The phototrophic metabolic behaviour of Candidatus accumulibacter.

The phototrophic capability of Candidatus Accumulibacter (Accumulibacter), a common polyphosphate accumulating organism (PAO) in enhanced biological phosphorus removal (EBPR) systems, was investigated in this study. Accumulibacter is phylogenetically related to the purple bacteria Rhodocyclus from the family Rhodocyclaceae, which belongs to the class Betaproteobacteria. Rhodocyclus typically exhibits both chemoheterotrophic and phototrophic growth, however, limited studies have evaluated the phototrophic potential of Accumulibacter. To address this gap, short and extended light cycle tests were conducted using a highly enriched Accumulibacter culture (95%) to evaluate its responses to illumination. Results showed that, after an initial period of adaptation to light conditions (approximately 4-5 h), Accumulibacter exhibited complete phosphorus (P) uptake by utilising polyhydroxyalkanoates (PHA), and additionally by consuming glycogen, which contrasted with its typical aerobic metabolism. Mass, energy, and redox balance analyses demonstrated that Accumulibacter needed to employ phototrophic metabolism to meet its energy requirements. Calculations revealed that the light reactions contributed to the generation of, at least more than 67% of the ATP necessary for P uptake and growth. Extended light tests, spanning 21 days with dark/light cycles, suggested that Accumulibacter generated ATP through light during initial operation, however, it likely reverted to conventional anaerobic/aerobic metabolism under dark/light conditions due to microalgal growth in the mixed culture, contributing to oxygen production. In contrast, extended light tests with an enriched Tetrasphaera culture, lacking phototrophic genes in its genome, clearly demonstrated that phototrophic P uptake did not occur. These findings highlight the adaptive metabolic capabilities of Accumulibacter, enabling it to utilise phototrophic pathways for energy generation during oxygen deprivation, which holds the potential to advance phototrophic-EBPR technology development.

Assessing the efficiency and microbial diversity of H2S-removing biotrickling filters at various pH conditions.

This study aimed to investigate the operation of three parallel biotrickling filters (BTFs) in removing H2S at different pH conditions (haloalkaliphilic, neutrophilic, and acidophilic) and their associated microbial population in the biodesulfurization process. BTF columns were inoculated with enriched inoculum and experiments were performed by gradually reducing Empty Bed Retention Time (EBRT) and increasing inlet concentration in which the maximum removal efficiency and maximum elimination capacity in EBRT 60 s reached their maximum level in haloalkaline condition (91% and 179.5 g S-H2S m-3 h-1). For visualizing the attached microbial biofilms on pall rings, Scanning Electron Microscopy (SEM) was used and microbial community structure analysis by NGS showed that the most abundant phyla in haBTF, nBTF, and aBTF belong to Gammaproteobacteria, Betaproteobacteria, and Acidithiobacillia, respectively. Shannon and Simpson indexes evaluation showed a lower diversity of bacteria in the aBTF reactor than that of nBTF and haBTF and beta analysis indicated a different composition of bacteria in haBTF compared to the other two filters. These results indicated that the proper performance of BTF under haloalkaliphilic conditions is the most effective way for H2S removal from air pollutants of different industries.

Computational Insights into the Catalysis of the pH Dependence of Bromite Decomposition Catalyzed by Chlorite Dismutase from Dechloromonas aromatica (DaCld).

