Disentangling microbial coupled fillers mechanisms for the permeable layer optimization process in multi-soil-layering systems.

The multi-soil-layering (MSL) systems is an emerging solution for environmentally-friendly and cost-effective treatment of decentralized rural domestic wastewater. However, the role of the seemingly simple permeable layer has been overlooked, potentially holding the breakthroughs or directions to addressing suboptimal nitrogen removal performance in MSL systems. In this paper, the mechanism among diverse substrates (zeolite, green zeolite and biological ceramsite) coupled microorganisms in different systems (activated bacterial powder and activated sludge) for rural domestic wastewater purification was investigated. The removal efficiencies performed by zeolite coupled with microorganisms within 3 days were 93.8% for COD, 97.1% for TP, and 98.8% for NH4+-N. Notably, activated sludge showed better nitrification and comprehensive performance than specialized nitrifying bacteria powder. Zeolite attained an impressive 89.4% NH4+-N desorption efficiency, with a substantive fraction of NH4+-N manifesting as exchanged ammonium. High-throughput 16S rRNA gene sequencing revealed that aerobic and parthenogenetic anaerobic bacteria dominated the reactor, with anaerobic bacteria conspicuously absent. And the heterotrophic nitrification-aerobic denitrification (HN-AD) process was significant, with the presence of denitrifying phosphorus-accumulating organisms (DPAOs) for simultaneous nitrogen and phosphorus removal. This study not only raises awareness about the importance of the permeable layer and enhances comprehension of the HN-AD mechanism in MSL systems, but also provides valuable insights for optimizing MSL system construction, operation, and rural domestic wastewater treatment.

Isolation of Aerobic Heterotrophic Bacteria from a Microbial Mat with the Ability to Grow on and Remove Hexavalent Chromium through Biosorption and Bioreduction.

Water pollution with toxic hexavalent chromium, Cr(VI), is an environmental threat that has a direct impact on living organisms. The use of microorganisms from microbial mats to remove Cr(VI) has scarcely been investigated. Here, we isolated aerobic heterotrophic bacteria from a Cr-polluted microbial mat found in a mining site in Oman, and investigated their ability to remove Cr(VI), and the underlying mechanism(s) of removal. All isolates fell phylogenetically into the genera Enterobacter, Bacillus, and Cupriavidus, and could completely remove 1 mg L-1 Cr(VI) in 6 days. The strains could tolerate up to 2000 mg L-1 Cr(VI), and exhibited the highest Cr(VI) removal rate at 100 ± 9 mg L-1 d-1. Using scanning electron microscopy (SEM) coupled with elemental analysis, the strains were shown to adsorb Cr(VI) at their cell surfaces. The functional groups OH, NH2, Alkyl, Metal-O, and Cr(VI)-O were involved in the biosorption process. In addition, the strains were shown to reduce Cr(VI) to Cr(III) with the involvement of chromate reductase enzyme. We conclude that the aerobic heterotrophic bacteria isolated from Cr-polluted microbial mats use biosorption and bioreduction processes to remove Cr(VI) from wastewater.

New insights into N2O emission and electron competition under different chemical oxygen demand to nitrogen ratios in a biofilm system.

Nitrous oxide (N2O) is a greenhouse gas that could accumulate during the heterotrophic denitrification process. In this study, the effects of different chemical oxygen demand to nitrogen ratio (COD/N) on N2O production and electron competition was investigated. The electron competition was intensified with the decrease of electron supply, and Nos had the best electron competition ability. The model simulation results indicated that the degradation of NOxNs was a combination of diffusion and biological degradation. As reaction proceeding, N2O could accumulate inside biofilm. A thinner biofilm and a longer hydraulic retention time (HRT) might be an effective way to control N2O emission. The application of mathematical model is an opportunity to gain deep understanding of substrate degradation and electron competition inside biofilm.

The regulation of the thermal stability and affinity of the HSPA5 (Grp78/BiP) by clients and nucleotides is modulated by domains coupling.

The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.

