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PubMed2003

Characterization of microbial communities and composition in constructed dairy wetland wastewater effluent.

Constructed wetlands have been recognized as a removal treatment option for high concentrations of contaminants in agricultural waste before land application. The goal of this study was to characterize microbial composition in two constructed wetlands designed to remove contaminants from dairy washwater. Water samples were collected weekly for 11 months from two wetlands to determine the efficiency of the treatment system in removal of chemical contaminants and total and fecal coliforms. The reduction by the treatment was greatest for biological oxygen demand, suspended solids, chemical oxygen demand, nitrate, and coliforms. There was only moderate removal of total nitrogen and phosphorus. Changes in the total bacterial community and ammonia-oxidizing bacterial composition were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified fragments of the gene carrying the alpha subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and DGGE bands. DGGE analysis of wetlands and manure samples revealed that the total bacterial community composition was dominated by bacteria from phylogenetic clusters related to Bacillus, Clostridium, Mycoplasma, Eubacterium, and Proteobacteria originally retrieved from the gastrointestinal tracts of mammals. The population of ammonia-oxidizing bacteria showed a higher percentage of Nitrosospira-like sequences from the wetland samples, while a higher percentage of Nitrosomonas-like sequences from manure, feces, raw washwater, and facultative pond was found. These results show that the wetland system is a natural process dependent upon the development of healthy microbial communities for optimal wastewater treatment.

Bacillus↗

PubMed2004

Dynamics of a pasture soil microbial community after deposition of cattle urine amended with [13C]urea.

Within grazed pastures, urine patches are hot spots of nitrogen turnover, since dietary N surpluses are excreted mainly as urea in the urine. This short-term experiment investigated 13C uptake in microbial lipids after simulated deposition of cattle urine at 10.0 and 17.1 g of urea C m(-2). Confined field plots without or with cattle urine amendment were sampled after 4 and 14 days, and soil from 0- to 5-cm and 10- to 20-cm depths was analyzed for content and composition of phospholipid fatty acids (PLFAs) and for the distribution of urea-derived 13C among individual PLFAs. Carbon dioxide emissions were quantified, and the contributions derived from urea were assessed. Initial changes in PLFA composition were greater at the lower level of urea, as revealed by a principal-component analysis. At the higher urea level, osmotic stress was indicated by the dynamics of cyclopropane fatty acids and branched-chain fatty acids. Incorporation of 13C from [13C]urea was low but significant, and the largest amounts of urea-derived C were found in common fatty acids (i.e., 16:0, 16:1omega7c, and 18:1omega7) that would be consistent with growth of typical NH4(+)-oxidizing (Nitrosomonas) and NO2(-)-oxidizing (Nitrobacter) bacteria. Surprisingly, a 20 per thousand depletion of 13C in the cyclopropane fatty acid cy17:0 was observed after 4 days, which was replaced by a 10 to 20 per thousand depletion of that in cy19:0 after 14 days. Possible reasons for this pattern are discussed. Autotrophic nitrifiers could not be implicated in urea hydrolysis to any large extent, but PLFA dynamics and the incorporation of urea-derived 13C in PLFAs indicated a response of nitrifiers which differed between the two urea concentrations.

Animals↗

PubMed2004

High-rate nitrification at low pH in suspended- and attached-biomass reactors.

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 +/- 0.1) and low bulk ammonium concentrations (9.3 +/- 3.3 mg.liter(-1)), a very high nitrification rate of 5.6 g of N oxidized.liter(-1).day(-1) was achieved. The specific nitrification rate (0.55 g of N.g of biomass(-1).day(-1)) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 +/- 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (15N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass.g of N oxidized(-1). Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.

Bacteria↗

PubMed2004

Long-chain N-acyltyrosine synthases from environmental DNA.

