Degradation Of Organic Nitrogen Research Articles

Characterization of a New M13 Metallopeptidase from Deep-Sea Shewanella sp. E525-6 and Mechanistic Insight into Its Catalysis.

Bacterial extracellular peptidases are important for bacterial nutrition and organic nitrogen degradation in the ocean. While many peptidases of the M13 family from terrestrial animals and bacteria are studied, there has been no report on M13 peptidases from marine bacteria. Here, we characterized an M13 peptidase, PepS, from the deep-sea sedimentary strain Shewanella sp. E525-6, and investigated its substrate specificity and catalytic mechanism. The gene pepS cloned from strain E525-6 contains 2085 bp and encodes an M13 metallopeptidase. PepS was expressed in Escherichia coli and purified. Among the characterized M13 peptidases, PepS shares the highest sequence identity (47%) with Zmp1 from Mycobacterium tuberculosis, indicating that PepS is a new member of the M13 family. PepS had the highest activity at 30°C and pH 8.0. It retained 15% activity at 0°C. Its half life at 40°C was only 4 min. These properties indicate that PepS is a cold-adapted enzyme. The smallest substrate for PepS is pentapeptide, and it is probably unable to cleave peptides of more than 30 residues. PepS prefers to hydrolyze peptide bonds with P1′ hydrophobic residues. Structural and mutational analyses suggested that His531, His535 and Glu592 coordinate the catalytic zinc ion in PepS, Glu532 acts as a nucleophile, and His654 is probably involved in the transition state stabilization. Asp538 and Asp596 can stablize the orientations of His531 and His535, and Arg660 can stablize the orientation of Asp596. These results help in understanding marine bacterial peptidases and organic nitrogen degradation.

Diversity of cultivable protease-producing bacteria in sediments of Jiaozhou Bay, China.

Although protease-producing bacteria are key players in the degradation of organic nitrogen and essential for the nitrogen recycling in marine sediments, diversity of both these bacteria and their extracellular proteases is still largely unknown. This study investigated the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the eutrophied Jiaozhou Bay, China through phylogenetic analysis and protease inhibitor tests. The abundance of the cultivable protease-producing bacteria was up to 104 cells/g in all six sediment samples. The cultivated protease-producing bacteria mostly belonged to the phyla Proteobacteria and Firmicutes with the predominant genera being Photobacterium (39.4%), Bacillus (25.8%), and Vibrio (19.7%). Protease inhibitor tests revealed that extracellular proteases secreted by the bacteria were mainly serine proteases and/or metalloproteases with relatively low proportions of cysteine proteases. This study represents the first comprehensive analysis on the diversity of protease-producing bacteria and their extracellular proteases in sediments of a eutrophic bay.

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP-01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP-01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate-binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP-01 displayed a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1' site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.

Structural and Functional Characterization of Mature Forms of Metalloprotease E495 from Arctic Sea-Ice Bacterium Pseudoalteromonas sp. SM495

E495 is the most abundant protease secreted by the Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. As a thermolysin family metalloprotease, E495 was found to have multiple active forms in the culture of strain SM495. E495-M (containing only the catalytic domain) and E495-M-C1 (containing the catalytic domain and one PPC domain) were two stable mature forms, and E495-M-C1-C2 (containing the catalytic domain and two PPC domains) might be an intermediate. Compared to E495-M, E495-M-C1 had similar affinity and catalytic efficiency to oligopeptides, but higher affinity and catalytic efficiency to proteins. The PPC domains from E495 were expressed as GST-fused proteins. Both of the recombinant PPC domains were shown to have binding ability to proteins C-phycocyanin and casein, and domain PPC1 had higher affinity to C-phycocyanin than domain PPC2. These results indicated that the domain PPC1 in E495-M-C1 could be helpful in binding protein substrate, and therefore, improving the catalytic efficiency. Site-directed mutagenesis on the PPC domains showed that the conserved polar and aromatic residues, D26, D28, Y30, Y/W65, in the PPC domains played key roles in protein binding. Our study may shed light on the mechanism of organic nitrogen degradation in the Arctic sea ice.