Transcriptome analysis of the degradation process of organic nitrogen by two heterotrophic nitrifying and aerobic denitrifying bacteria.

The heterotrophic nitrification aerobic denitrification bacteria (HNDS) can perform nitrification and denitrification at the same time. Two HNDS strains, Achromobacter sp. HNDS-1 and Enterobacter sp. HNDS-6 which exhibited an amazing ability to solution nitrogen (N) removal have been successfully isolated from paddy soil in our lab. When peptone or ammonium sulfate as sole N source, no significant difference in gene expression related to nitrification and denitrification of the strains was found according to the transcriptome analysis. The expression of phosphomethylpyrimidine synthase (thiC), ABC transporter substrate-binding protein, branched-chain amino acid ABC transporter substrate-binding protein, and RNA polymerase (rpoE) in HNDS-1 were significantly upregulated when used peptone as N source, while the expression of exopolysaccharide production protein (yjbE), RNA polymerase (rpoC), glutamate synthase (gltD) and ABC-type branched-chain amino acid transport systems in HNDS-6 were significantly upregulated. This indicated that these two strains are capable of using organic N and converting it into NH4+-N, then utilizing NH4+-N to synthesize amino acids and proteins for their own growth, and strain HNDS-6 can also remove NH4+-N through nitrification and denitrification.

Integrated overview of stramenopile ecology, taxonomy, and heterotrophic origin.

Stramenopiles represent a significant proportion of aquatic and terrestrial biota. Most biologists can name a few, but these are limited to the phototrophic (e.g., diatoms and kelp) or parasitic species (e.g., oomycetes, Blastocystis), with free-living heterotrophs largely overlooked. Though our attention is slowly turning towards heterotrophs, we have only a limited understanding of their biology due to a lack of cultured models. Recent metagenomic and single-cell investigations have revealed the species richness and ecological importance of stramenopiles-especially heterotrophs. However, our lack of knowledge of the cell biology and behaviour of these organisms leads to our inability to match species to their particular ecological functions. Because photosynthetic stramenopiles are studied independently of their heterotrophic relatives, they are often treated separately in the literature. Here, we present stramenopiles as a unified group with shared synapomorphies and evolutionary history. We introduce the main lineages, describe their important biological and ecological traits, and provide a concise update on the origin of the ochrophyte plastid. We highlight the crucial role of heterotrophs and mixotrophs in our understanding of stramenopiles with the goal of inspiring future investigations in taxonomy and life history. To understand each of the many diversifications within stramenopiles-towards autotrophy, osmotrophy, or parasitism-we must understand the ancestral heterotrophic flagellate from which they each evolved. We hope the following will serve as a primer for new stramenopile researchers or as an integrative refresher to those already in the field.

Marine bacteria Alteromonas spp. require UDP-glucose-4-epimerase for aggregation and production of sticky exopolymer.

The physiology and ecology of particle-associated marine bacteria are of growing interest, but our knowledge of their aggregation behavior and mechanisms controlling their association with particles remains limited. We have found that a particle-associated isolate, Alteromonas sp. ALT199 strain 4B03, and the related type-strain A. macleodii 27126 both form large (>500 µm) aggregates while growing in rich medium. A non-clumping variant (NCV) of 4B03 spontaneously arose in the lab, and whole-genome sequencing revealed a partial deletion in the gene encoding UDP-glucose-4-epimerase (galEΔ308-324). In 27126, a knock-out of galE (ΔgalE::kmr) resulted in a loss of aggregation, mimicking the NCV. Microscopic analysis shows that both 4B03 and 27126 rapidly form large aggregates, whereas their respective galE mutants remain primarily as single planktonic cells or clusters of a few cells. Strains 4B03 and 27126 also form aggregates with chitin particles, but their galE mutants do not. Alcian Blue staining shows that 4B03 and 27126 produce large transparent exopolymer particles (TEP), but their galE mutants are deficient in this regard. This study demonstrates the capabilities of cell-cell aggregation, aggregation of chitin particles, and production of TEP in strains of Alteromonas, a widespread particle-associated genus of heterotrophic marine bacteria. A genetic requirement for galE is evident for each of the above capabilities, expanding the known breadth of requirement for this gene in biofilm-related processes. IMPORTANCE: Heterotrophic marine bacteria have a central role in the global carbon cycle. Well-known for releasing CO2 by decomposition and respiration, they may also contribute to particulate organic matter (POM) aggregation, which can promote CO2 sequestration via the formation of marine snow. We find that two members of the prevalent particle-associated genus Alteromonas can form aggregates comprising cells alone or cells and chitin particles, indicating their ability to drive POM aggregation. In line with their multivalent aggregation capability, both strains produce TEP, an excreted polysaccharide central to POM aggregation in the ocean. We demonstrate a genetic requirement for galE in aggregation and large TEP formation, building our mechanistic understanding of these aggregative capabilities. These findings point toward a role for heterotrophic bacteria in POM aggregation in the ocean and support broader efforts to understand bacterial controls on the global carbon cycle based on microbial activities, community structure, and meta-omic profiling.