The heterologous expression of DNA extracted directly from environmental samples (environmental DNA [eDNA]) in easily cultured hosts provides access to natural products produced by previously inaccessible microorganisms. When eDNA cosmid libraries were screened in Escherichia coli for antibacterially active clones, long-chain N-acyltyrosine-producing clones were found in every eDNA library. These apparently common natural products have not been previously described from screening extracts of cultured bacteria for biologically active natural products. Of the 11 long-chain N-acyl amino acid synthases (NASs) that were characterized, 10 are unique sequences. A predicted protein of previously unknown function from Nitrosomonas europaea, a gram-negative nitrifying beta-proteobacterium, is 14 to 37% identical to eDNA NASs. When cloned into E. coli, this open reading frame confers the production of long-chain N-acyltyrosines to the host and is therefore the first NAS from a cultured bacterium to be functionally characterized. Understanding the role that long-chain N-acyl amino acids play in soil microbial communities should now be feasible with the identification of a cultured organism that has the genetic capacity to produce these compounds.

Acylation↗

PubMed2005

Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history.

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c(554); and cycB, cytochrome c(m)(552). The deduced protein sequences of HAO, c(554), and c(m)(552) were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c(m)(552), NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c(554) gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c(554) gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.

Amino Acid Sequence↗

PubMed2006

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances.

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

Ammonia↗

PubMed1967

Oxygen-nitrogen relationships in autotrophic nitrification.

Oxygen utilization by the autotrophic nitrifiers Nitrosomonas and Nitrobacter was studied. Experimental evidence is presented which reflects the effect of carbon dioxide fixation on overall oxygen utilization in autotrophic nitrification. Measurement of dissolved oxygen and inorganic nitrogen changes indicates that oxygen-nitrogen ratios in inorganic nitrogen oxidation are equal to 3.22 parts (expressed in milligrams per liter) of oxygen per part of ammonia nitrogen oxidized to nitrite nitrogen and 1.11 parts of oxygen per part of nitrite nitrogen oxidized to nitrate nitrogen. These values rather than the stoichiometric ratios should be used in nitrogenous oxygen demand calculations.

Ammonia↗

PubMed1974

Autecological study of the chemoautotroph Nitrobacter by immunofluorescence.

Fluorescent antibodies (FA) prepared for Nitrobacter agilis and N. winogradskyi were highly reactive in homologous staining. Low-level cross-reactions between the two species were removed by adsorption. All 15 pure-culture isolates of Nitrobacter tested reacted strongly with either N. agilis FA or N. winogradskyi FA. All pure-culture isolates from soils were determined to be N. winogradskyi; those from Mammoth Cave sediments and a cattle waste oxidation ditch were N. agilis. No cross-reaction was found in extensive tests that included five isolates of Nitrosomonas europaea and 668 heterotrophic aerobic and anaerobic bacteria isolated from soil, sewage, and cave sites. The FA preparations were used to detect Nitrobacter species in Mammoth Cave sediments, in a cattle waste oxidation ditch, and in surface waters and sediments of a river and to observe that N. winogradskyi can outgrow N. agilis in enrichment culture.

Aerobiosis↗

PubMed1971

Comparison of the morphology and deoxyribonucleic acid composition of 27 strains of nitrifying bacteria.

The gross morphology, fine structure, and per cent guanine plus cytosine (GC) composition of deoxyribonucleic acid of 27 strains of nitrifying bacteria were compared. Based on morphological differences, the ammonia-oxidizing bacteria were separated into four genera. Nitrosomonas species and Nitrosocystis species formed one homogenous group, and Nitrosolobus species and Nitrosospira species formed a second homogenous group in respect to their deoxyribonucleic acid GC compositions. Similarly, the nitrite-oxidizing bacteria were separated into three genera based on their morphology. The members of two of these nitrite-oxidizing genera, Nitrobacter and Nitrococcus, had similar GC compositions, but Nitrospina gracilis had a significantly lower GC composition than the members of the other two genera.

Ammonia↗

PubMed1972

Growth of obligate autotrophic bacteria on glucose in a continuous flow-through apparatus.

