Ammonium Assimilation Research Articles

Biological nitrogen removal characteristics and mechanisms of a novel salt-tolerant strain Vibrio sp. LV-Q1 and its application capacity for high-salinity nitrogen-containing wastewater treatment

High concentration of salts in wastewater can adversely affects the metabolic activities and growth of microorganisms, resulting in reduced efficiency of biological nitrogen removal. Screening high-efficiency heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria with salt tolerance is important for the wastewater treatment. In this study, a novel salt-tolerant strain LV-Q1 capable of HN-AD was isolated and identified as Vibrio sp. This strain demonstrates a salinity tolerance range of 0–8 %. Notably, strain LV-Q1 exhibited exceptional nitrogen removal efficiencies (at initial concentrations of 100 mg/L), achieving 97.97 % for NH4+-N, 100 % for NO3--N, and approximately 100 % for mixed nitrogen (NH4+-N and NO3--N) at a salinity of 5 %. Optimal ammonium removal parameters for LV-Q1 were established. The nitrogen removal pathway of LV-Q1 primarily involves direct assimilation of ammonium and its conversion into N2 through HN-AD. Analysis of the salt tolerance mechanism revealed LV-Q1's ability to equilibrate osmotic pressure through the accumulation of amino acids and betaine, production of antioxidative enzymes or extracellular protein, and utilization of its quorum sensing (QS) properties to withstand high-salinity environments. The strain LV-Q1 exhibited strong resistance to salinity shock in bioaugmentation within a moving bed biofilm reactor (MBBR), markedly enhancing nitrogen and carbon removal performance with an improved removal efficiency of 26.74 % and 10.15 % averagely at the end of the reaction at a salinity of 5 %. Moreover, Vibrio sp. LV-Q1 successfully proliferated and dominated, and its relative abundance gradually increased to 59.81 %. This study contributes new insights and resources for the treatment of nitrogen in saline effluents.

Transcriptomic analysis of nitrogen metabolism pathways in Klebsiella aerogenes under nitrogen-rich conditions.

The acceleration of the nitrogen cycle and the nitrogen excess observed in some coastal waters has increased interest into understanding the biochemical and molecular basis of nitrogen metabolism in various microorganisms. To investigate nitrogen metabolism of a novel heterotrophic nitrification and aerobic denitrification bacterium Klebsiella aerogenes strain (B23) under nitrogen-rich conditions, we conducted physiological and transcriptomic high-throughput sequencing analyses on strain B23 cultured on potassium nitrate-free or potassium nitrate-rich media. Overall, K. aerogenes B23 assimilated 82.47% of the nitrate present into cellular nitrogen. Further, 1,195 differentially expressed genes were observed between K. aerogenes B23 cultured on potassium nitrate-free media and those cultured on potassium nitrate-rich media. Gene annotation and metabolic pathway analysis of the transcriptome were performed using a series of bioinformatics tools, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Non-Redundant Protein Database annotation. Accordingly, the nitrogen metabolism pathway of K. aerogenes B23 was analyzed; overall, 39 genes were determined to be involved in this pathway. Differential expression analysis of the genes involved in the nitrogen metabolism pathway demonstrated that, compared to the control, FNR, NarK/14945, fdx, gshA, proB, proA, gapA, argH, artQ, artJ, artM, ArgR, GAT1, prmB, pyrG, glnS, and Ca1 were significantly upregulated in the nitrogen-treated K. aerogenes B23; these genes have been established to be involved in the regulation of nitrate, arginine, glutamate, and ammonia assimilation. Further, norV, norR, and narI were also upregulated in nitrogen-treated K. aerogenes B23; these genes are involved in the regulation of NO metabolism. These differential expression results are important for understanding the regulation process of key nitrogen metabolism enzyme genes in K. aerogenes B23. Therefore, this study establishes a solid foundation for further research into the expression regulation patterns of nitrogen metabolism-associated genes in K. aerogenes B23 under nitrogen-rich conditions; moreover, this research provides essential insight into how K. aerogenes B23 utilizes nutritional elements.

