In activated sludge, the antibiotic resistance genes (ARGs) can be present either in the intracellular (iDNA) or extracellular DNA fraction (exDNA). Recent advances in the exDNA extraction methodology allow a better profiling of the pool of ARGs. However, little is known about how stress conditions modify the distribution of ARGs between both DNA fractions. Here, we performed two batch tests for analyzing the effects of two different stress conditions, namely nutrient starvation and high concentrations of sulfamethoxazole (1, 10 and 150 mg L⁻¹) in activated sludge. We tracked by qPCR the resulting relative abundances of four target genes, namely the universal 16S rRNA gene, the class 1 integron-integrase gene intI1, and the sulfonamide resistance genes sul1 and sul2 in both the iDNA and exDNA fractions. In the exDNA pool, unlike starvation, which provoked a decrease of 1-2 log₁₀ [copies] per ng DNA in the concentration of sul1 and intI1, the presence of sulfamethoxazole did not influence the abundances of sul1 and sul2. However, high concentrations of sulfamethoxazole (150 mg L⁻¹) selected for microorganisms harboring sul1 and, more remarkably, sul2 genes in their iDNA during their exponential growth phase. The abundances of intI1 and sul1 were positively correlated in the exDNA fraction (r > 0.7), whereas no significant correlation (p < 0.05) between the abundance of these two genes was found in the iDNA fraction of the sludge. High SMX concentrations influenced the abundance of ARGs in the iDNA; their abundance in the exDNA was influenced by nutrient limitations. Further studies should consider the profiling of exDNA fractions because of the relationship between ARGs and mobile genetic elements. Besides, the surveillance of antimicrobial resistance is encouraged in wastewater treatment plants facing high antibiotic concentrations.

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Biomarkers such as functional gene mRNA (transcripts) and proteins (enzymes) provide direct proof of metabolic regulation during the reductive dechlorination (RD) of chlorinated ethenes (CEs). Yet, current models to simulate their spatiotemporal variability are not flexible enough to mimic the homologous behavior of RDase functional genes. To this end, we developed new enzyme-based kinetics to model the concentrations of CEs together with the transcript and enzyme levels during RD. First, the model was calibrated to existing microcosm data on RD of cis-DCE. The model mirrored the tceA and vcrA gene expression and the production of their enzymes in Dehalococcoides spp. Considering tceA and vcrA as homologous instead of nonhomologous improved fitting of the mRNA time series. Second, CEs and biomarker patterns were explored as a proof of concept under groundwater flow conditions, considering degraders occurring in immobile and mobile states. Under both microcosm and flow conditions, biomarker-rate relationships were nonlinear hysteretic because tceA and vcrA acted as homologous genes. The mobile biomarkers additionally undergo advective-dispersive transport, which increases the nonlinearity and makes the observed patterns even more challenging to interpret. The model offers a thorough mechanistic description of RD while also allowing simulation of spatiotemporal dynamic patterns of various key biomarkers in aquifers.@en

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L⁻¹ d⁻¹) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.

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BACKGROUND: Carboxylates such as volatile fatty acids (VFA) can be produced by acidogenic fermentation (AF) of dairy wastes including cheese whey, a massive residue produced at 160.67 million m³ of which 42% are not valorized and impact the environment. In mixed-culture fermentations, selection pressures can favor AF and halt methanogenesis. In this study, inoculum pre-treatment was evaluated as a selective pressure for AF demineralized cheese whey in batches. Alkaline (NaOH, pH 8.0, 6 h) and thermal (90 °C for 5 min, ice-bath until 23 °C) pre-treatments were tested with batch operations runs at initial pH 7.0 and 9.0, food-to-microorganism (F/M) ratios of 0.5 to 4.0 g COD g⁻¹ VS, and under pressurized (P) and nonpressurized (NP) headspace, in experiments duplicated in two different research institutes. RESULTS: Acetic acid was highly produced on both Unicamp and TU Delft samples (1.36 and 1.40 g COD_AcOH L⁻¹, respectively), at the expense of methanogenesis by combining a thermal pre-treatment of inoculum with a NP batch operation started at pH 9.0. Microbial communities comprising VFA and alcohol producers, such as Clostridium, Fonticella and Intestinimonas, and fermenters such as Longilinea and Leptolinea. The lipid-accumulating Candidatus microthrix was observed in both bulk material and foam. Despite the absence of methane production, Methanosaeta were detected within the microbial community. An F/M ratio of 0.5 g COD g⁻¹ VS led to the best VFA production of 1769.4 mg L⁻¹. CONCLUSION: Overall, inoculum thermal pre-treatment, initial pH 9.0 and NP headspace acted as a selective pressure for halting methanogenesis and producing VFAs, valorizing cheese whey via batch acidogenic fermentation.