Isolation and Identification of Ammonibacteria and Ammoniation Characteristic Analysis

Two strains of T1, Z1 ammonibacterium were isolated from the biofilm of the BAF reactor. According to the morphologic observation and physiological biochemistry test, the strains T1, Z1 were preliminarily identified asProteusandMicrococcus respectively. The research of the strains T1, Z1 for ammoniation characteristics indicated that the suitable temperature was 30°C, the suitable pH value was 7.0 and the best inoculum quantity was 5%. The strain T1 was strictly aerobic bacteria and Z1 was facultative aerobic bacteria. In these operating parameters, two strains of T1, Z1 were good of the degradation of organic nitrogen and the growth rate of ammonia nitrogen could respectively reach up to 86.91% and 69.98%. The strains T1, Z1 had a certainly practical value for treating the waste water with the high concentration of organic nitrogen.

Rates and regulation of nitrogen cycling in seasonally hypoxic sediments during winter (Boknis Eck, SW Baltic Sea): Sensitivity to environmental variables

This study investigates the biogeochemical processes that control the benthic fluxes of dissolved nitrogen (N) species in Boknis Eck – a 28 m deep site in the Eckernförde Bay (southwestern Baltic Sea). Bottom water oxygen concentrations (O 2−BW) fluctuate greatly over the year at Boknis Eck, being well-oxygenated in winter and experiencing severe bottom water hypoxia and even anoxia in late summer. The present communication addresses the winter situation (February 2010). Fluxes of ammonium (NH 4 +), nitrate (NO 3 −) and nitrite (NO 2 −) were simulated using a benthic model that accounted for transport and biogeochemical reactions and constrained with ex situ flux measurements and sediment geochemical analysis. The sediments were a net sink for NO 3 − (−0.35 mmol m −2 d −1 of NO 3 −), of which 75% was ascribed to dissimilatory reduction of nitrate to ammonium (DNRA) by sulfide oxidizing bacteria, and 25% to NO 3 − reduction to NO 2 − by denitrifying microorganisms. NH 4 + fluxes were high (1.74 mmol m −2 d −1 of NH 4 +), mainly due to the degradation of organic nitrogen, and directed out of the sediment. NO 2 − fluxes were negligible. The sediments in Boknis Eck are, therefore, a net source of dissolved inorganic nitrogen (DIN = NO 3 − + NO 2 − + NH 4 +) during winter. This is in large part due to bioirrigation, which accounts for 76% of the benthic efflux of NH 4 +, thus reducing the capacity for nitrification of NH 4 +. The combined rate of fixed N loss by denitrification and anammox was estimated at 0.08 mmol m −2 d −1 of N 2, which is at the lower end of previously reported values. A systematic sensitivity analysis revealed that denitrification and anammox respond strongly and positively to the concentration of NO 3 − in the bottom water (NO 3 − BW). Higher O 2−BW decreases DNRA and denitrification but stimulates both anammox and the contribution of anammox to total N 2 production (%R amx). A complete mechanistic explanation of these findings is provided. Our analysis indicates that nitrification is the geochemical driving force behind the observed correlation between %R amx and water depth in the seminal study of Dalsgaard et al. (2005). Despite remaining uncertainties, the results provide a general mechanistic framework for interpreting the existing knowledge of N-turnover processes and fluxes in continental margin sediments, as well as predicting the types of environment where these reactions are expected to occur prominently.

Assessing soluble organic nitrogen pools in horticultural soils: a case study in the suburbs of Shanghai (China)

