Efficient total nitrogen removal from wastewater by Paracoccus denitrificans DYTN-1.

Bioaugmentation is an effective treatment method to reduce nitrogenous pollutants from wastewater. A strain of DYTN-1, which could effectively remove TN from sewage, was isolated from the sludge of a wastewater treatment plant and was identified as Paracoccus denitrificans. The TN in wastewater reduced to <20 mg l-1 within 12 h under optimal conditions by free cells of P. denitrificans DYTN-1. To enhance the removal of TN, P. denitrificans DYTN-1 cells were immobilized in sodium alginate (SA) using different divalent metal ions as cross-linking agents. It was found that the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 to below 20 mg l-1 within 8 h. After the optimization of an orthogonal experiment, the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 mg l-1 to below 20 mg l-1 within 1 h and significantly reduce the fermentation cycle. These findings would provide an economical and effective method for the removal of total nitrogen in wastewater by immobilized cells of P. denitrificans DYTN-1. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified a new Paracoccus denitrificans strain (DYTN-1) for removal of the total nitrogen in wastewater. The total nitrogen could be removed effectively by P. denitrificans DYTN-1 within 12 h in wastewater. Using sodium alginate as the carrier and Ba2+ as cross-linking agent, the immobilized P. denitrificans DYTN-1 cells could improve the removal efficiency of total nitrogen in wastewater and significantly reduce the fermentation cycle. The assay has provided an economical and effective method for the removal of total nitrogen in wastewater by immobilized cell.

Autotrophic, Heterotrophic, and Mixotrophic Nitrogen Assimilation for Single-Cell Protein Production by Two Hydrogen-Oxidizing Bacterial Strains.

To recover a nitrogen resource from high-ammonia-nitrogen wastewater, two amphitrophic hydrogen-oxidizing bacteria (HOB), Paracoccus denitrificans Y5 and P. versutus D6, capable of nitrogen assimilation for single-cell protein (SCP) production were isolated. These two HOB strains could grow autotrophically with H2 as an electron donor, O2 as an electron acceptor, CO2 as a carbon source, and ammonia nitrogen (NH4+-N) as a nitrogen source. The cell molecular formulas of strains Y5 and D6 determined by autotrophic cultivation were C3.33H6.83O2.58N0.77 and C2.87H5.34O3.17N0.57, respectively. The isolated strains could synchronously remove NH4+-N and organic carbon and produce SCP via heterotrophic cultivation. The rates of removal of NH4+-N and soluble chemical oxygen demand reached 35.47 and 49.04%, respectively, for Y5 under mixotrophic cultivation conditions with biogas slurry as a substrate. SCP content of strains Y5 and D6 was 67.34-73.73% based on cell dry weight. Compared with soybean meal, the SCP of Y5 contained a variety of amino acids.

Sulfide oxidation and nitrate reduction for potential mitigation of H2S in landfills.

Because H2S emitted by landfill sites has seriously endangered human health, its removal is urgent. H2S removal by use of an autotrophic denitrification landfill biocover has been reported. In this process, nitrate-reducing and sulfide-oxidizing bacteria use a reduced sulfur source as electron donor when reducing nitrate to nitrogen gas and oxidizing sulfur compounds to sulfate. The research presented here was performed to investigate the possibility of endogenous mitigation of H2S by autotrophic denitrification of landfill waste. The sulfide oxidation bioprocess accompanied by nitrate reduction was observed in batch tests inoculated with mineralized refuse from a landfill site. Repeated supply of nitrate resulted in rapid oxidation of the sulfide, indicating that, to a substantial extent, the bioprocess may be driven by functional microbes. This bioprocess can be realized under conditions suitable for the autotrophic metabolic process, because the process occurred without addition of acetate. H2S emissions from landfill sites would be substantially reduced if this bioprocess was introduced.

Biodegradation of isopropanol by a solvent-tolerant Paracoccus denitrificans strain.

The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5×10(8) cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (µmax)=0.0277/hr, a saturation constant (KS)=0.7333 g/L, and an inhibition concentration (Ki)=8.9887 g/L. Paracoccus denitrificans GH3 is considered to be well used in degrading IPA in wastewater.

Determination of low concentration of Paracoccus denitrificans encapsulated in polyvinyl alcohol LentiKat's pellets.

The aim of this work was to compare three methods to determinate low concentrations of Paracoccus denitrificans encapsulated in polyvinyl alcohol pellets, which is important for evaluation and optimization of pellet production as well as for monitoring of biomass growth. Pellets with different and well-defined biomass concentrations were used for experiments. The following fast and simple methods were tested: (1) dissolution of polyvinyl alcohol in hot water followed by dry weight estimation, (2) dissolution of polyvinyl alcohol in hot water followed by optical density measurement, (3) and extraction and quantification of proteins. Dry weight estimation proved to be problematic as it was difficult to separate biomass from polymeric carrier. Optical density measurement showed good linearity of dependence of optical density on biomass content, but determined limits of detection and limits of quantification were not within the range necessary for intended application. The only tested method meeting the requirements for sensitivity was determination of protein concentration after protein extraction.

New insights into the biodegradation of thiodiglycol, the hydrolysis product of Yperite (sulfur mustard gas).

