Assessment of denitrifying bacterial composition in activated sludge

The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10(4)-10(5), nosZ with 10(4)-10(6) and 16S rRNA gene in the range 10(9)-10(10) copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

城市污水处理系统的菌群分析和除氮基因工程出发菌的选择

目的:对脱氮污水处理工艺的活性污泥的菌群组成进行分析,以期获得适合于脱氮基因工程改良的出发菌.方法:首先采用平板稀释法对活性污泥进行菌落计数,并对分离到的菌落进行详细的生化鉴定,对其中的优势菌-假单胞菌进行脱氮能力测定,并分析其对常作为筛选标志的抗生素的药物敏感性.结果:发现在采用该工艺的活性污泥中,优势菌为假单胞菌、肠杆菌、莫拉菌和不动杆菌,分别占总菌数的23%、16%、16%和12%.根据菌群分析的结果,从中选择了两株耐药性弱、脱氮能力强的菌作为基因改良的出发菌.结论:本研究阐明了活性污泥的菌群构成,获得了两株适合基因工程改良的菌株,为日后脱氮基因工程菌的构建奠定基础.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Evaluation of the denitrifying microbiota of anoxic reactors

Removal of inorganic nitrogen compounds from wastewaters can be accomplished by a combination of the biological processes of nitrification and denitrification. The information on the microbiota present in denitrifying reactors is still scarce. In the present work the evaluation of the denitrifying microbiota of different reactor sludges was performed by specific activity measurements and MPN count of denitrifiers. We also present the isolation and physiological and phylogenetic characterisation of denitrifying bacteria from the anoxic reactor of a combined system treating landfill leachate. Specific denitrifying activity measurements were faster to perform and more reliable than MPN enumerations. 16S rDNA characterisation of the isolates showed that they belonged to the genera Thauera, Acidovorax and Alcaligenes and were closely related to microorganisms retrieved from ecosystems rich in recalcitrant compounds. Two of the isolates could grow on aromatic compounds as sole carbon source.     

Denitrifying bacteria in bulk and maize-rhizospheric soil: diversity and N2O-reducing abilities

The aim of this study was to determine the effect of the rhizosphere of maize on the diversity of denitrifying bacteria. Community structure comparison was performed by constructing a collection of isolates recovered from bulk and maize planted soil. A total of 3240 nitrate-reducing isolates were obtained and 188 of these isolates were identified as denitrifiers based on their ability to reduce nitrate to N2O or N2. 16S rDNA fragments amplified from the denitrifying isolates were analysed by restriction fragment length polymorphism. Isolates were grouped according to their restriction patterns, and 16S rDNA of representatives from each group were sequenced. A plant dependent enrichment of Agrobacterium-related denitrifiers has been observed resulting in a modification of the structure of the denitrifying community between planted and bulk soil. In addition, the predominant isolates in the rhizosphere soil were not able to reduce N2O while dominant isolates in the bulk soil evolve N2 as a denitrification product.     

Polyphosphate-Accumulating and Denitrifying Bacteria Isolated from Anaerobic-Anoxic and Anaerobic-Aerobic Sequencing Batch Reactors

In this study, phosphate-accumulating bacteria achieved complete phosphate removal in two different systems: an anaerobic-anoxic sequencing batch reactor and an anaerobic-aerobic sequencing batch reactor. This result shows that phosphate-accumulating bacteria in the A₂ SBR can use nitrate as terminal electron acceptor instead of oxygen. Phosphate-accumulating bacteria accumulated phosphate with a rates between 30 and 70 mg P/L/h in the A/O SBR and between 15 and 32 mg P/L/h in the A₂ SBR. Twenty denitrifying isolates were screened from A₂ SBR and nine from A/O SBR. Identification of these isolates by the Biolog system and the API 20 NE identification kit revealed that the most active denitrifiers in both SBRs reactors were species of Ochrobactrum, Pseudomonas, Corynebacterium, Agrobacterium, Aquaspirillum, Haemophilus, Xanthomonas, Aeromonas, and Shewanella. The most active phosphate accumulating and denitrifying bacteria were identified as Agrobacterium tumefaciens B, Aquaspirillum dispar, and Agrobacterium radiobacter. This study showed that the active phosphate accumulating-bacteria were also the most efficient denitrifying bacteria in both reactors. Received: 24 February 1998 / Accepted: 21 July 1998     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge.

