Microbial nitrogen removal of ammonia wastewater in poly (butylenes succinate)-based constructed wetland: effect of dissolved oxygen

Constructed wetland (CW) is popular in wastewater treatment for its prominent advantage of low construction and operation cost. However, the nitrogen removal in conventional CW is usually limited by the low dissolved oxygen (DO) and insufficient electron donor. This paper investigated the nitrogen removal performance and mechanisms in the poly (butylenes succinate)-based CW (PBS-CW) while treating ammonia wastewater under different DO levels. The average DO contents in limited-aeration and full-aeration phases were 1.68 mg L⁻¹ and 5.71 mg L⁻¹, respectively. Results indicated that, with the ammonia nitrogen loading rate of 25 g N m⁻³ day⁻¹, total nitrogen removal ratios in the PBS-CW under the limited-aeration and full-aeration phases were 72% and 99%, respectively. Combined analyses revealed that simultaneous nitrification and denitrification (SND) via nitrite/nitrate were the main microbial nitrogen removal pathways in the aeration phase of the PBS-CW (> 89%). The microbial carrier of biodegradable material was believed to play a significant role in prompting SND performance while dealing with low C/N wastewater. Due to the coexistence of micro-anaerobic zone and carbon supply inside the coated biofilm, the high DO level in the PBS-CW increased the abundance of the nitrifying bacteria (amoA and nxrA), denitrifying bacteria (narG, nirK, nirS, and nosZ), and even anammox bacteria (anammox 16s rRNA). These features are beneficial to many microbial processes which require the simultaneous aerobic, anoxic, and anaerobic environment.

     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (<Emphasis Type="Italic">cnorB</Emphasis>) gene abundance and mRNA levels in <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis> inoculated into anoxic soil

The effect of glucose addition (0 and 500 μg C g⁻¹ soil) and nitrate (NO₃) addition (0, 10, 50 and 500 μg NO₃–N g⁻¹ soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB _p (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO₃ addition treatments. Without glucose addition, cnorB _p mRNA levels were higher when 500 μg NO₃–N g⁻¹ soil was added compared with other NO₃ additions. In treatments with glucose added, addition of 50 μg NO₃–N g⁻¹ soil resulted in higher cnorB _p mRNA levels than soil without NO₃ but was not different from the 10 and 500 μg NO₃–N g⁻¹ treatments. cnorB _p abundance in soils without glucose addition was significantly higher in soils with 500 μg NO₃–N g⁻¹ soil compared to lower N-treated soils. Conversely, addition of 500 μg NO₃–N g⁻¹ soil resulted in lower cnorB _p abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 μg glucose-C g⁻¹ soil, and 50 or 500 μg NO₃–N g⁻¹ was higher than all other treatments. There was a positive correlation between cnorB _p abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB _p abundance and mRNA levels than soils without glucose added, however response of cnorB _p abundance and mRNA levels to NO₃ supply depended on carbon availability.     

Highly efficient regeneration and medicinal component determination of Phellodendron chinense Schneid

Phellodendron chinense Schneid is an important Chinese herb with berberine and phellodendrine in stems and leaves, but with little information available on in vitro culture of this species. Disinfection of explants in 75% alcohol for 45 s, sterilization in 0.1% HgCl₂ for 20 min, and submersion in 1.0 mol L⁻¹ gibberellin3 (GA₃) solution for 24 h was the optimal condition for seed germination. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (6-BA) in combination with 1.5 mg L⁻¹ 1-naphthylacetic acid (NAA) was optimal for callus induction. MS medium supplemented with 2.0 mg L⁻¹ 6-BA was the appropriate medium for induction of adventitious shoots, and 1/2MS medium supplemented with 2.0 mg L⁻¹ indole-3-butytric acid (IBA) and 0.5% active carbon was the optimal medium for root induction. The 15-d survival rate of regenerated plantlets after transplanting to basins containing perlite and peat moss (1:4) was greater than 80%, and the berberine and phellodendrine accumulation was lower in callus compared with regenerated plantlets. The establishment of highly efficient regeneration system provides technical support for genetic breeding of Phellodendron chinense Schneid.