Soil soluble organic nitrogen plays a significantly important role in nitrogen biogeochemical cycling. The aim of this study was to investigate the amount of soluble organic and inorganic nitrogen pools extracted by water, salt solutions, and centrifugal-drainage technique from different horticultural management system soils. Approximately 5.4–16.6, 4.4–46.5, 7.1–18.2, 8.8–27.8, 1.8–5.6, and 1.7–4.4 mg kg − 1 soluble organic nitrogen were obtained by 1M KCl, 0.5M K2SO4, 10 mM CaCl2, 1/15 mM phosphate buffer (pH 7.0), water, and centrifugal-drainage technique, respectively, in the 0–20 cm horticultural soils. Large variation in soluble organic nitrogen pools was observed across the sites in the present study and the volumes of soluble organic nitrogen pools generally followed the order: organic production system soil > conventional production system soil > transitional production system soil. In relative contrast to soluble organic nitrogen, organic management, known primarily for the absence of artificial fertilizers and pesticides, resulted in significantly lower levels of both soluble inorganic nitrogen and total soluble nitrogen in the soil. The degree of soluble inorganic nitrogen recovery appears highly dependent on the different chemical extractants and generally followed the series: phosphate buffer (pH 7.0) > KCl≈K2SO4 > CaCl2 > water > centrifugal-drainage technique. The soluble organic carbon and nitrogen in all soil extracts were all positively related to soil total carbon, total nitrogen, and electric conductivity value. This suggested that soil total carbon and nitrogen played a central role in the retention of soluble organic nitrogen in soils. Further studies on investigating the soluble organic nitrogen degradation pathway and its bottleneck are warranted.

The complete genome of Zunongwangia profunda SM-A87 reveals its adaptation to the deep-sea environment and ecological role in sedimentary organic nitrogen degradation

BackgroundZunongwangia profunda SM-A87, which was isolated from deep-sea sediment, is an aerobic, gram-negative bacterium that represents a new genus of Flavobacteriaceae. This is the first sequenced genome of a deep-sea bacterium from the phylum Bacteroidetes.ResultsThe Z. profunda SM-A87 genome has a single 5 128 187-bp circular chromosome with no extrachromosomal elements and harbors 4 653 predicted protein-coding genes. SM-A87 produces a large amount of capsular polysaccharides and possesses two polysaccharide biosynthesis gene clusters. It has a total of 130 peptidases, 61 of which have signal peptides. In addition to extracellular peptidases, SM-A87 also has various extracellular enzymes for carbohydrate, lipid and DNA degradation. These extracellular enzymes suggest that the bacterium is able to hydrolyze organic materials in the sediment, especially carbohydrates and proteinaceous organic nitrogen. There are two clustered regularly interspaced short palindromic repeats in the genome, but their spacers do not match any sequences in the public sequence databases. SM-A87 is a moderate halophile. Our protein isoelectric point analysis indicates that extracellular proteins have lower predicted isoelectric points than intracellular proteins. SM-A87 accumulates organic osmolytes in the cell, so its extracelluar proteins are more halophilic than its intracellular proteins.ConclusionHere, we present the first complete genome of a deep-sea sedimentary bacterium from the phylum Bacteroidetes. The genome analysis shows that SM-A87 has some common features of deep-sea bacteria, as well as an important capacity to hydrolyze sedimentary organic nitrogen.

Methane and carbon dioxide emissions and nitrogen turnover during liquid manure storage

Animal slurry stored in-house and outside is a significant source of atmospheric methane (CH4). The CH4 source strength of stored slurry is greatly affected by temperature. To improve emission calculations on a global scale there is a need for knowledge about the relationship between production of CH4 in slurry and temperature. In this study, the filling of slurry channels was reproduced in the laboratory by gradually filling 1 m-high PVC vessels during 9 days followed by incubation for 100–200 days. A preliminary test showed that little CH4 was produced from animal slurry during 10 days of incubation at 20°C, if no inoculum (slurry incubated anaerobically at the test temperature for 1.5–2 months) was present. However, the addition of 7.6% inoculum supported an immediate production of CH4. Vessels amended with inoculum and gradually filled with cattle or pig slurry were then incubated at 10, 15 and 20°C. Methane production from stored pig and cattle slurry was not significant at temperatures below 15°C, where CO2 was the main product of decomposition processes. In contrast, the anaerobic production of CH4 was high and significant relative to the production of CO2 at 20°C. Peak emissions of CH4 averaging 0.012 and 0.02 g C h−1 kg−1 volatile solids (VS) were reached within about 10 days at 10 and 15°C, respectively. At 20°C, the emission of CH4 from pig slurry was about 0.01 g C h−1 kg−1 for 10 days, and thereafter emissions increased to about 0.10 g C h−1 kg−1 VS. For cattle slurry a peak emission of 0.08 g C h−1 kg−1 VS was measured after 180 days. Degradation of organic nitrogen (N) in cattle slurry was related to the reduction of organic material as reflected in CO2 and CH4 emission. The mineralization of organic N during storage represented 10–80% of organic N in cattle slurry, and 40–80% of the organic N in pig slurry.