Feasibility study on heterotrophic utilization of galactose by Chlorella sorokiniana and promotion of galactose utilization through mixed carbon sources culture.

BACKGROUND: The development of alternative carbon sources is important for reducing the cost of heterotrophic microalgae cultivation. Among cheap feedstocks, galactose is one of the most abundant sugars and can be easily obtained from many natural biomasses. However, it is generally difficult to be utilized by microalgae. In addition, the mechanism of its low utilization efficiency in heterotrophic cultivation is still unknown. RESULTS: Among seven tested carbon sources, only glucose and acetate could be efficiently utilized by C. sorokiniana in heterotrophic cultivation while there were no apparent signs of utilization of other carbohydrates, including galactose, in regular heterotrophic cultivation. However, galactose could be utilized in cultures with high inoculation sizes. This confirmed that C. sorokiniana has a complete pathway for transporting and assimilating galactose under dark conditions, but the rate of galactose utilization is quite low. In addition, the galactose utilization was greatly enhanced in mixotrophic cultures, which indicated that galactose utilization could be enhanced by additional pathways that can enhance cell growth. Based on above results, a mixed carbon source culture strategy was proposed to improve the utilization rate of galactose, and a significant synergistic effect on cell growth was achieved in cultures using a mixture of galactose and acetate. CONCLUSIONS: This study indicated that the galactose metabolism pathway may not be inherently deficient in Chlorophyta. However, its utilization rate was too low to be detected in regular heterotrophic cultivation. Mixed carbon source culture strategy was confirmed effective to improve the utilization rate of galactose. This study contributes to a deeper understanding of the utilization ability of difficultly utilized substrates in the heterotrophic cultivation of microalgae, which is of great significance for reducing the cost of heterotrophic cultivation of microalgae.

Hormesis and synergistic effects of disinfectants chloroxylenol and benzethonium chloride on highly efficient heterotrophic nitrification-aerobic denitrification functional strain: From performance to mechanism.

The heterotrophic nitrification-aerobic denitrification (HNAD) strain Exiguobacterium H1 (H1) was isolated in this study. The changes in nitrogen metabolism functions of H1 strain were discussed in presence of disinfectants chloroxylenol (PCMX) and benzethonium chloride (BEC) alone and combined pollution (PCMX+BEC). The H1 strain could use NH4+-N, NO2--N and NO3--N as nitrogen sources and had good nitrogen removal performance under conditions of C/N ratio 25, pH 5-8, 25-35 oC and sodium acetate as carbon. PCMX and BEC alone exhibited hormesis effects on H1 strain which promoted the growth of H1 strain at low concentrations but inhibited it at high concentrations, and combined pollution showed synergistic inhibitory on H1 strain. H1 strain owned a full nitrogen metabolic pathway according to functional genes quantification. PCMX encouraged nitrification process of H1, while BEC and combined pollution mostly blocked nitrogen removal. PCMX, but not BEC, mainly led to the enrichment of resistance genes. These findings will aid in systematic assessment of contaminant tolerance characteristics of HNAD strain and its application prospects.