AIMS: To isolate thiodiglycol (TDG)-degrading bacteria, the mustard gas hydrolysis product, and to characterize the metabolites formed and the enzymes involved in the degradation. METHODS AND RESULTS: Two strains, identified as Achromobacter xylosoxydans G5 and Paracoccus denitrificans E4, isolated from a petroleum-contaminated soil, utilized TDG as sole carbon and sulfur source. During the degradation of TDG by strain E4 [(2-hydroxyethyl)thio] acetic acid (HETA), thiodiglycolic acid (TDGA) and bis-(2-hydroxyethyl)disulfide (BHEDS) were identified by gas chromatography-mass spectrometry analysis, while HETA and TDGA were identified for strain G5. Two-dimensional isoelectric focussing-gel electrophoresis (2-D IEF/SDS-PAGE) maps of protein extracts of P. denitrificans E4 grown on TDG showed a spot identified as a methanol dehydrogenase. Increased expression of a putative iscS gene, involved in sulfur assimilation, was observed in TDG-grown cells of A. xylosoxydans G5. CONCLUSIONS: TDG degradation by P. denitrificans E4 occurred through two pathways: one involved cleavage of the C-S bond of HETA, yielding BHEDS and the other, oxidation of the alcoholic groups of TDG, yielding TDGA. The cleavage of the C-S bond of TDGA gave mercaptoacetic acid, further oxidized to acetate and sulfate. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased knowledge of TDG-degrading bacteria and the possibility of using them in a tailored-two-stage mustard gas destruction process.

RNA microarray for estimating relative abundance of 16S rRNA in microbial communities.

We developed an RNA microarray protocol in which total RNA from a microbial community was attached to a slide glass, and rRNA was detected by fluorescently labeled oligonucleotide probes. The RNA microarray requires only 4 h for hybridization and enables double staining and estimating relative abundance of rRNA.

Performance of calorimetric methods for the investigation of microbial systems in combination with additional sensors.

Calorimetric methods are used in combination with online oxygen measurement (using an amperometric sensor) and determination of the optical density (using a fibre optic sensor) to investigate microbial growth behaviour. The calorimetric curves of different batch experiments show a characteristic and reproducible course. Changes in the slope of the DeltaT-time curves indicate the effects of limiting factors on the microbial activity during the cultivation. A first limitation could be correlated with the depletion of oxygen in the medium; a second correlates with the depletion of the carbon source. Measurements of optical density in some cases provide reliable information about the growth of a microorganism culture. Our measurements show a good correlation of the universal calorimetric signal (heat-time curve) to the signal of the miniaturised photometric (OD) sensor.

In-situ characterization of microbial community in an A/O submerged membrane bioreactor with nitrogen removal.

The bacterial community involved in removing nitrogen from sewage and their preferred DO environment within an anoxic/oxic membrane bioreactor (A/O MBR) was investigated. A continuously operated laboratory-scale A/O MBR was maintained for 360 d. At a sludge age of 150 d and a C/N ratio of 3.5, the system was capable of removing 88% of the influent nitrogen from raw wastewater through typical nitrogen removal transformations (i.e. aerobic ammonia oxidation and anoxic nitrate reduction). Characterization of the A/O MBR bacterial community was carried out using fluorescence in situ hybridization (FISH) techniques. FISH results further showed that Nitrosospira spp. and Nitrospira spp. were the predominant groups of ammonia and nitrite oxidizing group, respectively. They constituted up to 11% and 6% of eubacteria at DO below 0.05 mg/l (low DO), respectively, and about 14% and 9% of eubacteria at DO between 2-5 mg/l (sufficient DO), respectively, indicating preference of nitrifiers for a higher DO environment. Generally low counts of the genus Paracoccus were detected while negative results were observed for Paracoccus denitrificans, Alcaligenes spp, and Pseudomonas stutzeri under the low and sufficient DO environments. The overall results indicate that Nitrosospira spp., Nitrospira spp. and members of Paracoccus spp. can be metabolically functional in nitrogen removal in the laboratory-scale A/O MBR system.

[Formation of a methylotrophic denitrifying biocenosis in a system of sewage treatment for nitrates]. / Formirovanie metilotrofnogo denitrifitsiruiushchego soobshchestva v sisteme ochistki stochnykh vod ot nitratov.

A methylotrophic denitrifying bioenosis composed of hyphomicrobes and paracocci was isolated from the active ooze in a system of sewage purification from nitrates. The morphological and physiological characteristics of the isolated Hyphomicrobium sp. Z-115 and Paracoccus denitrificans Z-100 and Z-121 strains differed from those of the type strains, which made it difficult to identify them and to isolate them as a pure culture. This should be taken into account while determining the agents operating in such purification systems. The rate of growth, the rate of nitrate reduction and the activity of enzymes involved in methanol assimilation are higher in the anabolic syntrophic bicenosis than in its components in pure culture. A combined culture composed of the collection Hyphomicrobium and Paracoccus strains was neither effective nor stable under the conditions of anaerobic growth with nitrate and methanol. Therefore, the natural biocenosis af the purification system cannot be substituted by an artificial one composed of the collection cultures.

Denitrification of high concentrations of nitrites and nitrates in synthetic medium with different sources of organic carbon. III. Methanol.

The denitrification of nitrites and nitrates (1000 mg N/l) in medium containing methanol as a source of organic carbon was studied. Continuous cultures of mixed population of autochtonic microflora from bottom sludge of nitrogenous wastewater reservoir were set up in a chemostat-type column and packed bed reactor. The efficiency of denitrification of nitrates in packed bed reactor was 506.7 mg N/l/h whereas denitrification of nitrites was from 8.7 to 16.0 mg N/l/h depending on the granulation of the filing material. In the latter case 83% nitrogen was removed from the medium. One of the factors causing low efficiency of denitrification of nitrites is excessive alkalization of the medium in the bed. The use of a three-step bed with adjusted pH resulted in complete denitrification of nitrites with efficiency 60 mg N/l/h. The bacteria inside the bed were dominated by Paracoccus denitrificans and by Pseudomonas aeruginosa when nitrates were present. The sensitivity of P. denitrificans to high concentrations of nitrites seems to be the second factor contributing to low efficiency of denitrification with methanol as organic substrate.