Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Microbial community structure in autotrophic nitrifying granules characterized by experimental and simulation analyses

This study evaluates the community structure in nitrifying granules (average diameter of 1600 μm) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 μm below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 μm, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH₄⁺, NO₂^–, NO₃^– and O₂ concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria–substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Populations of heavy fuel oil-degrading marine microbial community in presence of oil sorbent materials

Aims:  To investigate the feasibility of applying sorbent material X-Oil^® in marine oil spill mitigation and to survey the interactions of oil, bacteria and sorbent.
Methods and Results:  In a series of microcosms, 25 different treatments including nutrient amendment, bioaugmentation with Alcanivorax borkumensis and application of sorbent were tested. Microbial community dynamics were analysed by DNA fingerprinting methods, RISA and DGGE. Results of this study showed that the microbial communities in microcosms with highly active biodegradation were strongly selected in favour of A. borkumensis . Oxygen consumption measurements in microcosms and gas chromatography of oil samples indicated the fast and intense depletion of linear alkanes as well as high oxygen consumption within 1 week followed by consequent slower degradation of branched and polyaromatic hydrocarbons.
Conclusion:  Under given conditions, A. borkumensis was an essential organism for biodegradation, dominating the biofilm microbial community formation and was the reason of emulsification.
Significance and Impact of the Study:  This study strongly emphasizes the pivotal importance of A. borkumensis as an essential organism in the initial steps of marine hydrocarbon degradation. Interaction with the sorbent material X-Oil^® proved to be neutral to beneficial for biodegradation and also promoted the growth of yet unknown micro-organisms.     

Time-course of inorganic nitrogen uptake and incorporation by natural populations of freshwater phytoplankton

SUMMARY. 1. Time-course measurements of NH₄⁺ and NO₃⁻uptake were made on the natural phytoplankton populations in a eutrophic lake at a time when these nutrients were at their lowest annual concentration.
2. Both NH₄⁺ and NO₃⁻ uptake was increased at least five-fold during the first 5 min of incubation following near saturating pulses of these nutrients.
3. Elevated uptake was also observed following low level (∼2μg N 1⁻¹) pulses of NH₄⁺ and NO₃⁻, but substrate depletion during the first hour of incubation may have been partially responsible for this apparent enhancement.
4. Incorporation of ^I5N into TCA-insoluble material (protein) following the saturating NH₄⁺ pulse was increased less than total cellular ¹⁵N uptake, whereas no elevation of ¹⁵N incorporation into protein was observed following a saturating NO₃⁻pulse.
5. The percentage of ^I5N incorporated into protein, with respect to total cellular uptake, was ∼32% and ∼12% for NH₄⁺ and NO₃⁻, respectively, following 5 h of incubation.     

Growth, photosynthesis, and respiration of Chlorella sorokiniana after N-starvation. Interactions between light, CO2 and NH4+ supply

N-sufficient cells of Chlorella sorokiniana Shihira and Krauss, strain 211/8k, absorbed NH₄⁺ under light plus CO₂ conditions, when growth occurred, but not in darkness or in the absence of CO₂, when growth was inhibited. N-sufficient cells subjected to conditions of N-starvation for a 24-h period showed a marked loss of photosynthetic activity. Upon supply of NH₄⁺, N-starved cells sufflated with CO₂ air exhibited a time-dependent recovery of photosynthetic activity, both when suspended in light and in darkness. By contrast, growth only occurred in cells suspended in light. N-starved cells absorbed NH₄⁺ in darkness, but at a lower rate than in light. All of these data suggest that dark NH₄⁺ uptake is driven by N assimilation to recover from N-starvation and that the light-dependent NH₄⁺ uptake is driven by growth, being then influenced by conditions that affect recovery or growth. Unlike CO₂ conditions, in a CO₂-free atmosphere, absorption of NH₄⁺ by N-starved cells occurred at a higher rate in darkness than in light. Accordingly, resumption of photosynthetic potential after NH₄⁺ supply occurred in darkened cells, but not in illuminated cells. Respiratory activity of N-starved cells was enhanced up to 3-fold by NH₄⁺ and 2-fold by methylammonium, with different patterns, suggesting that respiratory enzymes were affected by N-metabolism, especially through short-term control mechanisms triggered by the expenditure of metabolic energy involved in N-metabolism.     