Outdoor tubular photobioreactor microalgae-microorganisms biofilm treatment of municipal wastewater: Enhanced heterotrophic assimilation and synergistic aerobic denitrogenation.

This research evaluated a microalgae consortium (MC) in a pilot-scale tubular photobioreactor for municipal wastewater (MWW) treatment, compared with an aeration column photobioreactor. Transitioning from suspended MC to a microalgae-microbial biofilm (MMBF) maintained treatment performance despite increasing influent from 50 L to 150 L in a 260 L system. Carbon and nitrogen removal were effective, but phosphorus removal varied due to biofilm shading and the absence of phosphorus-accumulating organisms. High influent flow caused MMBF detachment due to shear stress. Stabilizing and re-establishing the MMBF showed that a stable phycosphere influenced microbial diversity and interactions, potentially destabilizing the MMBF. Heterotrophic nitrification-aerobic denitrification bacteria were crucial for MC equilibrium. Elevated gene expression related to nitrogen fixation, organic nitrogen metabolism, and nitrate reduction confirmed strong microalgal symbiosis, highlighting MMBF's treatment potential. This study supports the practical application of microalgae in wastewater treatment.

Sponge iron enriches autotrophic/aerobic denitrifying bacteria to enhance denitrification in sequencing batch reactor.

Sponge iron (SFe) coupled with a sludge system has great potential for improving biological denitrification; however, the underlying mechanism is not yet fully understood. In this study, the denitrification performance and microbial characteristics of ordinary sludge and SFe-sludge systems were investigated. Overall, the SFe-sludge reactor had faster ammonium degradation rate (94.0 %) and less nitrate accumulation (1.5-53.3 times lower) than ordinary reactor during the complete operation cycle of sequencing batch reactors. The addition of SFe increased the activities of nitrate and nitrite reductases. The total relative abundance of autotrophic denitrifying bacteria (Acidovorax, Arenimonas, etc.) in the SFe-sludge system after 38 days of operation was found to be 10.6 % higher than that in the ordinary sludge reactor. The aerobic denitrifying bacteria (Dokdonella, Phaeodactylibacter, etc.) was 5.3 % higher than ordinary sludge. The SFe-sludge system improved denitrification by enriching autotrophic/aerobic denitrifying bacteria in low carbon-to-nitrogen ratio wastewater treatment.

From kitchen to crop: The efficacy and safety of the microbial consortium treated dishwasher wastewater for the Zero Mile system.

A microbial consortium, based on the functional integration of photosynthetic and heterotrophic microorganisms, is the core of the Zero Mile System. This system is designed for reusing and upcycling household greywaters, a still untapped water resource. The previous challenges of dishwasher wastewater bioremediation demonstrated the capability of an ad hoc consortium (including a photosynthetic cyanobacterium and three heterotrophic bacterial isolates from dishwasher wastewater) to reclaim the wastewater at small/medium scale. In this study the wastewater treatment demonstrated to be effective in nutrient recycling and upcycling at a larger scale, i.e. 4 L (in three replicates to treat the total amount of wastewater discharged by the dishwasher), by removing high percentage of N and P from the wastewater (70% nitrogen, 50% phosphorous, respectively). Again, the reclaimed wastewater successfully fertilized lettuce plants both indoor (in the Zero Mile System demonstrator) and outdoor (in open field). Plants showed a significant higher biomass productivity in fresh weight compared to control plants and comparable or better values of the pigments and quality indices (e.g., soluble solids, total phenols, total flavonoids). Furthermore, the safety of the reclaimed wastewater is demonstrated by the analysis of the metabolic/ecologically relevant functions of the microbial communities in both untreated and treated wastewater. Colonizers were mainly organic matter degraders and bacteria involved in nitrogen cycling. The human related genera are quite few and no pathogens or potential microbiological contaminants of water bodies (as E. coli), were found. Hence, the utilization of treated dishwasher wastewater does not imply biological risks to agricultural products, soil, or groundwater.

