.We monitored nitrification activity via concentration measurements of ammonium, nitrite and nitrate (Fig. S3) and conducted nanoSIMS analyses (Fig. 2) for two successive experiments using sludge collected from the same treatment plant on different days as replication of experimental results (referred to as E1 and E2). Additionally, we confirmed the reproducibility of the method with two further experiments, where nitrification activity was followed (Fig. S4). Details on the experimental setup are given in Fig. 1 and the Supplementary Text.Open in a separate windowFig. 2Single cell isotope probing of nitrifying activated sludge in batch, recirculated, and flow-through incubations.a–c Show representative FISH images of E2 (AOB in yellow; NOB in magenta; other cells counterstained by DAPI in gray) of batch, recirculated, and flow-through incubations, respectively, and d–f show the corresponding nanoSIMS images. Scale bar is 10 µm in all images. g–l Show ¹³C labeling of AOB, NOB, and other cells quantified by nanoSIMS at the single-cell level for E1 (g–i) and E2 (j–l) in batch, recirculated, and flow-through incubations, respectively. We used FISH probe sets targeting AOB (Nitrosomonas oligotropha cluster (Cl6a192), Nitrosomonas eutropha/europea/urea cluster (NEU)) and NOB (Nitrotoga (Ntoga122), Nitrospira Lineage 1 (Ntspa1431), and Nitrospira Lineage 2 (Ntspa1151)), respectively, for differential staining of the two nitrifier groups. In (g–l), dashed lines give the ¹³C natural abundance values of the filter surface. The number of cells analyzed per group is indicated below each boxplot. For each experiment, lower case letters indicate significant difference in ¹³C labeling between groups (AOB, NOB, other cells) within an incubation type and upper case letters indicate significant difference between incubation types for a given group (Kruskal–Wallis test followed by Dunn’s test; Statistics are given in Table S2). Boxplots depict the 25–75% quantile range, with the center line depicting the median (50% quantile) and whiskers encompass data points within 1.5× the interquartile range.In recirculated and batch control incubations, the consumption of ammonium, production of nitrite and nitrate (Fig. S3), and single cell ¹³C-incorporation (Fig. 2) indicated that both AOB and NOB were active. However, nitrification activity (i.e., nitrite and nitrate production) in recirculated incubations were reduced by 57% (E1) and 83% (E2) compared to batch incubations (Fig. S3). The reduced ammonia oxidation activity in the recirculated incubations compared to the batch incubations was also reflected by a 73–82% lower ¹³C-incorporation in AOB cells in the former incubations (Fig. 2, median AOB ¹³C-enrichment in recirculated setup was 3.7 and 3.9 ¹³C-atom%, in batch incubations 20.7 and 14.7 ¹³C-atom% for E1 and E2, respectively). AOB in the flow-through incubations also showed lower ¹³C-enrichment levels (8.2 and 8.5 atom% for E1 and E2, respectively) compared to batch incubations but higher enrichment than in the recirculated incubations. The lower enrichment of AOB in the recirculated compared to the flow-through incubations might be due to an accumulation of compounds leaching from the used tubing (PharMed^® Ismaprene, Table S1), which may negatively affect AOB. Indeed, nitrifiers have previously been reported to be sensitive to various organic compounds [19, 20]. Use of different rubber tubing or replacing rubber tubing by glass might alleviate these effects. AOB ¹³C-enrichment was highest in batch incubations, which could be due to both the lack of stress from the continuous medium flow and the observed reaggregation of the sonicated activated sludge into larger flocs—reminiscent of native activated sludge flocs.As expected, NOB were ¹³C-enriched in both the batch (13.3 and 4.9 atom% for E1 and E2, respectively) and recirculated incubations (7.2 and 4.7 atom% for E1 and E2, respectively). In contrast, as intended, the flow-through setup resulted in a substantial reduction in ¹³C-enrichment of NOB (2.0 atom% for both E1 and E2, respectively; with consistently low ¹³C-enrichment in all NOB cells measured). This