Nitrosomonas europaea, Nitrobacter agilis, Thiobacillus denitrificans, T. neapolitanus, and T. thioparus (all obligate autotrophic bacteria) have been grown in dialysis culture, on glucose salts media, in the absence of their specific inorganic energy source. Metabolic products for N. agilis grown on nitrite salts medium were identified as keto acids. Pyruvic acid inhibited this organism at 5 x 10(-5)m. Keto acids were not inhibitory for the thiobacilli grown on thiosulfate medium. However, when T. denitrificans was grown on glucose with dialysis, addition of 5 x 10(-4)m pyruvate inhibited growth. Thus, it appears pyruvate may be inhibitory for other autotrophs, as has been reported for T. thiooxidans.

Ammonia↗

PubMed1995

Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase activity in vitro.

We investigated the effects of bovine serum albumin (BSA) on both the assay and the stability of ammonia-oxidizing activity in cell extracts of Nitrosomonas europaea. Ammonia-dependent O2 uptake activity of freshly prepared extracts did not require BSA. However, a dependence on BSA developed in extracts within a short time. The role of BSA in the assay of ammonia-oxidizing activity apparently is to absorb endogenous free fatty acids which are present in the extracts, because (i) only proteins which bind fatty acids, e.g., BSA or beta-lactoglobulin, supported ammonia-oxidizing activity; (ii) exogenous palmitoleic acid completely inhibited ammonia-dependent O2 uptake activity; (iii) the inhibition caused by palmitoleic acid was reversed only by proteins which bind fatty acids; and (iv) the concentration of endogenous free palmitoleic acid increased during aging of cell extracts. Additionally, the presence of BSA (10 mg/ml) or CuCl2 (500 microM) stabilized ammonia-dependent O2 uptake activity for 2 to 3 days at 4 degrees C. The stabilizing effect of BSA or CuCl2 was apparently due to an inhibition of lipolysis, because both additives inhibited the increase in concentrations of free palmitoleic acid in aging extracts. Other additives which are known to modify lipase activity were also found to stabilize ammonia-oxidizing activity. These additives included HgCl2, lecithin, and phenylmethylsulfonyl fluoride.

Adsorption↗

PubMed2004

Inhibition of membrane-bound methane monooxygenase and ammonia monooxygenase by diphenyliodonium: implications for electron transfer.

Diphenyliodonium (DPI) is known to irreversibly inactivate flavoproteins. We have found that DPI inhibits both membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus and ammonia monooxygenase (AMO) of Nitrosomonas europaea. The effect of DPI on NADH-dependent pMMO activity in vitro is ascribed to inactivation of NDH-2, a flavoprotein which we proposed catalyzes reduction of the quinone pool by NADH. DPI is a potent inhibitor of type 2 NADH:quinone oxidoreductase (NDH-2), with 50% inhibition occurring at approximately 5 micro M. Inhibition of NDH-2 is irreversible and requires NADH. Inhibition of NADH-dependent pMMO activity by DPI in vitro is concomitant with inhibition of NDH-2, consistent with our proposal that NDH-2 mediates reduction of pMMO. Unexpectedly, DPI also inhibits pMMO activity driven by exogenous hydroquinols, but with approximately 100 micro M DPI required to achieve 50% inhibition. Similar concentrations of DPI are required to inhibit formate-, formaldehyde-, and hydroquinol-driven pMMO activities in whole cells. The pMMO activity in DPI-treated cells greatly exceeds the activity of NDH-2 or pMMO in membranes isolated from those cells, suggesting that electron transfer from formate to pMMO in vivo can occur independent of NADH and NDH-2. AMO activity, which is known to be independent of NADH, is affected by DPI in a manner analogous to pMMO in vivo: approximately 100 micro M is required for 50% inhibition regardless of the nature of the reducing agent. DPI does not affect hydroxylamine oxidoreductase activity and does not require AMO turnover to exert its inhibitory effect. Implications of these data for the electron transfer pathway from the quinone pool to pMMO and AMO are discussed.

