Silica-immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 degrade high concentrations of phenol

Aims: To immobilize Methylobacterium sp. NP3 and Acinetobacter sp. PK1 to silica and determine the ability of the immobilized bacteria to degrade high concentrations of phenol. Methods and Results: The phenol degradation activity of suspended and immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 bacteria was investigated in batch experiments with various concentrations of phenol. The bacterial cells were immobilized by attachment to or encapsulation in silica. The encapsulated bacteria had the highest phenol degradation rate, especially at initial phenol concentrations between 7500 and 10 000 mg l^?1. Additionally, the immobilized cells could continuously degrade phenol for up to 55 days. Conclusions: The encapsulation of a mixed culture of Methylobacterium sp. NP3 and Acinetobacter sp. PK1 is an effective and easy technique that can be used to improve bacterial stability and phenol degradation. Significance and Impact of the Study: Wastewater from various industries contains high concentrations of phenol, which can cause wastewater treatment failure. Silica‐immobilized bacteria could be applied in bioreactors to initially remove the phenol, thereby preventing phenol shock loads to the wastewater treatment system.     

Growth at Low Ammonium Concentrations and Starvation Response as Potential Factors Involved in Niche Differentiation among Ammonia-Oxidizing Bacteria

In nature, ammonia-oxidizing bacteria have to compete with heterotrophic bacteria and plants for limiting amounts of ammonium. Previous laboratory experiments conducted with Nitrosomonas europaea suggested that ammonia-oxidizing bacteria are weak competitors for ammonium. To obtain a better insight into possible methods of niche differentiation among ammonia-oxidizing bacteria, we carried out a growth experiment at low ammonium concentrations with N. europaea and the ammonia oxidizer G5-7, a close relative of Nitrosomonas oligotropha belonging to Nitrosomonas cluster 6a, enriched from a freshwater sediment. Additionally, we compared the starvation behavior of the newly enriched ammonia oxidizer G5-7 to that of N. europaea. The growth experiment at low ammonium concentrations showed that strain G5-7 was able to outcompete N. europaea at growth-limiting substrate concentrations of about 10 μM ammonium, suggesting better growth abilities of the ammonia oxidizer G5-7 at low ammonium concentrations. However, N. europaea displayed a more favorable starvation response. After 1 to 10 weeks of ammonium deprivation, N. europaea became almost immediately active after the addition of fresh ammonium and converted the added ammonium within 48 to 96 h. In contrast, the regeneration time of the ammonia oxidizer G5-7 increased with increasing starvation time. Taken together, these results provide insight into possible mechanisms of niche differentiation for the ammonia-oxidizing bacteria studied. The Nitrosomonas cluster 6a member, G5-7, is able to grow at ammonium concentrations at which the growth of N. europaea, belonging to Nitrosomonas cluster 7, has already ceased, providing an advantage in habitats with continuously low ammonium concentrations. On the other hand, the ability of N. europaea to become active again after longer periods of starvation for ammonium may allow better exploitation of irregular pulses of ammonium in the environment.     

High cell density cultivation of the chemolithoautotrophic bacterium <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Nitrosomonas europaea is a chemolithoautotrophic nitrifier, a gram-negative bacterium that can obtain all energy required for growth from the oxidation of ammonia to nitrite, and this may be beneficial for various biotechnological and environmental applications. However, compared to other bacteria, growth of ammonia oxidizing bacteria is very slow. A prerequisite to produce high cell density N. europaea cultures is to minimize the concentrations of inhibitory metabolic by-products. During growth on ammonia nitrite accumulates, as a consequence, N. europaea cannot grow to high cell concentrations under conventional batch conditions. Here, we show that single-vessel dialysis membrane bioreactors can be used to obtain substantially increased N. europaea biomasses and substantially reduced nitrite levels in media initially containing high amounts of the substrate. Dialysis membrane bioreactor fermentations were run in batch as well as in continuous mode. Growth was monitored with cell concentration determinations, by assessing dry cell mass and by monitoring ammonium consumption as well as nitrite formation. In addition, metabolic activity was probed with in vivo acridine orange staining. Under continuous substrate feed, the maximal cell concentration (2.79?×?10¹²/L) and maximal dry cell mass (0.895 g/L) achieved more than doubled the highest values reported for N. europaea cultivations to date.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