demonstrates that Flow-SIP efficiently removed the secondary substrate nitrite released by the AOB primary substrate consumers, thereby strongly limiting cross-feeding between AOB and NOB. The low ¹³C-enrichment of NOB in the flow-through incubations was statistically not significantly different to the ¹³C-enrichment of non-nitrifier cells (Table S2). It is unlikely that this low background ¹³C-enrichment was due to ¹³C-bicarbonate adsorption, as all samples were treated with acid before nanoSIMS analysis. It is, however, possible that at least some of the observed ¹³C-enrichment in NOB and other bacteria is due to anaplerotic reactions leading to C-fixation by background cellular activity rather than substrate-induced autotrophic C-fixation [e.g., 21, 22]. Transfer of ¹³C-labeled metabolites from the autotrophic nitrifiers to non-nitrifier cells was negligible in all incubations, including batch and recirculated incubations (Fig. 2), which was likely due to the short incubation time (24 h). In contrast, other SIP studies using incubation times of several days reported significant C-isotope transfer from nitrifiers to non-nitrifiers [11, 23].Our results demonstrate that Flow-SIP is a promising approach to significantly reduce cross-feeding in complex microbial communities and can be even successfully applied to highly aggregated samples like activated sludge flocs when they are dispersed prior to the experiment. Flow-SIP and conventional SIP are complementary to each other in the analysis of such aggregated or biofilm communities. In such systems, Flow-SIP enables microbial ecologists studying microbial physiologies with drastically reduced cross-feeding, but destroys the spatial arrangement of cells, while conventional SIP retains the 3D architecture, but its results are strongly influenced by cross-feeding. We expect that Flow-SIP is ideally suited for oligotrophic fresh- or seawater samples, which predominantly harbor planktonic cells or small aggregates, and thus do not require any sonication pretreatment before incubation. Furthermore, for such samples, tracer can be directly added to sterile filtered water without the need for using artificial medium, as used for the presented proof-of-principle experiments.Flow-SIP may, after upscaling to label more biomass, also be used in combination with DNA-, RNA- or protein-SIP, which should in comparison to conventional SIP, where cross-feeding is not inhibited, allow microbial ecologists to more precisely identify both previously known and yet unknown primary consumers of a supplied substrate. For example, Flow-SIP with ¹³C-bicarbonate and unlabeled ammonium would allow distinguishing comammox organisms from canonical NOB, as comammox but not the canonical NOB would be active under these conditions together with the canonical ammonia oxidizers. In addition, Flow-SIP has the potential to study direct use of chemically unstable substrates by microorganisms, by distinguishing it from microbial consumption of their chemically formed decomposition products. For example, cyanate, which abiotically decays relatively fast to ammonium and carbon dioxide [24, 25], has previously been shown to serve as energy and nitrogen source for ammonia-oxidizing archaea [25, 26]. Using Flow-SIP, cyanate could be constantly supplied, thereby strongly reducing abiotic decay. At the same time, any abiotically formed ammonium (and ammonium produced by other organisms) would be constantly removed, which should allow identifying ammonia-oxidizing microorganisms that directly use cyanate as a substrate. Furthermore, the presented approach may be coupled to fluorescence-based activity markers, where a substrate of interest and bioorthogonal noncanonical amino acids are supplied and, subsequently, translationally active cells are visualized on an epifluorescence microscope (BONCAT) [27]. In conclusion, Flow-SIP expands the toolbox of microbial ecologists interested in structure–function analyses of microbial communities and will contribute to a more precise understanding of the ecophysiology of bacteria and archaea catalyzing key processes in their natural environments.     