Nutrients recovery by coupled bioreactor of heterotrophic ammonia assimilation and microbial fuel cell in saline wastewater

Heterotrophic ammonia assimilation (HAA) process had been widely used in the treatment of high salt wastewater, but the electro enhanced coupling process and electron transfer process were rarely studied. In this study, a HAA process coupled microbial fuel cell (MFC) system was established to treat ammonia-containing wastewater under increasing salinity to achieve nitrogen recovery and electricity generation. Up to 95.4 % NH4+-N and 96.4 % COD removal efficiencies were achieved at 2 % salinity in HAA-MFC. The maximum power density and current density at 2 % salinity were 29.93 mW/m2 and 182.37 mA/m2, respectively. The residual organic matter in the cathode effluent was effectively removed by the anode. The increase of salinity not only enhanced the sludge settling performance and activity, but also promoted the enzyme activity and amino acid production of the ammonia assimilation pathway. Marinobacter and Halomonas were gradually enriched at the anode and cathode with increased salinity to promote ammonia assimilation and electron production. This research offered a promising solution to overcome salinity-related challenges in wastewater treatment and resource recovery.

Differences in regulation mechanisms of glutamine synthetases from methanogenic archaea unveiled by structural investigations

Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50ʹ-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

Incorporating microbial inoculants to reduce nitrogen loss during sludge composting by suppressing denitrification and promoting ammonia assimilation

To address the challenge of increasing nitrogen retention in compost, this study investigated the effects of microbial communities on denitrification and ammonia assimilation during sludge composting by inoculating microbial inoculants. The results showed that the retention rates of total Kjeldahl nitrogen (TKN) and humic acid (HA) in MIs group (with microbial inoculants) were 4.94 % and 18.52 % higher than those in the control group (CK), respectively. Metagenomic analysis showed that Actinobacteria and Proteobacteria were identified as main microorganisms contributing to denitrification and ammonia assimilation. The addition of microbial agents altered the structure of the microbial community, which in turn stimulated the expression of functional genes. During cooling period, the ammonia assimilation genes glnA, gltB and gltD in MIs were 15.98 %, 24.84 % and 32.88 % higher than those in CK, respectively. Canonical correspondence analysis revealed a positive correlation between the dominant bacterial genera from the cooling stage to the maturity stage and the levels of NO3−-N, NH4+-N, HA, and TKN contents. NH4+-N was positively correlated with HA, indicating NH4+-N might be incorporated into HA. Heat map and network analyses revealed NH4+-N as a key factor affecting functional genes of denitrification and ammonia assimilation, with Nitrospira identified as the core bacteria in the microbial network. Therefore, the addition of microbial agents could increase nitrogen retention and improve compost product quality.

The distinctive level of interaction between carbon and nitrogen metabolisms in the leaves of submerged macrophytes plays a key role in ammonium detoxification

Possible ammonium detoxification mechanisms have been proposed recently, on submerged macrophytes, evidently illustrating that glutamate dehydrogenase (GDH) plays a greater role in ammonium detoxification compared to the primary glutamine synthetase/glutamate oxaloacetate transaminase (GS/GOGAT) pathway. In the current investigation, we cultured three submerged macrophytes to extreme concentrations of [NH4+-N] of up to 50 mg/L with the aim of clarifying the interaction between carbon and nitrogen metabolisms. The activities of carboxylation enzymes pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPC), in lieu of Rubisco, increased almost two-fold for ammonium tolerant species P. maackianus and M. spicatum, compared with the sensitive species P. lucens. While these enzymes are well known for their central role in CO2 fixation, their inference in conferring resistance to ammonium stress has not been well elucidated before. In this study, we demonstrate that the overproduction of PEPC and PPDK led to improved photosynthesis, better ammonium assimilation and overall ammonium detoxification in M. spicatum and P. maackianus. These findings propose likelihood for the existence of a complementary ammonium detoxification pathway that targets carbon metabolism, thus, presenting a relatively efficient linkage between nitrogen and carbon metabolisms and identify candidate species for practical restoration of fresh water resources.

Mitochondrial aspartate aminotransferase ( maa1 ) inactivation causes glutamate-requiring glu1 mutation in Schizosaccharomyces pombe.