Significance of gaseous NO for ammonia oxidation by Nitrosomonas eutropha

Nitrification by the obligately lithoautotrophic ammonia oxidizer Nitrosomonas eutropha was significantly inhibited when nitric oxide was removed from the culture medium by means of intensive aeration and turbulence. Nearly complete recovery of ammonia oxidation could be achieved by adding 100 ppm NO to the supplied air. Inhibition of ammonia oxidation occurred also upon addition of the NO binding agens 2,3-Dimercapto-1-propane-sulfonic acid (DMPS). Recovery of ammonia oxidation occurred within 3 h in the presence of 100 ppm NO and within 76 h in the absence of externally added NO. In co-cultures of N. eutropha and the NO detoxifying bacterium Pseudomonas PS88, hardly any nitrification was detectable and release of NO was extremely low when the heterotroph was provided with an organic substrate. When cells of Pseudomonas PS88 were added to a mixotrophically nitrifying culture of N. eutropha the release of NO decreased drastically upon the addition and ammonia oxidation ceased. These results confirm for the first time the significance of NO in the course of ammonia oxidation by N. eutropha.     

Nanosilver inhibits nitrification and reduces ammonia‐oxidising bacterial but not archaeal amoA gene abundance in estuarine sediments

Silver nanoparticles (AgNPs) enter estuaries via wastewater treatment effluents, where they can inhibit microorganisms, because of their antimicrobial properties. Ammonia‐oxidising bacteria (AOB) and archaea (AOA) are involved in the first step of nitrification and are important to ecosystem function, especially where effluent discharge results in high nitrogen inputs. Here, we investigated the effect of a pulse addition of AgNPs on AOB and AOA ammonia monooxygenase (amoA) gene abundances and benthic nitrification potential rates (NPR) in low‐salinity and mesohaline estuarine sediments. Whilst exposure to 0.5 mg L^?1 AgNPs had no significant effect on amoA gene abundances or NPR, 50 mg L^?1 AgNPs significantly decreased AOB amoA gene abundance (up to 76% over 14 days), and significantly decreased NPR by 20‐fold in low‐salinity sediments and by twofold in mesohaline sediments, after one day. AgNP behaviour differed between sites, whereby greater aggregation occurred in mesohaline waters (possibly due to higher salinity), which may have reduced toxicity. In conclusion, AgNPs have the potential to reduce ammonia oxidation in estuarine sediments, particularly where AgNPs accumulate over time and reach high concentrations. This could lead to long‐term risks to nitrification, especially in polyhaline estuaries where ammonia‐oxidation is largely driven by AOB.     

A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O₂ uptake and NO₂⁻ production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA‐based stable isotope probing (SIP) and high‐throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea‐amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water‐amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time‐course incubations indicated that archaeal amoA genes were increasingly labelled by ¹³CO₂ in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the ¹³C‐DNA, and acetylene inhibition suggests that autotrophic growth of urease‐containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a‐associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Nitrogen-removal bioreactor capable of simultaneous nitrification and denitrification for application to industrial wastewater treatment

A bioreactor system with 30 packed gel envelopes was installed in a thermal power plant for the removal of nitrogen from ammonia-containing desulfurization wastewater. Each envelope consisted of double-sided plate gels containing Nitrosomonas europaea and Paracoccus denitrificans cells with an internal space in between for injecting an electron donor. The envelope can remove ammonia from wastewater in a single step. When the wastewater was continuously treated with the bioreactor system, it removed 95.0% of the total nitrogen in the inlet, and the total nitrogen concentration in the outlet was below 9.0 mg L⁻¹. The maximum nitrogen removal rate was 6.0 g day⁻¹ per square meter of the gel area. The maximum utilization efficiency of the injected ethanol for denitrification was 98.4%, and the total organic carbon concentration in the outflow was maintained at a low level. Since the bioreactor system could use the electron donor effectively, it was not necessary to use an additional aerobic tank to remove the electron donor and a settling tank to segregate the surplus sludge containing bacteria from wastewater. Our concept of using packed gel envelopes would be highly effective for constructing a simple and efficient nitrogen removal system capable of simultaneous nitrification and denitrification.     