Ammonium nitrogen and phosphorus removal by bacterial-algal symbiotic dynamic sponge bioremediation system in micropolluted water: Operational mechanism and transformation pathways.

Construct a bacteria-algae symbiotic dynamic sponge bioremediation system to simultaneously remove multiple pollutants under micro-pollution conditions. The average removal efficiencies of NH4+-N, PO43--P, total nitrogen (TN), and Ca2+ were 98.35, 78.74, 95.64, and 84.92 %, respectively. Comparative studies with Auxenochlorella sp. sponge and bacterial sponge bioremediation system confirmed that NH4+-N and TN were mainly removed by bacterial heterotrophic nitrification - aerobic denitrification (HN-AD). PO43--P was removed by algal assimilation and the generation of Ca3(PO4)2 and Ca5(PO4)3OH, and Ca2+ was removed by algal electron transfer formation of precipitates and microbially induced calcium precipitation (MICP) by bacteria. Algae provided an aerobic environment for the bacterial HN-AD process through photosynthesis, while respiration produced CO2 and adsorbed Ca2+ to promote the formation of calcium precipitates. Immobilization of Ca2+ with microalgae via bacterial MICP helped to lift microalgal photoinhibition. The bioremediation system provides theoretical support for research on micropolluted water treatment while increasing phosphorus recovery pathways.

Influence and mechanism of typical transition metal ions on the denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria.

To investigate the inhibitory effects of various transition metal ions on nitrogen removal and their underlying mechanisms, the single and combined effects of Cu2+ Ni2+ and Zn2+ on Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria Acinetobacter sp. TAC-1 were studied in a batch experiment system. The results revealed that increasing concentrations of Cu2+ and Ni2+ had a detrimental effect on the removal of ammonium nitrogen (NH4+-N) and total nitrogen (TN). Specifically, Cu2+ concentration of 10 mg/L, the TN degradation rate was 55.09%, compared to 77.60% in the control group. Cu2+ exhibited a pronounced inhibitory effect. In contrast, Zn2+ showed no apparent inhibitory effect on NH4+-N removal and even enhanced TN removal at lower concentrations. However, when the mixed ion concentration of Zn2++Ni2+ exceeded 5 mg/L, the removal rates of NH4+-N and TN were significantly reduced. Moreover, transition metal ions did not significantly impact the removal rates of chemical oxygen demand (COD). The inhibition model fitting results indicated that the inhibition sequence was Cu2+ > Zn2+ > Ni2+. Transcriptome analysis demonstrated that metal ions influence TAC-1 activity by modulating the expression of pivotal genes, including zinc ABC transporter substrate binding protein (znuA), ribosomal protein (rpsM), and chromosome replication initiation protein (dnaA) and DNA replication of TAC-1 under metal ion stress, leading to disruptions in transcription, translation, and cell membrane structure. Finally, a conceptual model was proposed by us to summarize the inhibition mechanism and possible response strategies of TAC-1 bacteria under metal ion stress, and to address the lack of understanding regarding the influence mechanism of TAC-1 on nitrogen removal in wastewater co-polluted by metal and ammonia nitrogen. The results provided practical guidance for the management of transition metal and ammonia nitrogen co-polluted water bodies, as well as the removal of high nitrogen.

Effects of Cd(II) on nitrogen removal by a heterotrophic nitrification aerobic denitrification bacterium Pseudomonas sp. XF-4.