Two genomic genes, which rescue ammonium assimilation defect in the glutamate-requiring Schizosaccharomyces pombe glu1 mutant, were identified. The maa1 , encoding a mitochondrial aspartate aminotransferase, is the causative gene of glu1 mutation because an inseparable linkage between maa1 and glu1 on the chromosome, and also the glu1 mutant strain has a nonsense mutation within the maa1 coding region, which is responsible for its defective phenotype. The yhm2 , a mitochondrial 2-oxoglutarate carrier, was also isolated as a weak multicopy suppressor gene. These findings reiterate the importance of the mitochondria in utilizing the amino acids for cellular nitrogen metabolism.

Novel insights into the co-metabolism of pyridine with different carbon substrates: Performance, metabolism pathway and microbial community

Pyridine is a widely employed nitrogen-containing heterocyclic organic, and the discharge of pyridine wastewater poses substantial environmental challenges due to its recalcitrance and toxicity. Co-metabolic degradation emerged as a promising solution. In this study, readily degradable glucose and the structurally analogous phenol were used as co-metabolic substrates respectively, and the corresponding mechanisms were thoroughly explored. To treat 400 mg/L pyridine, all reactors achieved remarkably high removal efficiencies, surpassing 98.5%. And the co-metabolism reactors had much better pyridine-N removal performance. Batch experiments revealed that glucose supplementation bolstered nitrogen assimilation, thereby promoting the breakdown of pyridine, and resulting in the highest pyridine removal rate and pyridine-N removal efficiency. The high abundance of Saccharibacteria (15.54%) and the enrichment of GLU and glnA substantiated this finding. On the contrary, phenol delayed pyridine oxidation, potentially due to its higher affinity for phenol hydroxylase. Nevertheless, phenol proved valuable as a carbon source for denitrification, augmenting the elimination of pyridine-N. This was underscored by the abundant Thauera (30.77%) and Parcubacteria (7.21%) and the enriched denitrification enzymes (narH, narG, norB, norC, and nosZ, etc.). This study demonstrated that co-metabolic degradation can bolster the simultaneous conversion of pyridine and pyridine-N, and shed light on the underling mechanism.

Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5T under conventional and alternative nitrogen fixation

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo‑nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SignificanceIn this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.

A microbial-explicit model with comprehensive nitrogen processes to quantify gaseous nitrogen production from agricultural soils

Agricultural soils are a major source of anthropogenic N2O, but their N2O emission estimates are highly uncertain, mainly due to the complexity of nitrogen (N) processes. Most soil N models include only primary N processes such as nitrification and denitrification, which limits their ability to realistically simulate N transformations in soils and accurately estimate N2O emissions from soils. This study introduces a Microbial-Explicit Model incorporating Comprehensive Nitrogen processes (MEMCN) to evaluate and quantify the influences of various N processes on the production of N2O as well as NO and N2, including nitrification, denitrification, anaerobic ammonium oxidation (ANAMMOX), dissimilatory nitrate reduction to ammonium (DNRA), mineralization, and microbial assimilation (i.e., growth) and death. The MEMCN was evaluated in laboratory experiments using agricultural soils with different levels of N additions under anaerobic and aerobic conditions, and reproduced well the dynamics of NH4+, NO3−, NO2−, NO, N2O, and N2. After nitrification and denitrification, ANAMMOX and assimilation were found to be most important in controlling N transformations in agricultural soils. ANAMMOX directly increased N2 emissions by 139% at the beginning of the simulations (i.e., 48 h) under anaerobic conditions, while microbial assimilation indirectly reduced NO, N2O, and N2 emissions by 88%, 54%, and 58%, respectively, at the end of the simulations (i.e., 336 h) under aerobic conditions. Correspondingly, the biomass of ANAMMOX bacteria increased significantly at the beginning of the simulations under anaerobic conditions, while the biomass of nitrite oxidizing bacteria increased substantially under aerobic conditions. In contrast, DNRA, mineralization and microbial death had minor effect on soil N transformations, and the biomass of DNRA bacteria and heterotrophs did not change significantly during the simulations. Our study shows that it is necessary to include ANAMMOX and microbial assimilation in soil N models, while explicit simulation of microbial biomass dynamics may only be necessary if microbial biomass pools change significantly.