The heme-containing chlorite dismutases catalyze the rapid and efficient decomposition of chlorite (ClO2-) to yield Cl- and O2, and the catalytic efficiency of chlorite dismutase from Dechloromonas aromatica (DaCld) in catalyzing the decomposition of bromite (BrO2-) was dependent on pH, which was supposed to be caused by the conversion of active Cpd I to the inactive Cpd II by proton-coupled electron transfer (PCET) from the pocket Tyr118 to the propionate side chain of heme at high pH. However, the direct evidence of PCET and how the pH affects the efficiency of DaCld, as well as whether Cpd II is really inactive, are still poorly understood. Here, on the basis of the high-resolution crystal structures, the computational models in both acidic (pH 5.0) and alkaline (pH 9.0) environments were constructed, and a series of quantum mechanical/molecular mechanical calculations were performed. On the basis of our calculation results, the O-Br bond cleavage of BrO2- always follows the homolytic mode to generate Cpd II rather than Cpd I. It is different from the O-O cleavage of O2/H2O2 or peracetic acid catalyzed by the other heme-containing enzymes. Thus, in the subsequent O-O rebound reaction, it is the Fe(IV)═O in Cpd II that combines with the O-Br radical. Because the porphyrin ring in Cpd II does not bear an unpaired electron, the previously suggested PCET from Tyr118 to the propionate side chain of heme was not theoretically recognized in an alkaline environment. In addition, the O-O rebound step in an alkaline solution corresponds to an energy barrier that is larger than that in an acidic environment, which can well explain the pH dependence of the activity of DaCld. In addition, the protonation state of the propionic acid side chains of heme and the surrounding hydrogen bond networks were calculated to have a significant impact on the barriers of the O-O rebound step, which is mainly achieved by affecting the reactivity of the Fe(IV)═O group in Cpd II. In an acidic environment, the relatively weaker coordination of the O2 atom to Fe leads to its higher reactivity toward the O-O rebound reaction. These observations may provide useful information for understanding the catalysis of chlorite dismutases.

Enzymatic synthesis of a skin active ingredient - glochidone by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans.

In this study, we applied AcmB2, sourced from Sterolibacterium denitrificans, to catalyze the oxidative dehydrogenation of 3-ketolupeol (lupenone), a derivative of lupeol, triterpene obtained from birch bark. This enzymatic Δ1-dehydrogenation catalyzed by AcmB2 yielded glochidone, a bioactive compound frequently obtained from medicinal plants like Salvia trichoclada and Maytenus boria. Glochidone is known for its broad biological activities, including antibacterial, antifungal, anti-inflammatory, anticancer, antidiabetic as well as acetylcholinesterase inhibition. Our research demonstrates >99% conversion efficiency with 100% regioselectivity of the reaction. The effective conversion to glochidone employed an electron acceptor e.g., potassium hexacyanoferrate III, in mild, environmentally friendly conditions: 8-16% 2-hydroxypropyl-ß-cyclodextrin, and 2-3% 2-methoxyethanol. AcmB2 reaction optimum was determined at pH 8.0 and 30 °C. Enzyme's biochemical attributes such as electron acceptor type, concentration and steroid substrate specificity were investigated. Among 4-, 5- and 6-ring steroid derivatives androst-4-en-3,17-dione and testosterone propionate were determined as the best substrates of AcmB2. Δ1-Dehydrogenation of substrates such as lupenone, diosgenone and 3-ketopetromyzonol was confirmed. We have assessed the antioxidant and rejuvenating characteristics of glochidone as an active component in formulations, considering its precursors, lupeol, and lupenone as well. Glochidone exhibited limited antioxidant and chelating capabilities compared to lupeol and reference compounds. However, it demonstrated robust rejuvenating properties, with a sirtuin induction level of 61.5 ± 1.87%, notably surpassing that of the reference substance, E-resveratrol (45.15 ± 0.09%). Additionally, glochidone displayed 26.5±0.67 and 19.41±0.76% inhibition of elastase and collagenase, respectively. The safety of all studied triterpenes was confirmed on skin reconstructed human Epidermis model. These findings provide valuable insights into the potential applications of glochidone in formulations aimed at addressing skin health concerns. This research presents the first example of an enzyme in the 3-ketosteroid dehydrogenase (KstD) family catalyzing the Δ1-dehydrogenation of a pentacyclic triterpene. We also explored structural differences between AcmB, AcmB2, and related KstDs pointing to G52 and P532 as potentially responsible for the unique substrate specificity of AcmB2. Our findings not only highlight the enzyme's capabilities but also present novel enzymatic pathways for bioactive compound synthesis.

Endosymbiont Tremblaya phenacola influences the reproduction of cotton mealybugs by regulating the mechanistic target of rapamycin pathway.