An alternative explanation for the post-disturbance NO3– flush in some forest ecosystems

The appearance of soil NO₃^– after forest disturbance is commonly ascribed to a higher availability of NH₄⁺ to autotrophic nitrifiers, or to a reduction in available-C resulting in lower microbial assimilation of NO₃^–. Alternatively, it has been proposed that increasing NH₄⁺ pools following disturbance could increase net nitrification by reducing microbial assimilation of NO₃^–. Forest floor material was collected from shelterwood harvest plots which displayed both low available-C and low NH₄⁺ pools, and where previous experiments had suggested the prevalence of heterotrophic nitrification. Subsamples were amended with incremental rates of glucose-C or NH₄⁺, and gross NO₃^– transformation rates were measured by isotope dilution. Glucose-C additions had little effect on the net difference between gross NO₃^– production and consumption rates. On the other hand, NH₄⁺ additions caused gross NO₃^– consumption processes to decrease sharply, while gross NO₃^– production processes remained constant. The results suggest that NH₄⁺ can have an immediate positive effect on net nitrification rates by suppressing NO₃^– assimilation and uptake systems.     

Response of nitrogen metabolism to boron toxicity in tomato plants

Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 m m and 2.0 m m B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR_L), concentration of B, nitrate (NO₃⁻), ammonium (NH₄⁺), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR_L, organic N, soluble proteins, and NR and NiR activities. The lowest NO₃⁻ and NH₄⁺ concentration in leaves was recorded when plants were supplied with 2.0 m m B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO₃⁻ reduction and increases NH₄⁺ assimilation in tomato plants.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Effect of darkness and CO2 starvation on NH4+ and NO3− assimilation in the unicellular alga Cyanidium caldarium

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO₂, assimilated NH₄⁺ at high rates both in the light and in the dark. The assimilation of NO₃⁻, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO₂-free air, NH₄⁺ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO₂ completely restored the capacity of the alga to assimilate NH₄⁺. NO₃⁻ assimilation, by contrast, was 33% higher in the absence of CO₂ and was linear with time. It is suggested that NO₃⁻ and NH₄⁺ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.     

Patterns of ammonium absorption and acetylene reduction during soybean developmental growth

Nodulated and unnodulated soybean plants ( Glycine max (L.) Merr. cv. Amsoy 71) were grown in nutrient solution either lacking or containing N. Nodulated plants, dependent on N₂ fixation, exhibited a generalized N-stress and were less vigorous than unnodulated plants dependent on inorganic N assimilation.
Starting at preflowering throughout mid pod-filling, NH₄⁺ absorption, expressed on the basis of root dry weight, was determined for intact nodulated and unnodulated plants in short-term kinetic experiments. Depletion of NH₄⁺ was measured from the liquid phase of a mist chamber. Maximum NH₄⁺ absorption occurred for both nodulated and unnodulated plants during vegetative growth. A pattern of progressive decrease in NH₄⁺ absorption was similar in nodulated and unnodulated plants, however. NH₄⁺ absorption was consistently greater in unnodulated plants. Simultaneous measurements of C₂H₂ reduction from the gas phase of the mist chamber revealed and 41-day-old plants, corresponding to late flowering and early pod-filling.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Reactor performance and microbial community of an EGSB reactor operated at 20 and 15°C

Aims:  To investigate the effects of low temperatures on the performance and microbial community of anaerobic wastewater treatment.
Methods and Results:  An expanded granular sludge bed (EGSB) reactor was employed to treat synthetic brewery wastewater at 20 and 15°C. Reactor performance was represented by chemical oxygen demand (COD) removal efficiency, while the microbial community was analysed using denaturing gradient gel electrophoresis (DGGE) and clone technology. When the hydraulic retention time (HRT) was maintained at 18 h, COD removal efficiencies above 85% were obtained at both 20 and 15°C, with influent COD concentrations up to 7300 and 4100 mg l⁻¹, respectively. At 15°C, the COD removal efficiency was more easily manipulated by increasing the influent COD concentration. DGGE and clone results for both temperatures revealed that Methanosaeta and Methanobacterium were two dominant methanogens, and that the majority of the eubacterial clones were represented by Firmicutes . When the temperature decreased from 20 to 15°C, both archaeal and eubacterial communities had higher diversity, and the proportion of Methanosaeta (acetate-utilizing methanogens) decreased markedly from 60·0% to 49·3%, together with an increase in proportions of hydrogen-utilizing methanogens (especially Methanospirillum ).
Conclusions:  The feasibility of psychrophilic anaerobic treatment of low and medium strength organic wastewaters was demonstrated, although lower temperature could significantly affect both reactor performance and the anaerobic microbial community.
Significance and Impact of the Study:  The findings enrich the theory involving the microbial community and the application of anaerobic treatment in a psychrophilic environment.     