Optimization of aeration strategies for intensified nitrogen and phosphorus removal in an aerated electrolysis-assisted submerged fixed bed reactor treating recirculating mariculture effluent

Nutrients such as nitrogen (N) and phosphorus (P) discharged from mariculture wastewater pose a serious threat to the environment. In this study, an electrochemically assisted submerged fixed bed bioreactor (E-SFBBR) with intermittent aeration was used to explore the effect of aeration strategies (aeration time, aeration rate and aeration position) on the removal of N and P. Phosphate (PO43--P) removal was maintained above 95% under different aeration strategies. Although PO43--P removal predominantly occurred in the cathode areas, the aeration position affected the main functional area for PO43--P removal by influencing the pattern of DO distribution within E-SFBBR; and chemical precipitation with iron ions was the main removal pathway as revealed by XPS analysis. E-SFBBR with upper aeration (i.e., UA-E-SFBBR), aerated for 6 h d−1 at 0.8 L min−1 showed the best N removal performance, i.e., 83.78 ± 4.67%. The cathode area mainly contributed to N removal through mixotrophic denitrification. Aeration position plays a greater role in affecting N removal compared to aeration time and rate. Upper aeration favored the growth of heterotrophic denitrification bacteria (f_Rhodobacteraceae and Marinicella) in the cathode through providing more anaerobic environment, resulting in a better N removal performance. Besides, results of N transformation pathway predicted by PICRUSt 2.0 revealed that upper aeration promoted the ammonia assimilation but inhibited ammonia oxidation and NO3--N reduction processes in the cathode area. This study provided a theoretical basis for the application of aerated E-SFBBR in mariculture wastewater treatment.

Functional redundancy is the key mechanism used by microorganisms for nitrogen and sulfur metabolism during manure composting

The microbial ecological functions associated with the nitrogen and sulfur cycles during composting have not been thoroughly elucidated. Using metagenomic sequencing, the microbial mechanisms underlying the nitrogen and sulfur metabolism during livestock and poultry manure composting were investigated in this study. The findings demonstrate that functional redundancy among microorganisms is a crucial factor for the nitrogen and sulfur cycling during livestock and poultry manure composting. Processes such as organic sulfur synthesis, assimilatory sulfate reduction, ammonia assimilation, and denitrification were found to be prevalent. Additionally, there was a certain degree of conservation in nitrogen and sulfur conversion functions among microorganisms at the phylum level. All high-quality metagenomic assembly genomes (MAGs) possessed carbon fixation potential, with 86.3 % of MAGs containing both nitrogen and sulfur conversion genes. Except for bin30, other MAGs encoding sulfur oxidation enzymes were found to be associated with at least one denitrification gene. This suggests a potential interplay between nitrogen and sulfur metabolism among microorganisms. 45, 19, 1, 31, 1, and 2 MAGs could completely regulate organic sulfur synthesis, assimilatory sulfate reduction, thiosulfate oxidation to sulfate, glutamine synthase-glutamate synthase pathway (GS-GOGAT), denitrification, and dissimilatory nitrate reduction, respectively by encoding the required enzymes. TN and pH were the key factors driving the functional redundancy in nitrogen and sulfur microbial community.

Microbial protein production under mixotrophic mode: ammonium assimilation pathway and C/N ratio optimization

Producing microbial protein (MP) is considered as a promising method to alleviate the demand for the global protein supply. The conversion of carbon dioxide (CO2) and organic carbon into MP by hydrogen oxidizing bacteria (HOB) is considered as a potent solution. In this study, under mixotrophic condition, MP production was 2.4 g/L, higher than the sum of that in autotrophic and heterotrophic mode (1.9 g/L), showing the mutualistic relationship of two pathways. According to functional enzyme analysis, the abundance of glutamate dehydrogenase and glutamine synthetase in the heterotrophic and mixotrophic modes was obviously enhanced (80%−800% increment), indicating that adding soluble organics could enhance both the direct (“NH4+ → L-Glutamate”) and the indirect pathway (“NH4+ → L-Glutamine → L-Glutamate”) of NH4+ assimilation in the mixed HOB consortium. For further enhancing MP production, different C/N ratio was evaluated based on mixotrophic mode, achieving amino acid production of 3.3 ± 0.1 g/L and high protein content (58.1%) in C/N-9 reactor. Corynebacterium was dominant (91.4%). In nitrogen assimilation pathway, the higher abundance of glutamine synthetase than glutamate dehydrogenase suggested that NH4+-N was mainly converted via the indirect pathway. With the increase of C/N ratio, 24–48% increment was obtained on glutamine synthetase, along with the decrement of glutamate dehydrogenase. Thus, high C/N ratio could boost the indirect pathway, while performing the negative effect on the direct pathway.

Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO-1 to deal with the toxicity of ammonium.

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2 OH → N2 , firstdefined in Alcaligenes ammonioxydans HO-1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO-1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO-1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO-1 was revealed. After the disruption of the Dirammox in HO-1, more consumed NH4 + was recovered as biomass-N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia-rich wastewater treatment.

Effects of nitrogen supply on hydrogen-oxidizing bacterial enrichment to produce microbial protein: Comparing nitrogen fixation and ammonium assimilation

To investigate the effects of nitrogen source supply on microbial protein (MP) production by hydrogen-oxidizing bacteria (HOB) under continuous feed gas provision, a sequencing batch culture comparison (N2 fixation versus ammonium assimilation) was performed. The results confirmed that even under basic cultivation conditions, N2-fixing HOB (NF-HOB) communities showed higher levels of CO2 and N2 fixation (190.45 mg/L Δ CODt and 11.75 mg/L Δ TNbiomass) than previously known, with the highest biomass yield being 0.153 g CDW/g COD-H2. Rich ammonium stimulated MP synthesis and the biomass accumulation of communities (increased by 7.4 ～ 14.3 times), presumably through the enhancement of H2 and CO2 absorption. The micro mechanism may involve encouraging the enrichment of species like Xanthobacter and Acinetobacter then raising the abundance of nitrogenase and glutamate synthase to facilitate the nitrogen assimilation. This would provide NF-HOB with ideas for optimizing their MP synthesis activity.

Nitrogen removal characteristics of novel bacterium Klebsiella sp. TSH15 by assimilatory/dissimilatory nitrate reduction and ammonia assimilation

A novel strain with heterotrophic nitrification and aerobic denitrification was screened and identified as Klebsiella sp. TSH15 by 16S rRNA. The results demonstrated that the ammonia-N and nitrate-N removal rates were 2.99 mg/L/h and 2.53 mg/L/h under optimal conditions, respectively. The analysis of the whole genome indicated that strain TSH15 contained the key genes involved in assimilatory/dissimilatory nitrate reduction and ammonia assimilation, including nas, nar, nir, nor, glnA, gltB, gdhA, and amt. The relative expression levels of key nitrogen removal genes were further detected by RT-qPCR. The results indicated that the N metabolic pathways of strain TSH15 were the conversion of nitrate or nitrite to ammonia by assimilatory/dissimilatory nitrate reduction (NO3−→NO2−→NH4+) and further conversion of ammonia to glutamate (NH4+-N → Glutamate) by ammonia assimilation. These results indicated that the strain TSH15 had the potential to be applied to practical sewage treatment in the future.

Disparities and mechanisms of carbon and nitrogen conversion during food waste composting with different bulking agents

The low C/N ratio, high moisture content, and low porosity of food waste require the addition of bulking agents for adjustment during the composting process. However, the effect and mechanism of different bulking agents on the reduction of carbon and nitrogen losses are unclear. Therefore, this study conducted experiments to evaluate and clarify the differences in carbon and nitrogen transformation between sawdust, rice husk and wheat bran in food waste composting. The results showed that the addition of bulking agents promoted the conversion of carbon and nitrogen into total organic carbon (TOC) and total organic nitrogen (TON) rather than CO2 and NH3. The carbon and nitrogen losses were reduced by 16.00–25.71% and 11.56–29.54%, respectively. Notably, the Sawdust group exhibited the highest carbon retention, whereas the Wheat_bran group demonstrated superior nitrogen retention. The succession of bacterial communities showed that sawdust enhanced the cellulolysis and xylanolysis functions while wheat bran promoted nitrogen fixation. Correlation analysis was further employed to speculate on potential interactions among carbon and nitrogen components. The incorporation of sawdust and rice husk improved humification partly due to the addition of lignocellulose and the accumulation of total dissolved nitrogen (DTN) in the substrate, respectively. In the process of ammonia assimilation, the addition of wheat bran promoted the accumulation of dissolved organic carbon (DOC), contributing to the synthesis of TON to a degree. These findings offer cost-effective strategies for conserving carbon and nitrogen from loss in food waste composting by selecting suitable bulking agents, ultimately producing high-quality fertilizer.