The intricate evolutionary dynamics of endosymbiotic relationships result in unique characteristics among the genomes of symbionts, which profoundly influence host insect phenotypes. Here, we investigated an endosymbiotic system in Phenacoccus solenopsis, a notorious pest of the subfamily Phenacoccinae. The endosymbiont, "Candidatus Tremblaya phenacola" (T. phenacola PSOL), persisted throughout the complete life cycle of female hosts and was more active during oviposition, whereas there was a significant decline in abundance after pupation in males. Genome sequencing yielded an endosymbiont genome of 221.1 kb in size, comprising seven contigs and originating from a chimeric arrangement between betaproteobacteria and gammaproteobacteria. A comprehensive analysis of amino acid metabolic pathways demonstrated complementarity between the host and endosymbiont metabolism. Elimination of T. phenacola PSOL through antibiotic treatment significantly decreased P. solenopsis fecundity. Weighted gene coexpression network analysis demonstrated a correlation between genes associated with essential amino acid synthesis and those associated with host meiosis and oocyte maturation. Moreover, altering endosymbiont abundance activated the host mechanistic target of rapamycin pathway, suggesting that changes in the amino acid abundance affected the host reproductive capabilities via this signal pathway. Taken together, these findings demonstrate a mechanism by which the endosymbiont T. phenacola PSOL contributed to high fecundity in P. solenopsis and provide new insights into nutritional compensation and coevolution of the endosymbiotic system.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase.

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4ß4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Contrasting response of comammox Nitrospira, ammonia oxidising bacteria, and archaea to soil pH and nitrogen inputs.

This study aimed to investigate the effect of soil pH change, and nitrogen amendment on ammonia oxidiser abundance and comammox Nitrospira community composition. The experimental design used soil mesocosms placed in a temperature-controlled incubator for 90 days. A Templeton silt loam was used as its physiochemical properties are typical of the region's dairy farms. The results showed that comammox Nitrospira clade B preferred the natural (pH 6.1-6.2) soil pH with no applied nitrogen. Furthermore, synthetic urine (N700) decreased the abundance of comammox Nitrospira clade B. This may have been because the large amounts of available ammonia in the N700 treatments inhibited the growth of comammox Nitrospira. These results suggest that while comammox Nitrospira clade B are present in New Zealand dairy farm soils, but their role in nitrification in the very high nitrogen environment under a urine patch in grazed pastures may be limited. Further research is needed to confirm this. In contrast to comammox, the AOB community (dominated by Nitrosospira) responded positively to the application of synthetic urine. The response was greatest in the high pH soil (7.1), followed by the natural and then the low pH (4.9) soils. This may be due to the difference in ammonia availability. At high pH, the ammonia/ammonium equilibrium favours ammonia production. Calculated ammonia availability in the N700 treatments accurately predicted the AOB amoA gene abundance. Interestingly, the AOA community abundance (which was predominantly made up of Thaumarchaeota group I.1b clade E) seemed to prefer the natural and high pH soils over the low pH. This may be due to the specific lineage of AOA present. AOA did not respond to the application of nitrogen.

The significant contribution of comammox bacteria to nitrification in a constructed wetland revealed by DNA-based stable isotope probing.

The discovery of Comammox bacteria (CMX) has changed our traditional concept towards nitrification, yet its role in constructed wetlands (CWs) remains unclear. This study investigated the contributions of CMX and two canonical ammonia-oxidizing microorganisms, ammonia-oxidizing bacteria (AOB) and archaea to nitrification in four regions (sediment, shoreside, adjacent soil, and water) of a typical CW using DNA-based stable isotope probing. The results revealed that CMX not only widely occurred in sediment and shoreside zones with high abundance (5.08 × 104 and 6.57 × 104 copies g-1 soil, respectively), but also actively participated in ammonia oxidation, achieving ammonia oxidation rates of 1.43 and 2.00 times that of AOB in sediment and shoreside, respectively. Phylogenetic analysis indicated that N. nitrosa was the dominant and active CMX species. These findings uncovered the crucial role of CMX in nitrification of sediment and shoreside, providing a new insight into nitrogen cycle of constructed wetlands.

Urea amendment decouples nitrification in hydrocarbon contaminated Antarctic soil.