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

好氧反硝化菌的研究进展

综述了好氧反硝化菌的种类和特性、好氧反硝化菌的反硝化作用机制和影响因素.好氧反硝化菌主要包括假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)、副球菌属(Para-coccus)和芽孢杆菌属(Bacillus)等,属好氧或兼性好氧异养微生物.好氧反硝化菌能在好氧条件下进行反硝化,其主要产物是N2O,并可将铵态氮直接转化成气态产物.催化好氧反硝化菌反硝化作用的硝酸盐还原酶是周质酶而不是膜结合酶.溶解氧和C/N往往是影响好氧反硝化菌反硝化作用的主要因素.介绍了间歇曝气法、选择性培养基法等好氧反硝化菌的主要分离筛选方法.概述了好氧反硝化菌在水产养殖、废水生物处理、降解有机污染物以及对土壤氮素损失的影响方面的研究进展.     

Numerically Dominant Denitrifying Bacteria from World Soils

Nineteen soils, three freshwater lake sediments, and oxidized poultry manure were examined to determine the dominant denitrifier populations. The samples, most shown or expected to support active denitrification, were from eight countries and included rice paddy, temperate agricultural, rain forest, organic, and waste-treated soils. Over 1,500 organisms that could grow anaerobically on nitrate agar were isolated. After purification, 146 denitrifiers were obtained, as verified by production of N₂ from NO₃^-. These isolates were characterized by 52 properties appropriate for the Pseudomonas-Alcaligenes group. Numerical taxonomic procedures were used to group the isolates and compare them with nine known denitrifier species. The major group isolated was representative of Pseudonomas fluorescens biotype II. The second most prevalent group was representative of Alcaligenes. Other Pseudomonas species as well as members of the genus Flavobacterium, the latter previously not known to denitrify, also were identified. One-third of the isolates could not utilize glucose or other carbohydrates as sole carbon sources. Significantly, none of the numerically dominant denitrifiers we isolated resembled the most studied species: Pseudomonas denitrificans, Pseudomonas perfectomarinus, and Paracoccus denitrificans. Denitrification appears to be a property of a very diverse group of gram-negative, motile bacteria, as shown by the large number (22.6%) of ungrouped organisms. The diversity of denitrifiers from a given sample was usually high, with at least two groups present. Denitrifiers, nitrite accumulators, and organisms capable of anaerobic growth were present in the ratio of 0.20±0.23:0.81±0.23:1. There were few correlations between their numbers and the sample characteristics measured. However, the temperatures at which isolates could grow were significantly related to the temperatures of the environments from which they were isolated. Regression analysis revealed few relationships between physical parameters and bacterial types, save for the anaerobe numbers, in which 94% of the variance could be accounted for.     

一株高效好氧反硝化菌的分离及特性

利用富集培养的方法从南昌市郊某养鱼塘采样分离出22株反硝化细菌,其中8株反硝化率较高,从中选择一株效果最好的作为研究对象,命名为HS-N62,对其生长特性进行了深入研究。结果表明:硝酸盐氮初始浓度为140mg/L,菌株HS-N62在12h内对硝酸盐氮的去除率可达96%,而且没有亚硝酸盐氮的积累。该菌最适生长温度范围为30°C-37°C,最适生长pH范围6.0-8.0,最适C/N比为10:1,并能利用多种碳源生长。运用正交试验探讨了该菌株最适的反硝化条件。反硝化菌株HS-N62还具有较好的除磷能力,12h除磷率达到67.7%(初始磷酸盐浓度57mg/L)。通过形态学特性和生理生化分析以及16S rRNA基因序列分析,菌株HS-N62与Pseudomonas sp.亲缘关系最为接近,相似性达99%,初步鉴定该菌为假单胞菌属(Pseudomonas sp.)。     

Identification of denitrifying rhizobacteria from bentgrass and bermudagrass golf greens

AIMS: As high rates of nitrogen fertilization are used in turfgrass management, there is a great potential for nitrogen loss. Research on identification of denitrifiers in turfgrass has been limited. Therefore, the aim was to identify denitrifier species and genes from turfgrass roots. METHODS AND RESULTS: Rhizobacteria were isolated from roots of bentgrass and bermudagrass in sand-based United States Golf Association (USGA) golf greens and used for denitrification biochemical analysis. Seventeen per cent (34 isolates) were identified as denitrifiers, 47% were classified as nitrate-reducers and 36% were nondenitrifiers. Identification of species of the denitrifiers was performed by chromatography fatty acid methyl ester (GC-FAME) and16S rDNA analyses. Bacillus and Pseudomonas were the major turfgrass denitrifiers. The two methods showed a 60% agreement at the genus level. Nitrite reductase genes nirK and nirS were detected in 74 and 15% of the denitrifiers, respectively, but not in nondenitrifiers. The nosZ gene encoding nitrous oxide reductase was detected in all the denitrifiers, but also in some nondenitrifiers. CONCLUSIONS: To our knowledge, this is the first report for identification of denitrifiers and denitrification-related genes associated with turfgrass roots. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide valuable data for future denitrification studies that seek to improve turfgrass nitrogen management for maximum efficiency.     