     

An efficient callus-based in vitro regeneration protocol for Warburgia Ugandensis Sprague,an important medicinal plant in Africa

Warburgia ugandensis Sprague is a woody species in the family Canellaceae and an important source of medicines in Africa. Natural propagation of W. ugandensis is problematic due to its recalcitrant seeds and lack of an efficient in vitro regeneration system for this species. This study describes an efficient regeneration protocol. Petiole bases and shoot tips were used as explants. Callus tissue developed when the explants were cultured on Murashige and Skoog medium containing 30 g L⁻¹ sucrose and 7 g L⁻¹ agar (MS30 medium), supplemented with 1.0 mg L⁻¹ indole-3-butyric acid (IBA), 1.6 mg L⁻¹ 6-benzylaminopurine (BA), and 0.1 mg L⁻¹ thidiazuron (TDZ). Adventitious buds were efficiently induced from the callus when the MS30 medium was supplemented with 0.8 mg L⁻¹ BA and 0.2 mg L⁻¹ IBA. Root induction occurred within 7–10 d on half-strength MS30 medium supplemented with 0.8–1.0 mg L⁻¹ 1-napthalene acetic acid (NAA), 0.2 mg L⁻¹ IBA, and 0.03% (w/v) activated charcoal (AC). Roots were followed by root elongation on the same medium but lacking NAA and IBA. Approximately 50% of the plantlets cultured produced roots, while more than 80% of the plantlets survived and successfully grew to maturity.

     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.     

Investigation of Heavy Metal Level and Mineral Nutrient Status in Widely Used Medicinal Plants’ Leaves in Turkey: Insights into Health Implications

The use of plants in treatments has been as old as humanity and it has preserved its popularity for centuries til now because of their availability, affordability and safeness. However, despite their widespread use, safety and quality issues have been major concerns in the world due to industrial- and anthropogenic-based heavy metal contamination risks. Thus, this study was attempted to analyze the heavy metal levels and mineral nutrient status of widely used medicinal plants in Turkey to have insights about their health implications on humans. The plant concentrations of B, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn were analyzed by ICP-OES in the leaves of 44 medical plants purchased from herbal markets of three different districts of Istanbul/Turkey. The measured lowest to highest concentrations were 0.065–79.749 mg kg⁻¹ B, 921.802–12,854.410 mg kg⁻¹ Ca, 0.020–0.558 mg kg⁻¹ Cd, 0.015–4.978 mg kg⁻¹ Cr, 0.042–8.489 mg kg⁻¹ Cu, 34.356–858.446 mg kg⁻¹ Fe, 791.323–15,569.349 mg kg⁻¹ K, 102.236–2837.836 mg kg⁻¹ Mg, 4.915–91.519 mg kg⁻¹ Mn, 10.224–3213.703 mg kg⁻¹ Na, 0.001–5.589 mg kg⁻¹ Ni, 0.003–3.636 mg kg⁻¹ Pb and 2.601–36.102 mg kg⁻¹ Zn. Those levels in plants were in acceptable limits though some elements in some plants have high limits which were not harmful. Variations (above acceptable limits) in element concentrations also indicated that these plants could be contaminated with other metals and that genetic variations may influence accumulation of these elements at different contents. Overall, analyzed medicinal plants are expected not to pose any serious threat to human health.

     

Effect of Vanadium on Testa,Seed Germination,and Subsequent Seedling Growth of Alfalfa (Medicago sativa L.)