Hydrocarbon contaminated soils resulting from human activities pose a risk to the natural environment, including in the Arctic and Antarctic. Engineered biopiles constructed at Casey Station, Antarctica, have proven to be an effective strategy for remediating hydrocarbon contaminated soils, with active ex-situ remediation resulting in significant reductions in hydrocarbons, even in the extreme Antarctic climate. However, the use of urea-based fertilisers, whilst providing a nitrogen source for bioremediation, has also altered the natural soil chemistry leading to increases in pH, ammonium and nitrite. Monitoring of the urea amended biopiles identified rising levels of nitrite to be of particular interest, which misaligns with the long term goal of reducing contaminant levels and returning soil communities to a 'healthy' state. Here, we combine amplicon sequencing, microfluidic qPCR on field samples and laboratory soil microcosms to assess the impact of persistent nitrite accumulation (up to 60 months) on nitrifier abundances observed within the Antarctic biopiles. Differential inhibition of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) Nitrobacter and Nitrospira in the cold, urea treated, alkaline soils (pH 8.1) was associated with extensive nitrite accumulation (76 ± 57 mg N/kg at 60 months). When the ratio of Nitrospira:AOB dropped below ∼1:1, Nitrobacter was completely inhibited or absent from the biopiles, and nitrite accumulated. Laboratory soil microcosms (incubated at 7 °C and 15 °C for 9 weeks) reproduced the pattern of nitrite accumulation in urea fertilized soil at the lower temperature, consistent with our longer-term observations from the Antarctic biopiles, and with other temperature-controlled microcosm studies. Diammonium phosphate amended soil did not exhibit nitrite accumulation, and could be a suitable alternative biostimulant to avoid excessive nitrite build-up.

Comparative genomic analysis of symbiotic and free-living Fluviibacter phosphoraccumulans strains provides insights into the evolutionary origins of obligate Euplotes-bacterial endosymbioses.

Endosymbiosis is a widespread and important phenomenon requiring diverse model systems. Ciliates are a widespread group of protists that often form symbioses with diverse microorganisms. Endosymbioses between the ciliate Euplotes and heritable bacterial symbionts are common in nature, and four essential symbionts were described: Polynucleobacter necessarius, "Candidatus Protistobacter heckmanni," "Ca. Devosia symbiotica," and "Ca. Devosia euplotis." Among them, only the genus Polynucleobacter comprises very close free-living and symbiotic representatives, which makes it an excellent model for investigating symbiont replacements and recent symbioses. In this article, we characterized a novel endosymbiont inhabiting the cytoplasm of Euplotes octocarinatus and found that it is a close relative of the free-living bacterium Fluviibacter phosphoraccumulans (Betaproteobacteria and Rhodocyclales). We present the complete genome sequence and annotation of the symbiotic Fluviibacter. Comparative analyses indicate that the genome of symbiotic Fluviibacter is small in size and rich in pseudogenes when compared with free-living strains, which seems to fit the prediction for recently established endosymbionts undergoing genome erosion. Further comparative analysis revealed reduced metabolic capacities in symbiotic Fluviibacter, which implies that the symbiont relies on the host Euplotes for carbon sources, organic nitrogen and sulfur, and some cofactors. We also estimated substitution rates between symbiotic and free-living Fluviibacter pairs for 233 genes; the results showed that symbiotic Fluviibacter displays higher dN/dS mean value than free-living relatives, which suggested that genetic drift is the main driving force behind molecular evolution in endosymbionts. IMPORTANCE: In the long history of symbiosis research, most studies focused mainly on organelles or bacteria within multicellular hosts. The single-celled protists receive little attention despite harboring an immense diversity of symbiotic associations with bacteria and archaea. One subgroup of the ciliate Euplotes species is strictly dependent on essential symbionts for survival and has emerged as a valuable model for understanding symbiont replacements and recent symbioses. However, almost all of our knowledge about the evolution and functions of Euplotes symbioses comes from the Euplotes-Polynucleobacter system. In this article, we report a novel essential symbiont, which also has very close free-living relatives. Genome analysis indicated that it is a recently established endosymbiont undergoing genome erosion and relies on the Euplotes host for many essential molecules. Our results provide support for the notion that essential symbionts of the ciliate Euplotes evolve from free-living progenitors in the natural water environment.

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

Chitiniphilus purpureus sp. nov., a novel chitin-degrading bacterium isolated from crawfish pond sediment.