Simultaneous heterotrophic nitrification and aerobic denitrification (SND) is gaining tremendous attention due to its high efficiency and low cost in water treatment. However, SND on an industrial scale is still immature since effects of coexisting pollutants, for example, heavy metals, on nitrogen removal remains largely unresolved. In this study, a HNAD bacterium (Pseudomonas sp. XF-4) was isolated. It could almost completely remove ammonium and nitrate at pH 5-9 and temperature 20 ℃-35 ℃ within 10 h, and also showed excellently simultaneous nitrification and denitrification efficiency under the coexistence of any two of inorganic nitrogen sources with no intermediate accumulation. XF-4 could rapidly grow again after ammonium vanish when nitrite or nitrate existed. There was no significant effects on nitrification and denitrification when Cd(II) was lower than 10 mg/L, and 95 % of Cd(II) was removed by XF-4. However, electron carrier and electron transport system activity was inhibited, especially at high concentration of Cd(II). Overall, this study reported a novel strain capable of simultaneous nitrification and denitrification coupled with Cd(II) removal efficiently. The results provided new insights into treatment of groundwater or wastewater contaminated by heavy metals and nitrogen.

Pooled CRISPR Interference Screening Identifies Crucial Transcription Factors in Gas-Fermenting Clostridium ljungdahlii.

Gas-fermenting Clostridium species hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) in Clostridium ljungdahlii, a representative species of gas-fermenting Clostridium, with a special focus on TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth of C. ljungdahlii, respectively. We focused our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleoside triphosphates (dNTPs) supply, carbon fixation, and product synthesis in C. ljungdahlii, thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression of deoR in C. ljungdahlii by adjusting its promoter strength, leading to an improved growth rate and ethanol synthesis of C. ljungdahlii when utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermenting Clostridium species, expanding the horizons for functional genomic research in these industrially important bacteria.

Effect of carbon source on carbon and nitrogen metabolism of common heterotrophic nitrification-aerobic denitrification pathway.

Pseudomonas sp. ZHL02, removing nitrogen via ammonia nitrogen (NH4+) → hydroxylamine (HN2OH) → nitrite (NO2-) → nitrate (NO3-) → NO2- → nitric oxide (NO) → nitrous oxide (N2O) pathway was employed for getting in-depth information on the heterotrophic nitrification-aerobic denitrification (HNAD) pathway from carbon oxidation, nitrogen conversion, electron transport process, enzyme activity, as well as gene expression while sodium succinate, sodium citrate, and sodium acetate were utilized as the carbon sources. The nitrogen balance analysis results demonstrated that ZHL02 mainly removed NH4+-N through assimilation. The carbon source metabolism resulted in the discrepancies in electron transport chain and nitrogen removal between different HNAD bacteria. Moreover, the prokaryotic strand-specific transcriptome method showed that, amo and hao were absent in ZHL02, and unknown genes may be involved in ZHL02 during the HNAD process. As a fascinating process for removing nitrogen, the HNAD process is still puzzling, and the relationship between carbon metabolism and nitrogen metabolism among different HNAD pathways should be studied further.

Machine Learning-Assisted Optimization of Mixed Carbon Source Compositions for High-Performance Denitrification.

Appropriate mixed carbon sources have great potential to enhance denitrification efficiency and reduce operational costs in municipal wastewater treatment plants (WWTPs). However, traditional methods struggle to efficiently select the optimal mixture due to the variety of compositions. Herein, we developed a machine learning-assisted high-throughput method enabling WWTPs to rapidly identify and optimize mixed carbon sources. Taking a local WWTP as an example, a mixed carbon source denitrification data set was established via a high-throughput method and employed to train a machine learning model. The composition of carbon sources and the types of inoculated sludge served as input variables. The XGBoost algorithm was employed to predict the total nitrogen removal rate and microbial growth, thereby aiding in the assessment of the denitrification potential. The predicted carbon sources exhibited an enhanced denitrification potential over single carbon sources in both kinetic experiments and long-term reactor operations. Model feature analysis shows that the cumulative effect and interaction among individual carbon sources in a mixture significantly enhance the overall denitrification potential. Metagenomic analysis reveals that the mixed carbon sources increased the diversity and complexity of denitrifying bacterial ecological networks in WWTPs. This work offers an efficient method for WWTPs to optimize mixed carbon source compositions and provides new insights into the mechanism behind enhanced denitrification under a supply of multiple carbon sources.