Seed germination is the critical initial phase in the life cycle of plant and it is affected by various exogenous factors, including heavy metals. Seed germination and subsequent seedling growth of alfalfa (Medicago sativa L.) incubated in glass Petri dish in presence of elevated concentrations of pentavalent vanadium V(V) solution (0, 0.1, 0.5, 2, 4, 10, 50 mg L⁻¹ V, supplied as NaVO₃·2H₂O) were evaluated. Results showed that vanadium did not (P > 0.05) affect seed germination, final survival rate, and seedling height of alfalfa when exogenously treated dosages were ≤ 10 mg L⁻¹ V, whereas the root vitality and root elongation were distinctly inhibited at ≥ 0.5 mg L⁻¹ V treatments. A progressively deepened testa color at increasing vanadium concentrations during germination and an apparent modified structure of the seed coat at 50 mg L⁻¹ V compared to control in alfalfa were noted. Alfalfa seeds showed rapid and almost synchronous radicle emergence, independently of the vanadium concentration in the medium. The accumulation of vanadium in testa is beneficial to alleviate its toxicity to the seed germination of alfalfa. Leaf proline content was dramatically increased at ≥ 0.5 mg L⁻¹ V treatments compared with the control. Emerged seedlings displayed enough vigor and health to potentially colonize in the vanadium-contained matrix. Thus, alfalfa represents a good candidate for phytoremediation approach aimed at decontaminating environments when vanadium concentrations are within the determined thresholds.

     

Nitric Oxide Reductase-Targeted Real-Time PCR Quantification of Denitrifier Populations in Soil

The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (cnorB_P [Pseudomonas mandelii and closely related strains] and cnorB_B [Bosea, Bradyrhizobium, and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorB_P population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorB_B guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorB_P populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorB_P population densities increased significantly with increasing rates of glucose addition. cnorB_B guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.     

Response of denitrification rate associated with wetting and drying cycles in a littoral wetland area of Lake Biwa,Japan

To clarify the relationship between denitrification activity and dry–wet levels in the littoral wetland sediments of Lake Biwa, Japan, denitrification rates and their regulating parameters (degree of dryness, redox potential, nitrate concentration) were measured on different moisture sediments. Redox potential in sediments was higher in the exposed region in contact with atmosphere than the flooded region covered with water. The nitrate concentration in interstitial waters was undetectable in the flooded region. On the other hand, concentration in the exposed region increased with increase in the degree of sediment dryness. The denitrification rate ranged from <0.001 to 0.88 μg N cm⁻³ h⁻¹ in the exposed region and increased with the increase in the degree of dryness. In the flooded region, on the other hand, no detectable rate (<0.001 μg N cm⁻³ h⁻¹) was observed. This indicates that the rates in the exposed region were mainly influenced by nitrate concentration in the interstitial waters accumulated by desiccation of sediments, whereas rates in the flooded region were strongly limited by no accumulation of nitrate in the anaerobic conditions. The potential denitrification rate, under the application condition of nitrate, ranged from 0.13 to 0.26 μg N cm⁻³ h⁻¹ in the flooded region and from 0.77 to 1.5 μg N cm⁻³ h⁻¹ in the exposed region. The potential rates in the flooded region had a tendency to be lower than those in the exposed region, implying that the number of denitrifying bacteria in the flooded region was low due to inactivation of aerobic respiration and denitrification in the denitrifying bacteria community. Kinetic parameters, maximum rate (V _max) and half-saturation constant (K _s) for denitrification were calculated on the experimental procedures of the wetting–drying cycles of sediments. Both parameters decreased by the wetting treatment and increased by the drying treatment. The fluctuation of V _max values with wetting–drying cycles indicated that the number of denitrifying bacteria was influenced by aerobic respiration and denitrification in the denitrifying bacteria community similar to the potential rates, and denitrifying enzyme was induced by the nitrate supplied by nitrification accelerated through the drying process. On the other hand, the fluctuation of K _s values implied that members of denitrifying bacteria were shifted to members of high nitrate affinity by wetting treatment and of low nitrate affinity by drying treatment.     

Genetic homogeneity and high shoot proliferation in banana (Musa acuminata Colla) by altering medium thiamine level and sugar type