A novel Gram-stain-negative, curved rod-shaped, motile and chitin-degrading strain, designated CD1T, was isolated from crawfish pond sediment in Caidian District (30° 58' N 114° 03' E), Wuhan City, Hubei Province, PR China. Growth of this strain was observed at 15-40°C (optimum between 28 and 30 °C), at pH 7.0-9.0 (optimum between pH 7.0 and 8.0) and with 0-1 % (w/v) NaCl (optimum at 0 %). With respect to the 16S rRNA gene sequences, strain CD1T had the highest similarity (96.91-97.25 %) to four type strains of the genera 'Chitinolyticbacter' and Chitiniphilus within the family Chitinibacteraceae. The phylogenetic trees based on genome sequences and 16S rRNA gene sequences indicated that strain CD1T was close to members of these two genera, in particular to the genus Chitiniphilus. The genomic DNA G+C content of strain CD1T was 64.8 mol%. The average nucleotide identity and the Genome-to-Genome Distance Calculator results showed low relatedness (below 95 and 70 %, respectively) between strain CD1T and the closely related type strains. Ubiquinone-8 was the predominant quinone. The major cellular fatty acids were C10 : 0, C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The polar lipid profile was composed of a mixture of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified lipids, two unidentified phospholipids, two unidentified aminolipids and an unidentified aminoglycolipid. On the basis of the evidences presented in this study, strain CD1T represents a novel species of the genus Chitiniphilus, for which the name Chitiniphilus purpureus sp. nov. is proposed, with strain CD1T (=CCTCC AB 2022395T=KCTC 92850T) as the type strain.

Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

The regulation effect of preventing soil nitrogen loss using microbial quorum sensing inhibitors.

Preventing soil nitrogen (N) losses driven by microbial nitrification and denitrification contributes to improving global environmental concerns caused by NO3--N leaching and N2O emission. Quorum sensing (QS) signals regulate nitrification and denitrification of N-cycling bacteria in pure culture and water treatment systems, and mediate the composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in activated sludge. However, whether disrupting QS could prevent soil N losses remains unclear. This study explored the feasibility of applying quorum sensing inhibitors (QSIs) as an innovative strategy to reduce N losses from agricultural soils. The two QSIs, penicillic acid and 4-iodo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-benzeneacetamide (4-iodo PHL), were more effective in reducing N losses than traditional inhibitors, including N-(n-butyl) thiophosphoric triamide and 3,4-dimethylpyrazole phosphate. After 36 days of aerobic incubation, penicillic acid and 4-iodo PHL inhibited nitrification by 39% and 68%, respectively. The inhibitory effects are attributed to the fact that 4-iodo PHL decreased the abundance of archaeal and bacterial amoA genes, as well as the relative abundance of Candidatus Nitrocosmicus (AOA), Candidatus Nitrososphaera (AOA), and Nitrospira (nitrite-oxidizing bacteria/comammox), while penicillic acid reduced archaeal amoA abundance and the relative abundance of Nitrosospira (AOB) and the microbes listed above. Penicillic acid also strongly inhibited denitrification (33%) and N2O emissions (61%) at the peak of N2O production (day 4 of anaerobic incubation) via decreasing nitrate reductase gene (narG) abundance and increasing N2O reductase gene (nosZ) abundance, respectively. Furthermore, the environmental risks of QSIs to microbial community structure and network stability, CO2 emissions, and soil animals were acceptable. Overall, QSIs have application potential in agriculture to reduce soil N losses and the associated effect on climate change. This study established a new method to mitigate N losses from the perspective of QS, and can serve as important basis of decreasing the environmental risks of agricultural non-point source pollution.

Efficient nitrogen removal from real municipal wastewater and mature landfill leachate using partial nitrification-simultaneous anammox and partial denitrification process.

