The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Methylamine,an inhibitor of ammonium oxidation and chemoautotrophic growth in the marine nitrifying bacteriumNitrosococcus oceanus

Methylamine (CH₃NH ₃ ⁺ ) appeared to utilize the same transport mechanism as ammonium (NH ₄ ⁺ ) to enter cells ofNitrosococcus oceanus. Methylamine uptake did not show clear evidence of saturable kinetics and was not fully saturated at 20 mM. Assimilated CH₃NH ₃ ⁺ was not incorporated into macromolecular constituents, but inhibited rates of nitrification, chemoautotrophic CO₂ fixation and growth. The degree of inhibition was dependent on the relative concentrations of NH ₄ ⁺ and CH₃NH ₃ ⁺ . Rates of CO₂ fixation and growth were inhibited four times more than the rate of nitrification.     

Phosphate requirements of the nitrifying bacteria

The influence of the phosphate concentration on the specific growth rate and the duration of lag has been studied inNitrobacter winogradskyi andNitrosomonas europaea.The optimum phosphate concentration range for the specific growth rate was 10 to 30mm forNitrobacter and 10 to 100mm forNitrosomonas. In this range the lag was least. Depletion of the cell-P does not affect the relation between specific growth rate and phosphate concentration while the lag seems to increase as cell-P depletion proceeds.     

The survival of nitrifying bacteria in the soil

Summary Nitrification as measured by the survival of nitrifying bacteria, was studied in 25 different soils from Argentine Patagonia, maintained in sterilized glass flasks in darkness for 5 years.Survival of nitrifying bacteria was related to environmental factors and to the level of mineral elements in the soils.The temperatures to which soils were submitted and the joint action of Ca and K in adequate quantities, were shown to be more important factors governing the survival of the nitrifying bacteria.P, Mg, and Cu did not influence in the initial number nor the survival of nitrifying bacteria nor did the content or organic matter and organic N and pH of the soil samples.     

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH(4)(+) as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (alpha-Proteobacteria), 23%; gamma-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. alpha- and gamma-Proteobacteria dominated the utilization of [(14)C]acetic acid and (14)C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1-(14)C]glucosamine (NAG). The GNSB accounted for 9% of the (14)C-amino acid-consuming bacteria and 27% of the [(14)C]NAG-consuming bacteria but did not utilize [(14)C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.     

Determination of the decay rate of nitrifying bacteria

The growth and decay of nitrifying organisms determines the amount of nitrifying bacteria in activated sludge systems. The growth rate of the nitrifying organisms is reasonable, well defined, and studied, while the decay rate is still rather uncertain. Experiments in previous studies were over periods up to 14 days and obtained results were not confirmed. Contradicting decay rates of nitrifiers in different bacterial communities is reported. No differentiation between ammonia and nitrite oxidizers was made. Therefore, in this studyper day the decay rate of the nitrifying organisms was studied. The starvation condition (aerobic, anoxic, or anaerobic), temperature, type of bacterial community, and the presence of higher organisms are the main aspects that were investigated. A simple and reliable method (adapted from previous studies) for determining the decay rate of nitrifying organisms under different starvation conditions and different temperatures was developed. The test procedure has been used for determining the decay rate of ammonium and nitrite oxidizing bacteria in an enriched nitrifying culture and in activated sludge. The test was successfully applied at starvation periods up to 30 days. The decay rate of the enriched culture of nitrifiers was very low compared to values for nitrifiers in activated sludge. The decay rate of the nitrifiers in activated sludge was found to be to 0.2, 0.1, and 0.06 per day for aerobic, anoxic, and anaerobic conditions, respectively. The decay rate of ammonia oxidizers and nitrite oxidizers was the same at the corresponding conditions.     

Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms

The cross-feeding of microbial products derived from 14C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [14C]bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of 14C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of 14C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized 14C-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of 14C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Transformations of ammonia and the environmental impact of nitrifying bacteria

In the sequence of events leading from ammonia to N₂ during the process of biotransformation of inorganic nitrogen compounds, the weakest link, with respect to our knowledge and understanding of the organisms involved, is nitrification. In particular, this is true for the oxidation of ammonia to nitrite. The enzymes have not been thoroughly studied, and the enzymatic mechanisms have not been identified. Almost any biochemical and physiological aspect studied proved to be controversial, and major ecological questions still remain unanswered. Unless the structure and function of the various components of the process are worked out, progress in developing means for controlling nitrification will depend mainly on laborious trial and error and not on knowledgeable manipulation of this group of bacteria.Abbreviations AMO ammonia monooxygenase - HAO hydroxylamine oxidoreductase - MPN most probable number - TCE trichloroethylene     

Characterization of nitrifying bacteria immobilized in calcium alginate

Activated sludge has been fed with a medium containing ammonium ions as the sole nitrogen source. Biomass collected from this continuous culture was immobilized in calcium alginate. The influence of pH, temperature, and the size and cell load of the biocatalyst beads on the nitrifying activity was determined, as well as the storage and operational stability of the system. The results are compared with those obtained with Nitrosomonas europaea. It has been concluded that the mixed culture is more difficult to work with than the pure strain and that the reproducibility of the results is lower. The trends found, however, were largely similar, except for the operational stability which was poorer in the case of the immobilized mixed culture.     

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea -like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1–2 orders of magnitude lower than those of N. europaea . Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic–anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.     

Visualization of pseudogenes in intracellular bacteria reveals the different tracks to gene destruction

Background  

Pseudogenes reveal ancestral gene functions. Some obligate intracellular bacteria, such as Mycobacterium leprae and Rickettsia spp., carry substantial fractions of pseudogenes. Until recently, horizontal gene transfers were considered to be rare events in obligate host-associated bacteria.     

Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification

A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification. A mixture of NaHCO₃ and CH₃COONa was selected as the appropriate carbon source for cell growth and nitrogen removal, the concentrations of carbon and nitrogen sources were also examined. Ammonia could be oxidized aerobically to nitrite by the mixed culture, and the intermediate nitrite was then reduced to dinitrogen gas. No nitrite was detected during the process. 0.212 g of ammonia/l could be removed in 30 h and nitrate could not be utilized aerobically by the mixed culture. Nitrite could be degraded aerobically as well as anaerobically. Very little ammonia was degraded anaerobically, but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42 h.     

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.     

Steady state and transient growth of autotrophic nitrifying bacteria

Growth of the autotrophic nitrifying bacteria Nitrosomonas europaea and Nitrobacter sp. was studied in continuous culture. Steady state growth kinetics of both organisms conformed with that predicted by chemostat theory, modified to account for maintenance energy requirement. Steady state data were used to calculate the maximum specific growth rate, the saturation constant for growth, the true growth yield and the maintenance coefficient. Transient growth was studied by imposing step changes in dilution rate. Step increases resulted in overshoots and oscillations in substrate concentration before establishment of a new steady state while step decreases in dilution rate were followed by monotonic changes in substrate concentration. The size of overshoots in substrate concentration following step increases in dilution rate was dependent on both the magnitude of the increase and of the dilution rate prior to the change.