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Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly ¹³C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. ¹³C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d⁻¹ respectively) was higher than the expected value based on the solid removal rate (0.132 d⁻¹), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d⁻¹. Key Points: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

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Purple phototrophic bacteria (PPB) show an underexplored potential for resource recovery from wastewater. Raceway reactors offer a more affordable full-scale solution on wastewater and enable useful additional aerobic processes. Current mathematical models of PPB systems provide useful mechanistic insights, but do not represent the full metabolic versatility of PPB and thus require further advancement to simulate the process for technology development and control. In this study, a new modelling approach for PPB that integrates the photoheterotrophic, and both anaerobic and aerobic chemoheterotrophic metabolic pathways through an empirical parallel metabolic growth constant was proposed. It aimed the modelling of microbial selection dynamics in competition with aerobic and anaerobic microbial community under different operational scenarios. A sensitivity analysis was carried out to identify the most influential parameters within the model and calibrate them based on experimental data. Process perturbation scenarios were simulated, which showed a good performance of the model.

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Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by circa 2.5 log gene copies mL−1, despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs enriched for bacteria containing genes linked to antibiotic resistance. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs.@en

Fermentative chemoorganoheterotrophic bacteria (FCB) and purple photoorganoheterotrophic bacteria (PPB) are two interesting microbial guilds to process carbohydrate-rich wastewaters. Their metabolic interactions have been studied in pure cultures or co-cultures, but little is known about mixed cultures. We studied the effect of reactor regimes (batch/chemostat) and illumination modes (continuous infrared light, dark, or light/dark cycles) on glucose conversions and process ecology of the interactions between FCB and PPB in mixed cultures. In batch, FCB (>80 % of sequencing read counts) outcompeted PPB, under any light conditions. In chemostat under continuous and alternating irradiance, three FCB populations were enriched (>70 %), while Rhodobacteraceae (PPB) made 30 % of the community. Glucose fermentation products were linked to the dominant FCB. Continuous culturing helped maintaining FCB and PPB in syntrophy: PPB grew on glucose metabolites produced by FCB. Engineering the association between FCB and PPB in mixed-culture processes can help to treat and valorize carbohydrate-rich aqueous waste.

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Whey has applications in food, beverages, personal care products, pharmaceuticals, and the medical sector. However, it remains a massive dairy residue worldwide (160.7 million m³ year⁻¹), with high organic and nutrient loads. About 42% is used for low-value products such as animal feed and fertilizers or is even directly discharged into water streams, leading to ecosystem damage via eutrophication. We reviewed the uses and applications of cheese whey, along with associated environmental impacts and innovative ways to mitigate them using affordable and scalable technologies. Recycling and repurposing whey remain challenges for remote locations and poor communities with limited access to expensive technology. We propose a closed-loop biorefinery strategy to simultaneously mitigate environmental impacts and valorize whey resources. Anaerobic digestion utilizes whey to produce biogas and/or carboxylates. Alternative processes combining anaerobic digestion and low-cost open photobioprocesses can valorize whey and capture organic, nitrogenous, and phosphorous nutrients into microalgal biomass that can be used as food and crop supply or processed into biofuels, pigments, and antioxidants, among other value-added products. The complete valorization of cheese whey also depends on facilitating access to relevant information on whey production, identifying stakeholders, reducing technology gaps among countries, enforcing legislation and compliance, and creating subsidies and fostering partnerships with industries and between countries.

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This study investigates the effects, conversions, and resistance induction, following the addition of 150 μg·L⁻¹ of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), in a laboratory-scale micro-aerated anaerobic membrane bioreactor (MA-AnMBR). TMP and SMX were removed at 97 and 86%, indicating that micro-aeration did not hamper their removal. These antibiotics only affected the pH and biogas composition of the process, with a significant change in pH from 7.8 to 7.5, and a decrease in biogas methane content from 84 to 78%. TMP was rapidly adsorbed onto the sludge and subsequently degraded during the long solids retention time of 27 days. SMX adsorption was minimal, but the applied hydraulic retention time of 2.6 days was sufficiently long to biodegrade SMX. The levels of three antibiotic-resistant genes (ARGs) (sul1, sul2, and dfrA1) and one mobile genetic element biomarker (intI1) were analyzed by qPCR. Additions of the antibiotics increased the relative abundances of all ARGs and intI1 in the MA-AnMBR sludge, with the sul2 gene folding 15 times after 310 days of operation. The MA-AnMBR was able to reduce the concentration of antibiotic-resistant bacteria (ARB) in the permeate by 3 log.@en

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3′’)-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.@en

Purple bacteria (PPB), anoxygenic photoorganoheterotrophic organisms with a hyper-versatile metabolism and high biomass yields over substrate, are promising candidates for the recovery of nutrient resources from wastewater. Infrared light is a pivotal parameter to control and design PPB-based resource recovery. However, the effects of light intensities on the physiology and selection of PPB in mixed cultures have not been studied to date. Here, we examined the effect of infrared irradiance on PPB physiology, enrichment, and growth over a large range of irradiance (0 to 350 W m−2) in an anaerobic mixed-culture sequencing batch photobioreactor. We developed an empirical mathematical model that suggests higher PPB growth rates as response to higher irradiance. Moreover, PPB adapted to light intensity by modulating the abundances of their phototrophic complexes. The obtained results provide an in-depth phylogenetic and metabolic insight the impact of irradiance on PPB. Our findings deliver the fundamental information for guiding the design of light-driven, anaerobic mixed-culture PPB processes for wastewater treatment and bioproduct valorization.@en

Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.