Anaerobic ammonia oxidation (anammox) of municipal wastewater is a research focus, especially the combined treatment with mature landfill leachate is a current research hotspot. In this study, municipal wastewater was treated by partial nitrification via sequencing batch reactor (SBR), and its effluent and mature landfill leachate were then mixed into an up-flow anaerobic sludge blanket (UASB) for simultaneous anammox and partial denitrification reaction. Through partial nitrification, a high nitrite accumulation rate (93.0 ± 3.8 %) was achieved by low dissolved oxygen (0.5-1.6 mg/L) and controlled aerobic time (3.5 h) in SBR. The UASB system was responsible for 78.8 ± 2.1 % nitrogen removal of the entire system with a hydraulic reaction time (HRT) of 3.8 h, accompanied by the anammox contribution up to 89.4 ± 6.0 %. The overall partial nitrification-simultaneous anammox and partial denitrification (PN-SAPD) system was controlled at a total COD/TIN of 2.8 ± 0.3 and a total HRT of only 10.2 h, achieving the nitrogen removal efficiency and effluent TIN were 95.2 ± 2.2 % and 3.4 ± 1.5 mg/L, respectively. The qPCR results showed functional genes (hzsA(B), hdh) associated with anaerobic ammonia-oxidizing bacteria (AnAOB), whose high gene copy abundance and transcription expression ensured the removal of major nitrogen from municipal wastewater and mature landfill leachate. 16S amplicon sequencing showed that the Ca. Brocadia (9.72-12.6 %) was further enrichment after sodium acetate was added, and the transcription expression of Thauera (0.5-7.0 %) caused nitrate to nitrite. The high abundance of related enzymes (hao, hzs, hdh, narGHI) involved in anammox and partial denitrification processes were found in the macrogenomic sequencing, and only Ca. Brocadia was involved in multi-pathway nitrogen metabolism in AnAOB. Based on the efficient nitrogen removal by AnAOB and denitrifying bacteria, this modified PN-SAPD process provides a new option for the co-treatment of mature landfill leachate in municipal wastewater treatment plants.

Multidrug-resistant plasmid RP4 inhibits the nitrogen removal capacity of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and comammox in activated sludge.

In wastewater treatment plants (WWTPs), ammonia oxidation is primarily carried out by three types of ammonia oxidation microorganisms (AOMs): ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox (CMX). Antibiotic resistance genes (ARGs), which pose an important public health concern, have been identified at every stage of wastewater treatment. However, few studies have focused on the impact of ARGs on ammonia removal performance. Therefore, our study sought to investigate the effect of the representative multidrug-resistant plasmid RP4 on the functional microorganisms involved in ammonia oxidation. Using an inhibitor-based method, we first evaluated the contributions of AOA, AOB, and CMX to ammonia oxidation in activated sludge, which were determined to be 13.7%, 41.1%, and 39.1%, respectively. The inhibitory effects of C2H2, C8H14, and 3,4-dimethylpyrazole phosphate (DMPP) were then validated by qPCR. After adding donor strains to the sludge, fluorescence in situ hybridization (FISH) imaging analysis demonstrated the co-localization of RP4 plasmids and all three AOMs, thus confirming the horizontal gene transfer (HGT) of the RP4 plasmid among these microorganisms. Significant inhibitory effects of the RP4 plasmid on the ammonia nitrogen consumption of AOA, AOB, and CMX were also observed, with inhibition rates of 39.7%, 36.2%, and 49.7%, respectively. Moreover, amoA expression in AOB and CMX was variably inhibited by the RP4 plasmid, whereas AOA amoA expression was not inhibited. These results demonstrate the adverse environmental effects of the RP4 plasmid and provide indirect evidence supporting plasmid-mediated conjugation transfer from bacteria to archaea.

Feast/famine ratio regulates the succession of heterotrophic nitrification-aerobic denitrification and autotrophic ammonia oxidizing bacteria in halophilic aerobic granular sludge treating saline wastewater.

Heterotrophic nitrification-aerobic denitrification (HN-AD) shows innovation potential of wastewater treatment process in a single tank. However, how to enrich HN-AD bacteria in activated sludge to enhance their contribution remained unknown. This study explored the impact of the feast/famine (F/F) ratio on the succession of autotrophic ammonia oxidizing bacteria (AOB) and HN-AD bacteria in a halophilic aerobic granular sludge (HAGS) system. As the F/F ratio decreased from 1/9 to 1/15, the total inorganic nitrogen (TIN) removal performance significantly decreased. The proportion of heterotrophic bacteria was dropped from 79.0 % to 33 %. Accordingly, the relative abundance of Paracoccus decreased from 70.8 % to 25.4 %, and the copy number of the napA gene was reduced from 2.2 × 1010 copies/g HAGS to 8.1 × 109 copies/g HAGS. It found the F/F ratio regulated the population succession of autotrophic AOB and HN-AD bacteria, thereby providing a solution to achieve the enrichment of HN-AD bacteria in HAGS.

Addition of cellulose and hemicellulose degrading microorganisms intensified nitrous oxide emission during composting.