Enzyme immobilized reactor design for ammonia removal from waste water

Removal of nitrogen compound from waste, water is essential and often accomplished by biological process. To prevent washout and to develop an efficient bioreactor, immobilization of suitable microorganisms could be sensible approach. Strains and permeabilized cells encapsulated in cellulose nitrate microcapsules and immobilized on polystyrene, films were prepared by the method described in the previous study. In the wastewater, treatment system, nitrification of ammonia component is generally known as rate controlling step. To enhance the rate of nitrification, firstly nitrifying strainsNitrosomonas europaea (IFO 14298), are permeabilized chemically, and immobilized on polystyrene, films and secondly oxidation rates of strain system and permeabilized strain system are compared in the same condition. With 30 minute permeabilized cells, it took about 25 hours to oxidize 70% of ammonia in the solution, while it took about 40 hours to treat same amount of ammonia with untreated cells. All the immobilization procedures did not harm to the enzyme activity and no mass transfer resistance through the capsule wall was shown. In the durability test of immobilized system, the system showed considerable activity for the repeated operation for 90 days. With these results, the system developed in this study showed the possibility to be used in the actual waste water treatment system.     

Activity of Nitrifying Biofilms Constructed on Low-Density Polyester Enhances Bioremediation of a Coastal Wastewater Effluent

Summary Nitrifying biofilms were constructed on low density polyester Dacron for the bioremediation of nitrogen from wastewater effluent of a municipal treatment plant. Dacron disks were inoculated with wastewater sludge enriched for 15 days for either ammonia- or nitrite-oxidizing bacteria (AOB or NOB, respectively) and packed into glass bioreactors. Wastewater effluent containing high levels of ammonia, nitrite, and phosphate was collected and fed to inoculated and uninoculated bioreactors. Both inoculated bioreactors showed stable nitrification efficiencies, removing 96 and 76% of the ammonia and 12 and 35% of the nitrite for AOB- and NOB-inoculated bioreactors, respectively. Efficiencies of phosphate removal were similar in both inoculated and uninoculated bioreactors, indicating that nitrifiers were not required for this process. AOB-inoculated bioreactors accumulated nitrite mid-way through the experiment and had low rates of conversion to nitrate, suggesting slow nitrite oxidizer growth. DGGE and sequence analysis of AOB 16S rRNA genes showed enrichment of Nitrosomonas spp. in both inoculated bioreactors, and a dominance of Nitrosospira spp. in non-inoculated bioreactors. This study describes an inexpensive and efficient technology for removing ammonia and nitrite from wastewater effluents of municipal treatment plants before its release to the environment.     

Efficacy of Acinetobacter sp. B9 for simultaneous removal of phenol and hexavalent chromium from co-contaminated system

The present study shows the feasibility of a newly isolated strain Acinetobacter sp. B9 for concurrent removal of phenol and Cr (VI) from wastewater. The experiments were conducted in a batch reactor under aerobic conditions. Initially, when mineral salt solution was used as the culture medium, the strain was found to utilize phenol as sole carbon and energy source while no Cr (VI) removal was observed. However, the addition of glucose as co-carbon source resulted in the removal of both toxicants. This co-removal efficiency of the strain was further improved with nutrient-rich media (NB). Optimum co-removal was determined at 188 mg L^?1 of phenol and 3.5 mg L^?1 of Cr (VI) concentrations at pH 7.0. Strain B9 followed the orthometabolic pathway for phenol degradation. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) studies showed sorption of chromium as one of the major mechanisms for Cr (VI) removal by B9 cells. Acinetobacter sp. B9 was later on checked for bioremediation of real tannery wastewater. After 96 h of batch treatment of tannery effluent containing an initial 47 mg L^?1 phenol and 16 mg L^?1 Cr (VI), complete removal of phenol and 87 % reduction of Cr (VI) were attained, showing high efficiency of the bacterial strain for potential application in industrial pollution control.     