To enhance the multiplication rate in Musa acuminata Colla (banana; ‘Grand Nain’) organogenesis, higher amounts of thiamine along with different sugar types and concentrations were evaluated at the proliferation phase. Thiamine at 1, 10, 50, 100, and 200 mg L⁻¹ was compared with 0.1 mg L⁻¹ thiamine found in conventional Murashige and Skoog (MS) medium. Maximum proliferation of banana was induced with 100 mg L⁻¹ thiamine. Additionally, 15, 30, and 45 g L⁻¹ sucrose, glucose, fructose, and sorbitol combined with regular and optimal levels of thiamine were tested. Glucose at 30 g L⁻¹ most improved shoot proliferation alone and enhanced shoot proliferation further, when combined with 100 mg L⁻¹ thiamine, followed by sucrose and fructose, whereas sorbitol completely inhibited growth and caused tissue browning. All evaluated vegetative traits were significantly affected by sugar type and concentration, and thiamine levels, unlike the photosynthetic pigments. Moreover, genetic stability of the plants recovered from the enhanced protocol was confirmed by inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. A total of 230 bands generated by both marker types were monomorphic for the randomly selected regenerated plants, compared with their mother plant. Thus, the proliferation medium supplemented with 30 g L⁻¹ glucose and 100 mg L⁻¹ thiamine could be recommended for banana organogenesis. Results herein are of great importance and helpful in enhancing the commercial in vitro propagation protocols of banana, without the need of increasing the number of subcultures, which can cause somaclonal variation.

     

Microbial degradation and metabolic pathway of pyridine by a <Emphasis Type="Italic">Paracoccus</Emphasis> sp. strain BW001

A bacterial strain using pyridine as sole carbon, nitrogen and energy source was isolated from the activated sludge of a coking wastewater treatment plant. By means of morphologic observation, physiological characteristics study and 16S rRNA gene sequence analysis, the strain was identified as the species of Paracoccus. The strain could degrade 2,614 mg l⁻¹ of pyridine completely within 49.5 h. Experiment designed to track the metabolic pathway showed that pyridine ring was cleaved between the C₂ and N, then the mineralization of the carbonous intermediate products may comply with the early proposed pathway and the transformation of the nitrogen may proceed on a new pathway of simultaneous heterotrophic nitrification and aerobic denitrification. During the degradation, NH₃-N occurred and increased along with the decrease of pyridine in the solution; but the total nitrogen decreased steadily and equaled to the quantity of NH₃-N when pyridine was degraded completely. Adding glucose into the medium as the extra carbon source would expedite the biodegradation of pyridine and the transformation of the nitrogen. The fragments of nirS gene and nosZ gene were amplified which implied that the BW001 had the potential abilities to reduce NO₂ ⁻ to NO and/or N₂O, and then to N₂.     

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. cv. Poinsett76) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L⁻¹ benzyladenine (BA), 20 mg L⁻¹ l-glutamine and 2 mg L⁻¹ phosphinothricin (PPT) or 100 mg L⁻¹ kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L⁻¹) containing MS media with 0.5 mg L⁻¹ gibberellic acid (GA₃) and 0.6 mg L⁻¹ indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T₀ and the progenies. T₁ segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.     

Abundance and Structure Composition of nirK and nosZ Genes as Well as Denitrifying Activity in Heavy Metal-polluted Paddy Soils

Denitrification is an important microbial process in soils and leads to the emission of nitrous oxide (N₂O). However, studies about the microbial community involved in denitrification processes in polluted paddy fields are scarce. Here, we studied two rice paddies which had been polluted for more than three decades by metal mining and smelter activities. Abundance and community composition were determined using real-time polymerase chain reaction (PCR) assay and denaturing gradient gel electrophoresis of nitrite reductase and nitrous oxide reductase gene amplicons (nirK and nosZ), while denitrifying activities were assessed by measuring potential denitrifier enzyme activity. We found that the community structure of both nirK and nosZ containing denitrifiers shifted under pollution in the two rice paddies. All the retrieved nirK sequences did not group into either α- or β-proteobacteria, while most of the nosZ species were affiliated with α-proteobacteria. While the abundance of both nirK and nosZ was significantly reduced in the polluted soils at “Dexing” (with relatively higher Cu levels), these parameters did not change significantly at “Dabaoshan” (polluted with Cd, Pb, Cu, and Zn). Furthermore, total denitrifying activity and N₂O production and reduction rates also only decreased under pollution at “Dexing.” These findings suggest that nirK and nosZ containing denitrifier populations and their activities could be sensitive to considerable Cu pollution, which could potentially affect N₂O release from polluted paddy soils.     