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Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.@en

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC–HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10–37 °C. “Candidatus Brocadia” appeared to be the dominant organism in all but two laboratory enrichments of “Ca. Scalindua” and “Ca. Kuenenia”. At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. “Ca. Scalindua” and “Ca. Kuenenia” had distinct profile of ladderane lipids compared to “Ca. Brocadia” biomasses with potential implications for adaptability to low temperatures. “Ca. Brocadia” membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for “Ca. Scalindua” at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of “Ca. Scalindua” in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

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Abstract: Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that “Candidatus Accumulibacter” can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a “Ca. Accumulibacter” enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of “Ca. Accumulibacter” contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with “Ca. Accumulibcater” and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. Key points: • NulOs in “Ca. Accumulibacter” are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.@en

Environmental microorganisms evolve constantly under various stressors using different adaptive mechanisms, including horizontal gene transfer. Microorganisms benefit from transferring genetic information that code for antibiotic resistance via mobile genetic elements (plasmids). Due to the complexity of natural microbial ecosystems, quantitative data on the transfer of genetic information in microbial communities remain unclear. Two 1-L chemostats (one control and one test) were inoculated with activated sludge, fed with synthetic wastewater, and operated for 45 days at a hydraulic retention time of 1 day to study the transformation capacity of a rolling-circle plasmid encoding GFP and kanamycin resistance genes, at increasing concentrations of kanamycin (0.01-2.5-50-100 mg L ⁻¹ ) representing environmental, wastewater, lab-selection, and gut or untreated pharmaceutical wastewater discharge environments. The plasmid DNA was spiked daily at 5 µg L ⁻¹ in the test chemostat. The evolution of the microbial community composition was analyzed by 16S rRNA gene amplicon sequencing and metagenomics, and the presence of the plasmid by quantitative PCR. We used Hi-C sequencing to identify natural transformant microorganisms under steady-state conditions with low (2.5 mg L ⁻¹ ) and high (50 mg L ⁻¹ ) concentrations of kanamycin. Both chemostats selected for the same 6 predominant families of Spirosomaceae, Comamonadaceae, Rhodocyclaceae, Rhizobiaceae, Microbacteriaceae , and Chitinophagaceae , while biomass formation in the presence of kanamycin was higher with the plasmid. Hence, the antibiotic exerted the main pressure on microbial selection, while the plasmid helped these populations better resist the antibiotic treatment and grow. The kanamycin resistance gene increased in both reactors (log 7 gene copies g VSS ⁻¹ ). When higher antibiotic concentrations were applied, the GFP/16S ratio was increased, highlighting plasmids accumulation in the test reactor over time. The plasmid transformed mainly inside populations of Bosea sp ., Runella spp ., and Microbacterium sp .. This study made one significant step forward by demonstrating that microorganisms in enrichments from activated sludge biomasses can acquire exogenous synthetic plasmids by transformation. Graphical abstract@en

The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions and cell physiology affect anammox performance at psychrophilic temperatures below 15 °C remains poorly understood. We tested 14 anammox communities, including 8 from globally-installed PN/A processes, for (i) specific activity at 10–30 °C, (ii) composition of membrane lipids, and (iii) microbial community structure. We observed that membrane composition and cultivation temperature were closely related to the activity of anammox biomasses. The size of ladderane lipids and the content of bacteriohopanoids were key physiological components related to anammox performance at low temperatures. We also indicate that the adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases can take up to 5 years. Interestingly, biomass enriched in the marine genus “Candidatus Scalindua” displayed outstanding potential for nitrogen removal from cold streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation mechanism, will enable more accurate modelling and suggests highly promising target anammox genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs.

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Anammox bacteria enable efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to ≤15 °C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of ‘cold shocks’ is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic “Ca. Kuenenia”). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LC–HRMS/MS) and the structure of membrane lipids (UPLC–HRMS/MS). The shocked culture was more active (0.66±0.06 vs 0.48±0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient expression of potential cold shock proteins (e.g. ppiD, UspA, pqqC), while putative cold shock proteins CspB and TypA were upregulated in both cultures. Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures; this confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures.

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