Kinetics of ammonia oxidation by a marine nitrifying bacterium: Methane as a substrate analogue

In pure culture, the marine ammonia oxidizer,Nitrosococcus oceanus, exhibits normal Michaelis Menten kinetics with respect to its primary substrate, ammonia.N. oceanus also exhibits a kinetic response to methane. In the absence of methane, oxidation of ammonia is first order with respect to ammonia concentration under atmospheric oxygen concentrations at seawater pH. In the presence of methane, ammonia oxidation is inhibited, and the amount of inhibition is related to the relative concentrations of methane and ammonia. Using semicontinuous batch cultures as a source of organisms for short-term kinetic experiments, I investigated the relationship between ammonia and methane oxidation inN. oceanus by varying the absolute and relative concentration of both substrates. Methane appeared to act as a substrate analogue, and its effect on ammonia oxidation was modeled as a permutation of competitive inhibition involving a cooperative enzyme system. Methane was oxidized byN. oceanus, even in the absence of measurable ammonia oxidation, but the process was inhibited at increasing methane concentrations. Of the two product pools analyzed, an average of 37% of methane oxidized was detected in particulate (cell) material and the remainder was detected in¹⁴CO₂. The contribution of methane to total carbon assimilation varied with the ratio [CH₄]/[NH₃] and may be significant under substrate concentrations typical of a dilute aquatic environment.     

Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen

Ammonia oxidation, the first step in nitrification, is performed by autotrophic bacteria and thaumarchaea, whose relative contributions vary in different soils. Distinctive environmental niches for the two groups have not been identified, but evidence from previous studies suggests that activity of thaumarchaea, unlike that of bacterial ammonia oxidizers, is unaffected by addition of inorganic N fertilizer and that they preferentially utilize ammonia generated from the mineralization of organic N. This hypothesis was tested by determining the influence of both inorganic and organic N sources on nitrification rate and ammonia oxidizer growth and community structure in microcosms containing acidic, forest soil in which ammonia oxidation was dominated by thaumarchaea. Nitrification rate was unaffected by the incubation of soil with inorganic ammonium but was significantly stimulated by the addition of organic N. Oxidation of ammonia generated from native soil organic matter or added organic N, but not added inorganic N, was accompanied by increases in abundance of the thaumarchaeal amoA gene, a functional gene for ammonia oxidation, but changes in community structure were not observed. Bacterial amoA genes could not be detected. Ammonia oxidation was completely inhibited by 0.01% acetylene in all treatments, indicating ammonia monooxygenase-dependent activity. The findings have implications for current models of soil nitrification and for nitrification control strategies to minimize fertilizer loss and nitrous oxide production.     

Simplified Modeling of Simultaneous Reaction Kinetics of Carbon Oxidation and Nitrification in Biofilm Processes

Batch experiments with varying initial substrate concentrations and biomass volumes were performed in a three‐phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the simultaneous carbon oxidation and nitrification in the biofilm process. A simplified mass balance equation for the biofilm was proposed and five different kinetic rate equations were used to match the actual data. The kinetic parameters were obtained by nonlinear regression analysis on a set of two differential equations representing the simultaneous carbon oxidation and nitrification. The competitive inhibition model incorporating the effects of total organic carbon (TOC) concentrations on nitrification rates was the best‐suited model based on the average r². In this model, oxygen concentration and its affinity constants were not included. Instead, it was assumed that the rate of carbon oxidation is independent of the NH₄⁺‐N, while nitrification is affected by TOC. The number of parameters was successfully minimized without reducing its ability to accurately predict the bulk concentration time course, which would reduce computational complexity and possibly enhance the availability for an actual wastewater treatment process.