Microbial community of acetate utilizing denitrifiers in aerobic granules

Nitrite accumulates during biological denitrification processes when carbon sources are insufficient. Acetate, methanol, and ethanol were investigated as supplementary carbon sources in the nitrite denitrification process using biogranules. Without supplementary external electron donors (control), the biogranules degraded 200 mg l⁻¹ nitrite at a rate of 0.27 mg NO₂–N g⁻¹ VSS h⁻¹. Notably, 1,500 mg l⁻¹ acetate and 700 mg l⁻¹ methanol or ethanol enhanced denitrification rates for 200 mg l⁻¹ nitrite at 2.07, 1.20, and 1.60 mg NO₂–N g⁻¹ VSS h⁻¹, respectively; these rates were significantly higher than that of the control. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nitrite reductase (NiR) enzyme identified three prominent bands with molecular weights of 37–41 kDa. A linear correlation existed between incremental denitrification rates and incremental activity of the NiR enzyme. The NiR enzyme activity was enhanced by the supplementary carbon sources, thereby increasing the nitrite denitrification rate. The capacity of supplementary carbon source on enhancing NiR enzyme activity follows: methanol > acetate > ethanol on molar basis or acetate > ethanol > methanol on an added weight basis.     

Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil

Analyses of the complete genomes of sequenced denitrifying bacteria revealed that approximately 1/3 have a truncated denitrification pathway, lacking the nosZ gene encoding the nitrous oxide reductase. We investigated whether the number of denitrifiers lacking the genetic ability to synthesize the nitrous oxide reductase in soils is important for the proportion of N₂O emitted by denitrification. Serial dilutions of the denitrifying strain Agrobacterium tumefaciens C58 lacking the nosZ gene were inoculated into three different soils to modify the proportion of denitrifiers having the nitrous oxide reductase genes. The potential denitrification and N₂O emissions increased when the size of inoculated C58 population in the soils was in the same range as the indigenous nosZ community. However, in two of the three soils, the increase in potential denitrification in inoculated microcosms compared with the noninoculated microcosms was higher than the increase in N₂O emissions. This suggests that the indigenous denitrifier community was capable of acting as a sink for the N₂O produced by A. tumefaciens. The relative amount of N₂O emitted also increased in two soils with the number of inoculated C58 cells, establishing a direct causal link between the denitrifier community composition and potential N₂O emissions by manipulating the proportion of denitrifiers having the nosZ gene. However, the number of denitrifiers which do not possess a nitrous oxide reductase might not be as important for N₂O emissions in soils having a high N₂O uptake capacity compared with those with lower. In conclusion, we provide a proof of principle that the inability of some denitrifiers to synthesize the nitrous oxide reductase can influence the nature of the denitrification end products, indicating that the extent of the reduction of N₂O to N₂ by the denitrifying community can have a genetic basis.     

Stimulating denitrification in a stream mesocosm with elemental sulfur as an electron donor

The heavy use of fertilizers in agricultural lands can result in significant nitrate (NO₃⁻) loadings to the aquatic environment. We hypothesized that biological denitrification in agricultural ditches and streams could be enhanced by adding elemental sulfur (S^o) to the sediment layer, where it could act as a biofilm support and electron donor. Using a bench-scale stream mesocosm with a bed of S^o granules, we explored NO₃⁻ removal fluxes as a function of the effluent NO₃⁻ concentrations. With effluent NO₃⁻ ranging from 0.5 mg N L⁻¹ to 4.1 mg N L⁻¹, NO₃⁻ removal fluxes ranged from 228 mg N m⁻² d⁻¹ to 708 mg N m⁻² d⁻¹. This is as much as 100 times higher than for agricultural drainage streams. Sulfate (SO₄²⁻) production was high due to aerobic sulfur oxidation. Molecular studies demonstrated that the S^o amendment selected for Thiobacillus species, and that no special inoculum was required for establishing a S^o-based autotrophic denitrifying community. Modeling studies suggested that denitrification was diffusion limited, and advective flow through the bed would greatly enhance NO₃⁻ removal fluxes. Our results indicate that amendment with S^o is an effective means to stimulate denitrification in a stream environment. To minimize SO₄²⁻ production, it may be better to place S^o deeper in the sediment layer.