Identification of Effective Microbes for Ammonia and Odor Removal from Animal Manure

In intensive livestock husbandry, the issue with ammonia (NH3) emissions and odors is still only par¬tially solved. The main reason is the insufficiently detailed knowledge of processes taking place in animal wastes due to the large number of involved microorganisms. The emitted NH3 is directly implicated in the formation of PM2.5 (“fine particles”) in the atmosphere, water eutrophication and soil acidification, contrib¬utes indirectly to N2O emissions, has an adverse impact on animal health and productivity and decreases manure fertilizing value. The development of technologies for control of emissions from livestock opera¬tions requires investigations on mechanisms of these reductions, in particular with respect to microbial ad¬ditives for manure treatment. A number of microbial strains of great practical importance for mitigation of animal excreta odor are studied. The mechanisms of inhibition of ammonia production and odors include: 1) assimilation of ammonia and odorous components; 2) oxidation of ammonia to non-volatile nitrogen compounds (nitrification); 3) reduction of manure pH by organic acids production; 4) аntimicrobial activity against ammonifying and odor-generating microorganisms. The effects of different microbial communities and proportions on odor removal should be studied in order to develop efficient odor eliminators.

Imbalanced metabolism induced NH4+ accumulation and its effect on the central metabolism of Methylomonas sp. ZR1.

Nitrogen and carbon are the two most essential nutrient elements, and their metabolism is tightly coupled in single carbon metabolic microorganisms. However, the nitrogen metabolism and the nitrogen/carbon (N/C) metabolic balance in single-carbon metabolism is poorly studied. In this study, the nitrogen metabolism pattern of the fast growing methanotrophs Methylomonas sp. ZR1 grown in methane and methanol was studied. Effect study of different nitrogen sources on the cell growth of ZR1 indicates that nitrate salts are the best nitrogen source supporting the growth of ZR1 using methane and methanol as carbon source. However, its metabolic intermediate ammonium was found to accumulate with high N/C ratio in the medium and consequently inhibit the growth of ZR1. Studies of carbon and nitrogen metabolic kinetic under different N/C ratio conditions indicate that the accumulation of NH4+ is caused by the imbalanced nitrogen and carbon metabolism in ZR1. Feeding carbon skeleton α-ketoglutaric acid could effectively relieve the inhibition effect of NH4+ on the growth of ZR1, which further confirms this assumption. qPCR analysis of the expression level of the central metabolic key enzyme gene indicates that the nitrogen metabolic intermediate ammonium has strong regulation effect on the central nitrogen and carbon metabolism in ZR1. qPCR-combined genomic analysis confirms that a third ammonium assimilation pathway glycine synthesis system is operated in ZR1 to balance the nitrogen and carbon metabolism. Based on the qPCR result, it was also found that ZR1 employs two strategies to relieve ammonium stress in the presence of ammonium: assimilating excess ammonium or cutting off the nitrogen reduction reactions according to the available C1 substrate. Validating the connections between single-carbon and nitrogen metabolism and studying the accumulation and assimilation mechanism of ammonium will contribute to understand how nitrogen regulates cellular growth in single-carbon metabolic microorganisms.

Diurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals

Uncoupling of biological nitrogen fixation from ammonia assimilation is a prerequisite step for engineering ammonia excretion and improvement of plant-associative nitrogen fixation. In this study, we have identified an amino acid substitution in glutamine synthetase, which provides temperature sensitive biosynthesis of glutamine, the intracellular metabolic signal of the nitrogen status. As a consequence, negative feedback regulation of genes and enzymes subject to nitrogen regulation, including nitrogenase is thermally controlled, enabling ammonia excretion in engineered Escherichia coli and the plant-associated diazotroph Klebsiella oxytoca at 23 °C, but not at 30 °C. We demonstrate that this temperature profile can be exploited to provide diurnal oscillation of ammonia excretion when variant bacteria are used to inoculate cereal crops. We provide evidence that diurnal temperature variation improves nitrogen donation to the plant because the inoculant bacteria have the ability to recover and proliferate at higher temperatures during the daytime.