The biogeochemical consequences of litter transformation by insect herbivory in the Subarctic: a microcosm simulation experiment

Warming may increase the extent and intensity of insect defoliations within Arctic ecosystems. A thorough understanding of the implications of this for litter decomposition is essential to make predictions of soil-atmosphere carbon (C) feedbacks. Soil nitrogen (N) and C cycles naturally are interlinked, but we lack a detailed understanding of how insect herbivores impact these cycles. In a laboratory microcosm study, we investigated the growth responses of heterotrophic soil fungi and bacteria as well as C and N mineralisation to simulated defoliator outbreaks (frass addition), long-term increased insect herbivory (litter addition at higher background N-level) and non-outbreak conditions (litter addition only) in soils from a Subarctic birch forest. Larger amounts of the added organic matter were mineralised in the outbreak simulations compared to a normal year; yet, the fungal and bacterial growth rates and biomass were not significantly different. In the simulation of long-term increased herbivory, less litter C was respired per unit mineralised N (C:N of mineralisation decreased to 20?±?1 from 38?±?3 for pure litter), which suggests a directed microbial mining for N-rich substrates. This was accompanied by higher fungal dominance relative to bacteria and lower total microbial biomass. In conclusion, while a higher fraction of foliar C will be respired by insects and microbes during outbreak years, predicted long-term increases in herbivory linked to climate change may facilitate soil C-accumulation, as less foliar C is respired per unit mineralised N. Further work elucidating animal-plant-soil interactions is needed to improve model predictions of C-sink capacity in high latitude forest ecosystems.     

The Role of the NADPH Oxidase NOX2 in Prion Pathogenesis

Prion infections cause neurodegeneration, which often goes along with oxidative stress. However, the cellular source of reactive oxygen species (ROS) and their pathogenetic significance are unclear. Here we analyzed the contribution of NOX2, a prominent NADPH oxidase, to prion diseases. We found that NOX2 is markedly upregulated in microglia within affected brain regions of patients with Creutzfeldt-Jakob disease (CJD). Similarly, NOX2 expression was upregulated in prion-inoculated mouse brains and in murine cerebellar organotypic cultured slices (COCS). We then removed microglia from COCS using a ganciclovir-dependent lineage ablation strategy. NOX2 became undetectable in ganciclovir-treated COCS, confirming its microglial origin. Upon challenge with prions, NOX2-deficient mice showed delayed onset of motor deficits and a modest, but significant prolongation of survival. Dihydroethidium assays demonstrated a conspicuous ROS burst at the terminal stage of disease in wild-type mice, but not in NOX2-ablated mice. Interestingly, the improved motor performance in NOX2 deficient mice was already measurable at earlier stages of the disease, between 13 and 16 weeks post-inoculation. We conclude that NOX2 is a major source of ROS in prion diseases and can affect prion pathogenesis.     

Sleep deprivation sensitizes human craniofacial muscles

It is unknown whether and how sleep deprivation influences craniofacial muscle sensitivity in healthy humans. We investigated whether total sleep deprivation (TSD) and one night of recovery sleep (RS) can alter mechanical pain sensitivity in temporal and masseter muscles. Fifteen healthy volunteers participated in three consecutive sessions. Pressure pain thresholds were measured on the temporal and masseter muscles. Both temporal and masseter muscles became sensitized after 24?h of TSD. RS reversed the muscle sensitization.     

Placental 11β-hydroxysteroid dehydrogenase and the programming of hypertension

Excessive foetal exposure to glucocorticoids retards growth and “programmes” adult hypertension in rats. Placental 11β-hydroxysteroid dehydrogenase (11β-HSD), which catalyses the conversion of corticosterone and cortisol to inert 11 keto-products, normally protects the foetus from excess maternal glucocorticoids. In both rats and humans there is considerable natural variation in placental 11β-HSD, and enzyme activity correlates with birth weight. Moreover, inhibition of placental 11β-HSD in the rat reduces birth weight and produces hypertensive adult offspring, many months after prenatal treatment with enzyme inhibitors; these effects are dependent upon maternal adrenal products. These data suggest that placental 11β-HSD, by regulating foetal exposure to maternal glucocorticoids, crucially determines foeto-placental growth and the programming of hypertension. Maternal protein restriction during pregnancy also produces hypertensive offspring and selectively attenuates placental 11β-HSD activity. Thus, deficiency of the placental barrier to maternal glucocorticoids may represent a common pathway between the maternal environment and foeto-placental programming of later disease. These data may, at least in part, explain the human epidemiological observations linking early life events to the risk of subsequent hypertension. The recent characterization, purification and cDNA cloning of a distinct human placental 11β-HSD (type 2) will aid the further study of these intriguing findings.