Mycobiont-cyanobiont interactions during dark nitrogen fixation by the lichen Peltigera aphthosa

Acetylene reduction (nitrogenase activity) by excised cephalodia of Peltigera aphthosa Willd. slowly declined on transfer of the cephalodia from light to darkness. The decline was more rapid in the absence of CO₂ or when phosphoenolpyruvate carboxylase activity was inhibited by adding maleic acid or malonic acid. When glutamine synthetase (GS) activity was totally inhibited by adding l -methionine- dl -sulphoximine (MSX) the decline in nitrogenase activity in the absence of CO₂ still occurred. However, this loss of activity did not occur when the mycobiont was disrupted using digitonin (0.01 % w/v) and the fixed NH₄⁺ was released into the medium. The data suggest that dark CO₂ fixation by the fungus supplies carbon skeletons which remove newly fixed NH₄⁺ produced by the cyanobacterium. When such carbon skeletons are not available MH₄⁺ accumulates and inhibits nitrogenase activity even in the absence of GS activity. It is probable that NH₄⁺ and a product of GS exert independent inhibitory effects on nitrogenase activity.     

Changes in abundance, composition and controls within the plankton of a fertilised arctic lake

1. An oligotrophic arctic lake was fertilised with inorganic nitrogen and phosphorus as (NH₄)₂ NO₃ and H₃PO₄ for five summers. The loading rate was 1.7–2.5 mmol N m^–2 day^–1 and 0.136–0.20 mmol P m^–2 day^–1 which is two to three times the annual loading of lakes in the area. The heterotrophic microzooplankton community was enumerated during the experiment as well as 1 year pre- and post-treatment.
2. The structure of the microplankton community changed from a nutrient limited system, dominated by oligotrich protozoans and small-particle feeding rotifers, to a system dominated by a succession of peritrich protozoans and predatory rotifers. These peritrich protozoans and predatory rotifers were not present prior to fertilisation and never constituted more than a small fraction of the biomass in other lakes at the research site. The average biomass of the rotifers and protozoans was more than seven and a half times larger by the end of fertilisation than it was initially.
3. Because of the increases in numbers of individuals in these new taxa, the structure of the microbial food web changed. When fertilisation stopped, most parameters returned to prefertilisation levels within 1 year.     

Analysis and succession of nitrifying bacteria community structure in sequencing biofilm batch reactor

To reveal the succession procedure of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) community structure in sequencing biofilm batch reactor (SBBR), the molecular biological techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and real-time PCR were applied. DGGE showed that the structural diversity of the bacterial community increased during the biofilm formation period, and some kinds of populations had been highly preponderant consistently. The results of cloning and sequencing revealed that Nitrosomonas was the dominant species. The real-time PCR analysis indicated that the amount of the AOB increased significantly after the cultivation period, and the NOB gradually decreased. The AOB content on the 25th day was 17 times that of the 6th day. It also showed the biofilm formed successfully with accumulating nitrite and prepared to achieve the achievement of simultaneous nitrification and denitrification in SBBR. Furthermore, the ammonia-oxidizing rate was in correspondence with the NH₄ ⁺-N removal efficiency.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.     

A reaction diffusion model of C-N-S-O species in some arctic sediments

Abstract A reaction diffusion model was used to simulate the mineralization processes in an Arctic sediment. The simulation and the actual sediment were compared in relation to profiles of O₂, NO₃ and NH₄⁺. The site of particulate organic matter (POM) degradation was the single most important factor in fitting the simulation profiles to those of the sediment. It was deduced that most POM degradation occurred close to the sediment surface. When a reasonably good simulation had been obtained, the sensitivity of the model to changes in other parameters was investigated. Increases in POM degradation in the upper sediment resulted in increases in concentration of NH₄⁺ and NO₃⁻, but further increases in POM degradation created anoxic conditions below 3 mm, resulting in decreases in NO₃⁻ concentrations. The model was relatively intensive to changes in POM degradation in the lower sediment layers; increases led to more anoxic conditions and to less NO₃⁻. Increases in the C/N ratio of the POM in the lower sediment layers had little effect; increases in C/N in the upper layers led to a decrease in NH₄⁺ and NO₃⁻. The model was sensitive to changes in the first order rate constant for nitrification, but not for denitrification. Decreases in the K _m for O₂ of the nitrifying bacteria had no effect on the profiles.     

Isolation and characterization of a metronidazole-resistant (Mtn-R) mutant strain of Nostoc muscorum affected in ammonium regulation of heterocyst formation and uptake hydrogenase activity

Abstract The metronidazole-resistant ( Mtn-R ) mutant strain of N. muscorum produced drug-resistant NADPH: ferredoxin (Fd) oxidoreductase and showed derepression of heterocyst formation and uptake hydrogenase activity in NH₄⁺-medium. The observation of NH₄⁺-repression in regulation of nitrogenase activity alone in the mutant strain suggests, that heterocyst formation and nitrogenase activity are regulated by two separate NH₄⁺-repression control systems, one specific for heterocyst and uptake hydrogenase and the other for nitrogenase. The partial drug-resistant NADPH: Fd oxidoreductase enzymatic activity seems to be the reason for drug-resistant growth of the cyanobacterium in N₂-medium and NH₄⁺-medium.