Patterns of prokaryotic activity along the marine planktonic matter continuum.

Prokaryotic abundance and activity are commonly assessed by dividing them into two size-fractions: free-living and attached to particles. Nevertheless, organic matter, essential for the growth of heterotrophic prokaryotes, is present in the environment in a continuum of sizes, from purely dissolved to large particles. Therefore, defining the activity of the prokaryotic community would be more accurate by considering all the distinct size fractions. To achieve this, we measured prokaryotic abundance (PA), heterotrophic prokaryotic activity (as leucine incorporation) and extracellular enzyme activities at a coastal site in the NW Mediterranean Sea. We conducted measurements in both bulk seawater and size fractionated samples sequentially passing through 5 different filter types: 0.2-0.8-3-5-10 µm pore size. Our results indicate that the fraction <0.8 µm contained the highest percentage of cells (91.6 ± 1.1 %) and leucine incorporation rates (72.2 ± 3.5 %). Most of the extracellular enzyme activity appeared in the dissolved fraction (<0.2 µm; 19.8-79.4 %), yet the specific activity of the enzymes (per cell activity) was 100-1000 times higher in the particulate (>0.8 µm) than in the free-living (0.2-0.8 µm) fraction. The size fraction with highest specific activities for leucine incorporation and most of the enzyme activities (ß-glucosidase, esterase, Leu-aminopeptidase and alkaline phosphatase) was the 5-10 µm fraction. In contrast, the higher specific chitobiase activity in the >10 µm fraction, suggests that the prokaryotic community colonizing large particles might be more specialized in the hydrolysis of organic matter of zooplanktonic origin than the community colonizing smaller particles.

Estuarine mangrove niches select cultivable heterotrophic diazotrophs with diverse metabolic potentials-a prospective cross-dialog for functional diazotrophy.

Introduction: Biological nitrogen fixation (BNF), an unparalleled metabolic novelty among living microorganisms on earth, globally contributes ~88-101 Tg N year-1 to natural ecosystems, ~56% sourced from symbiotic BNF while ~22-45% derived from free-living nitrogen fixers (FLNF). The success of symbiotic BNF is largely dependent on its interaction with host-plant, however ubiquitous environmental heterotrophic FLNFs face many limitations in their immediate ecological niches to sustain unhindered BNF. The autotrophic FLNFs like cyanobacteria and oceanic heterotrophic diazotrophs have been well studied about their contrivances acclimated/adapted by these organisms to outwit the environmental constraints for functional diazotrophy. However, FLNF heterotrophs face more adversity in executing BNF under stressful estuarine/marine/aquatic habitats. Methods: In this study a large-scale cultivation-dependent investigation was accomplished with 190 NCBI accessioned and 45 non-accessioned heterotrophic FLNF cultivable bacterial isolates (total 235) from halophilic estuarine intertidal mangrove niches of Indian Sundarbans, a Ramsar site and UNESCO proclaimed World Heritage Site. Assuming ~1% culturability of the microbial community, the respective niches were also studied for representing actual bacterial diversity via cultivation-independent next-generation sequencing of V3-V4 rRNA regions. Results: Both the studies revealed a higher abundance of culturable Gammaproteobacteria followed by Firmicutes, the majority of 235 FLNFs studied belonging to these two classes. The FLNFs displayed comparable selection potential in media for free nitrogen fixers and iron-oxidizing bacteria, linking diazotrophy with iron oxidation, siderophore production, phosphorus solubilization, phosphorus uptake and accumulation as well as denitrification. Discussion: This observation validated the hypothesis that under extreme estuarine mangrove niches, diazotrophs are naturally selected as a specialized multidimensional entity, to expedite BNF and survive. Earlier metagenome data from mangrove niches demonstrated a microbial metabolic coupling among C, N, P, S, and Fe cycling in mangrove sediments, as an adaptive trait, evident with the co-abundant respective functional genes, which corroborates our findings in cultivation mode for multiple interrelated metabolic potential facilitating BNF in a challenging intertidal mangrove environment.