Influence of Two Plant Species (Flax and Tomato) on the Distribution of Nitrogen Dissimilative Abilities within Fluorescent Pseudomonas spp

The distribution of nitrogen-dissimilative abilities among 317 isolates of fluorescent pseudomonads was studied. These strains were isolated from an uncultivated soil and from the rhizosphere, rhizoplane, and root tissue of two plant species (flax and tomato) cultivated on this same soil. The isolates were distributed into two species, Pseudomonas fluorescens (45.1%) and Pseudomonas putida (40.4%), plus an intermediate type (14.5%). P. fluorescens was the species with the greatest proportion of isolates in the root compartments and the greatest proportion of dissimilatory and denitrifying strains. According to their ability to dissimilate nitrogen, the isolates have been distributed into nondissimilatory and dissimilatory strains, nitrate reducers and true denitrifiers with or without N(inf2)O reductase. The proportion of dissimilatory isolates was significantly enhanced in the compartments affected by flax and tomato roots (55% in uncultivated soil and 90 and 82% in the root tissue of flax and tomato, respectively). Among these strains, the proportion of denitrifiers gradually and significantly increased in the root vicinity of tomato (44, 68, 75, and 94% in uncultivated soil, rhizosphere, rhizoplane, and root tissue, respectively) and was higher in the flax rhizoplane (66%) than in the uncultivated soil. A higher proportion of N(inf2)O reducers was also found in the root compartments. This result was particularly clear for tomato. It is hypothesized that denitrification could be a selective advantage for the denitrifiers in the root environment and that this process could contribute to modify the specific composition of the bacterial communities in the rhizosphere.     

Comparative typing of Pseudomonas species isolated from the aquatic environment in Greece by SDS-PAGE and RAPD analysis

AIMS: Three broadly used typing methods were employed in order to assess and compare the identification and classification of environmental Pseudomonas strains. The reproducibility, typeability and discriminatory power of the methods were also compared to evaluate their application. Finally, the potential impact on public health of the isolates is to be discussed. METHODS AND RESULTS: Pseudomonas strains (160) isolated from the aquatic environment in Greece and identified by a rapid identification commercially available system (API20NE), were subjected to whole-cell protein electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Randomly Amplified Polymorphic DNAs (RAPD) using two 10-mer primers. In general, the obtained results were in agreement. Twenty isolates that could not be identified by the API20NE system were classified by the other methods. CONCLUSIONS: Rapid identification systems may serve only for a first rough identification of environmental Pseudomonads. In order to acquire further information, so that conclusions about their role in the ecosystem and human health could be drawn, other phenotypic or genotypic methods have to be applied. SIGNIFICANCE AND IMPACT OF STUDY: It is important, from a public health point of view, to monitor the identities of environmental Pseudomonas isolates using specific methods due to their ubiquity, heterogeneity and their pathogenicity, either established or potential.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

Denitrifying potential of methanogenic sludge

Summary A methanogenic sludge showed denitrifying activity for acetate, glucose and effluents from methanogenic treatments as substrates; denitrifiers were present in a relatively high number. When glucose was used as substrate dissimilatory reduction of nitrate to ammonium occurred. Methane production from acetate was inhibited by denitrification and resumed after nitrite and nitrous oxide depletion.     

A comparison of three identification kits for the confirmation of Aeromonas spp.

A comparative study was made of the ability of three commercial identification kits to confirm the identity of motile aeromonads isolated from foods. The kits included the API 20E, API 20NE and Microbact 24E. The results showed that both the API 20NE and Microbact 24E correctly identified 97.5% of isolates but the API 20E only 72.5%.     

Phosphate uptake kinetics by Acinetobacter isolates

Acinetobacter isolates from activated sludge treatment plants of forest industry were used as model organisms for polyphosphate accumulating bacteria to study excess phosphate uptake by the overplus phenomenon as well as luxury uptake of phosphate during growth. The initial, rapid phosphate uptake by the phosphorus-starved Acinetobacter isolates (the overplus phenomenon) followed the Michaelis-Menten model (maximum initial phosphate uptake rate 29 mg P g(-1) dry mass (DM) h(-1), half-saturation constant for excess phosphate uptake 17 mg P L(-1)). During the rapid uptake no growth was observed, but most cells contained polyphosphate granules. Also growth and luxury uptake of phosphate could be modeled with the Michaelis-Menten equation (maximum phosphate uptake rate 3.7-12 mg P g(-1) DM h(-1), half-saturation constant for growth 0.47-6.0 mg P L(-1), maximum specific growth rate 0.15-0.55 h(-1)). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 304-309, 1997.