This study aims to clarify the mechanisms underlying effects of inoculating cellulose and hemicellulose-degrading microorganisms on nitrous oxide (N2O) emissions during composting with silkworm excrement and mulberry branches. Inoculation with cellulose and hemicellulose-degrading microorganisms resulted in significant increases of total N2O emission by 10.4 ± 2.0 % (349.1 ± 6.2 mg N kg-1 dw) and 26.7 ± 2.1 % (400.6 ± 6.8 mg N kg-1 dw), respectively, compared to the control (316.3 ± 3.6 mg N kg-1 dw). The stimulation of N2O emission was attributed to the enhanced contribution of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria to N2O production, as evidenced by the increased AOB amoA and denitrifying nirK gene abundances. Moreover, microbial inoculation stimulated N2O reduction to N2 owing to increased abundances of nosZⅠ and nosZⅠⅠ genes. These findings highlight the necessity to develop cost-effective and environmentally friendly strategies to reduce N2O emissions when cellulose and hemicellulose-degrading microorganisms are inoculated during composting.

Shedding light on the electron transfer chain of a moderately acidophilic iron oxidizer: characterization of recombinant HiPIP-41, CytC-18 and CytC-78 derived from Ferrovum sp. PN-J47-F6.

Efficient electron transfer from the donor to the acceptor couple presents a necessary requirement for acidophilic and neutrophilic iron oxidizers due to the low energy yield of aerobic ferrous iron oxidation. Involved periplasmic electron carriers are very diverse in these bacteria and show adaptations to the respective thermodynamic constraints such as a more positive redox potential reported for extreme acidophilic Acidithiobacillus spp. Respiratory chain candidates of moderately acidophilic members of the genus Ferrovum share similarities with both their neutrophilic iron oxidizing relatives and the more distantly related Acidithiobacillus spp. We examined our previous omics-based conclusions on the potential electron transfer chain in Ferrovum spp. by characterizing the three redox protein candidates CytC-18, CytC-78 and HiPIP-41 of strain PN-J47-F6 which were produced as recombinant proteins in Eschericha coli. UV/Vis-based redox assays suggested that HiPIP-41 has a very positive redox potential while redox potentials of CytC-18 and CytC-78 are more negative than their counterparts in Acidithiobacillus spp. Far Western dot blotting demonstrated interactions between all three recombinant redox proteins while redox assays showed the electron transfer from HiPIP-41 to either of the cytochromes. Altogether, CytC-18, CytC-78 and HiPIP-41 indeed represent very likely candidates of the electron transfer in Ferrovum sp. PN-J4-F6.

From metagenomes to metabolism: Systematically assessing the metabolic flux feasibilities for "Candidatus Accumulibacter" species during anaerobic substrate uptake.

With the rapid growing availability of metagenome assembled genomes (MAGs) and associated metabolic models, the identification of metabolic potential in individual community members has become possible. However, the field still lacks an unbiassed systematic evaluation of the generated metagenomic information to uncover not only metabolic potential, but also feasibilities of these models under specific environmental conditions. In this study, we present a systematic analysis of the metabolic potential in species of "Candidatus Accumulibacter", a group of polyphosphate-accumulating organisms (PAOs). We constructed a metabolic model of the central carbon metabolism and compared the metabolic potential among available MAGs for "Ca. Accumulibacter" species. By combining Elementary Flux Modes Analysis (EFMA) with max-min driving force (MDF) optimization, we obtained all possible flux distributions of the metabolic network and calculated their individual thermodynamic feasibility. Our findings reveal significant variations in the metabolic potential among "Ca. Accumulibacter" MAGs, particularly in the presence of anaplerotic reactions. EFMA revealed 700 unique flux distributions in the complete metabolic model that enable the anaerobic uptake of acetate and its conversion into polyhydroxyalkanoates (PHAs), a well-known phenotype of "Ca. Accumulibacter". However, thermodynamic constraints narrowed down this solution space to 146 models that were stoichiometrically and thermodynamically feasible (MDF > 0 kJ/mol), of which only 8 were strongly feasible (MDF > 7 kJ/mol). Notably, several novel flux distributions for the metabolic model were identified, suggesting putative, yet unreported, functions within the PAO communities. Overall, this work provides valuable insights into the metabolic variability among "Ca. Accumulibacter" species and redefines the anaerobic metabolic potential in the context of phosphate removal. More generally, the integrated workflow presented in this paper can be applied to any metabolic model obtained from a MAG generated from microbial communities to objectively narrow the expected phenotypes from community members.