Biphenyl Compounds↗

PubMed2004

Influence of soil moisture on linear alkylbenzene sulfonate-induced toxicity in ammonia-oxidizing bacteria.

Moisture affects bioavailability and fate of pollutants in soil, but very little is known about moisture-induced effects on pollutant toxicity. We here report on a modifying effect of moisture on degradation of linear alkylbenzene sulfonates (LASs) and on their toxicity towards ammonia-oxidizing bacteria (AOB) in agricultural soil. In soil spiked with two LAS levels (250 or 1,000 mg/kg) and incubated at four different moisture levels (9-100% of water-holding capacity), degradation was strongly affected by both soil moisture and initial LAS concentration, resulting in degradation half-lives ranging from 13 to more than 160 d. Toxicity towards AOB assessed by a novel Nitrosomonas europaea luxAB-reporter assay was correlated to total LAS concentration, indicating that LAS remained bioavailable over time without accumulation of toxic intermediates. Toxicity towards indigenous AOB increased with increasing soil moisture. The results indicate that dry soil conditions inhibit LAS degradation and provide protection against toxicity within the indigenous AOB, thus allowing for a rapid recovery of this population when LAS degradation is resumed and completed after rewetting. We propose that the protection of microbial populations against toxicity in dry soil may be a general phenomenon caused primarily by limited diffusion and thus a low bioavailability of the toxicant.

Alkanesulfonic Acids↗

PubMed2006

The expanded bed biofilter: combined nitrification, solids destruction, and removal of bacteria.

Developed for tertiary nitrification, this biofilter also removed carbonaceous BOD (cBOD) and (SS). Because the biofilter is expanded, it cannot clog, and therefore does not require backflushing; yet, it removed a significant proportion of the influent SS. This unanticipated capability was due to the activities of heterotrophic bacteria, protozoa, and metazoa (nematode and oligochaete worms). The expanded bed is an intensified process, which is based on natural immobilization of microbes to small support particles. Using glassy coke as the support material, an attached layer of microbes develops, forming particulate biofilms having a superficial surface area of 1 800 m2 m(-3)(expandedbed). Autotrophic nitritifiers (Nitrosomonas spp.) were detected in the biofilm using rRNA-based molecular methods and were likely responsible, at least in part, for reducing the ammonia concentration by up to 99% (to 0.1 mg L(-1)), while the other organisms reduced cBOD and SS by up to 56% and 62%, respectively. Furthermore, the influent concentrations of Escherichia coli, coliform and heterotrophic bacteria were reduced by over 80%. It thereby provides a single process solution for combined tertiary nitrification and solids removal. Operating the process to consistently achieve < 0.5mg NH3N L(-1) and at the same time removing a significant fraction of cBOD and SS, it can replace processes such as SAFs or NTFs followed by a sandfilter.

Ammonia↗

PubMed2006

Study on performance of granular ANAMMOX process and characterization of the microbial community in sludge.

Anaerobic Ammonium Oxidation (ANAMMOX) is a novel biological nitrogen removal process, which is regarded as the most economical process at present. In this paper, two lab-scale UASB reactors, one of which was inoculated with the mixture of anaerobic sludge and aerobic sludge, the other with river sediments, were started up, using the inorganic synthetic water containing ammonium and nitrite as influent. After 421 days' and 356 days operation respectively, the ammonium removal efficiencies in two reactors reached 94% and 86% respectively, the total nitrogen volumetric loading rates were 2.5 and 1.6 kgN/m3 x d. ANAMMOX granules were obtained in both reactors; the color of most granules was brown, but some of them were red. Based on the observation and studies on the microstructure of the granules, three kinds of ANAMMOX granular sludge formation mechanisms were proposed: adhering biofilm and disintegrated granular core mechanism, adhering biofilm and inorganic core mechanism and the self-coherence mechanism. For phylogenetic characterization of anaerobic ammonium oxidizers, 16S rDNA approach was performed using Planctomycetales-specific PCR amplification. The dominant anammox bacteria occupied more than 90% of Planctomycetales-specific bacteria, and 27% of all bacteria in reactors. The dominant anammox bacteria distantly related to all currently reported candidate anammox genera. Functional gene of amoA was analyzed to investigate the 'aerobic' ammonium-oxidizing bacteria in beta-Proteobacteria. The 'aerobic' ammonium-oxidizing bacteria were more diverse than anammox bacteria, but most of them clustered in anoxic ammonium-oxidizing Nitrosomonas eutropha/europaea groups. The composition of 'aerobic' ammonium-oxidizing bacteria is only 2% of all of bacteria in reactors.