An integrated approach of solar photocatalytic and biological treatment of N-containing organic compounds in wastewater*

In the present study the integration of TiO2-assisted solar photocatalysis and biological treatment for the degradation of organic nitrogen containing pyrazinamide (antituberculosis drug) synthesis process effluent is investigated. The experimental results showed that a considerable increase in the degradation efficiency of N-containing compounds is obtained by integration of photocatalysis and biological treatment when compared to either biological or photocatalysis treatment alone. The optimization parameter like amount of photocatalyst (TiO2 Degussa-P-25) was performed for photocatalytic treatment under solar irradiation. Furthermore BOD5/COD ratios were also evaluated. The optimum catalyst (wt.%) of 0.35 TiO2, and 40 times dilution are found to be optimum for achieving a maximum of 76, 83, 80% of COD, color and TOC removals respectively and an improved BOD5/COD ratio of 0.3 to 0.7 in a duration of 24 h. Also the formation of increased from 10 to 44 mg L−1 within 20 h of photocatalytic treatment. The effluent subjected to biological treatment for five days (120 h) has resulted 43% COD removal only, whereas the treatment method using integrated approach resulted a 91% COD removal in a duration of 44 h.

Nitrogen Isotopic Composition of Ammonium-rich Illite in Anthracites and Organic-rich Shales from Eastern Pennsylvania

Previous studies (Daniels and Altaner 1990; Sucha et al, 1994) have shown that ammonium-bearing illite/ muscovite can be formed, at temperature above 200~ by reaction of kaolinite with ammonia, derived from the thermal degradation of organic nitrogen. In the part icular case of Eastern Pennsylvania, the present work reports, for the first time, the isotopic composition of nitrogen in ammonium rich illite from low grade metamorphic rocks associated with anthracites ranking above Rmax: 4%.

Rates of organic carbon and nitrogen degradation in intensive fish ponds

Water quality in intensive aquaculture systems is to a large extent controlled by the microbial biodegradation of organic residues. The ability to simulate processes in such systems depends on the availability of data on the rates of these processes. The first order kinetic rate constants for both organic carbon and organic nitrogen degradation in fish ponds were evaluated. The rate constants were computed by either relating the measured rate of disappearance of the substrate concentration or by the comparison of the measured with a theoretically derived evolution pattern of organic carbon or nitrogen concentrations in model ponds. The organic carbon degradation rate constant was found to be 0.15/day. The organic nitrogen degradation rate constant was expected, theoretically to be about 0.06/day and was found experimentally to be in the range of 0.05–0.09/day. These values seem to be reasonable approximations for constantly aerated-mixed ponds, at a temperature range of 20–30 °C. Organic matter previously metabolized in anoxic sites at the pond (e.g. resuspended sediments), has a degradation rate of ca. 0.6/day, and can thus consume large amounts of oxygen in the water body.

Modeling of nitrogen transformation in intensively aerated fish ponds

A model describing the nitrogen sub-system in a completely mixed and aerated pond is proposed. Inputs of nitrogen include feed and influent water; outputs of nitrogen include uptake by fish, removal of sediment from the pond and discharge water. Internal transformations between the inorganic and organic pools are also computed. The organic nitrogen that is introduced with the feed is mineralized through either ammonia excretion by fish or the microbial degradation of organic nitrogen. The opposite process—immobilization of nitrogen—occurs through algal nitrogen uptake or by microbial protein synthesis. Computed results may enable the design of an optimal water exchange rate. The simulation of ponds fed with different feed compositions and rates, having variable water exchange regimes, gave results close to the measured ones.