Ammonia↗

PubMed1999

Water quality management for low temperature marine fishes in space.

Vestibular Function Experiment Unit (VFEU), one of the Spacelab facility flown in Neurolab mission (STS-90) in April, 1998, was to support neurophysiological research using a marine fish, Opsanus tau (oyster toadfish). The functions of the VFEU were primarily a quality management of environmental water during the mission at 14 degrees C and for acquiring physiological signals from implanted micro-electrodes in the otolith nerves as well as the spatial acceleration of the fish. A key element of the life support system was a balanced biological filter containing two types of nitrifying bacteria, Nitrosomonas for ammonia oxidization and Nitrobacter for nitrite oxidization. Although the 16 days mission was successful, two toadfishes died in late phase of the mission. Ammonium concentration in those two life support systems elevated to remarkably high level at the end of the mission whereas the other two indicated very low. This report summarizes the results of the water quality management of the VFEU during the Neurolab mission based on analysis of water samples taken during the flight and those taken prior and just after the flight.

Animals↗

PubMed2001

Implementation, study and calibration of a modified ASM2d for the simulation of SBR processes.

An enhanced process model for SBRs has been developed. Though the basic mechanism largely draws on the Activated Sludge Model n. 2d, its new features are the splitting of the nitrification stage in a two-step process, according to the well known Nitrosomonas-Nitrobacter oxidation sequence, and an improved XPAO dynamics, involved in the anaerobic/aerobic phosphorus removal process. The model was implemented through the DLL technique allowing complied C++ modules to be linked to an ordinary Simulink block diagram. The static sensitivity study revealed that if the parameter vector is partitioned into subsets of biologically related parameters and calibrated separately, the calibration procedure does not present particularly difficult aspects. Trajectory sensitivity showed also to which extent data collection could be optimised in order to improve calibration accuracy. The study of the shape of the error functional generated by parameters couples allows a much more effective calibration strategy.

Bioreactors↗

PubMed2001

Enhancement of nitrifying biofilm formation using selected EPS produced by heterotrophic bacteria.

The possibility of enhancing nitrifying biofilm formation rate with the aid of selected EPS produced by heterotrophic bacteria was investigated. When EPS production characteristics were examined for four kinds of heterotrophs isolated from a domestic wastewater treatment reactor, two strains obtained from biofilms (B1, B2) exhibited a higher polysaccharide production rate than those from suspended flocs (A1, A2). Among EPS components, the concentration of uronic acids gave a good correlation with flocculation ability, which suggests that acidic polysaccharides play a major role in bioaggregate formation. Addition of 1 g/L D-glucuronic acid as an EPS substitute enhanced the homocoagulation rate of autotrophic Nitrosomonas europaea and altered its zeta potential from ñ30.4 mV to +4.3 mV, which indicates a possibility that particular EPS components produced by heterotrophs are utilized as neutralising reagents for nitrifying biofilm formation. Moreover, when heterotrophic isolates with Nitrobacter winogradskyi were cultured in batch with fabric supports, biofilm formed on the substratum. These experimental results suggest the application of selected EPS for enhancing nitrifying biofilm formation.

Bacteria↗