Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.)

The present work deals with optimization of adventitious shoot culture of Bacopa monnieri for the production of biomass and bacoside A and has investigated the effects of macro elements (NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄) and nitrogen source [NH₄ ⁺/NO₃ ⁻] of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) on accumulation of biomass and bacoside A content. Optimum number of adventitious shoots (99.33 shoots explant⁻¹), fresh weight (1.841 g) and dry weight (0.150 g) were obtained in the medium with 2.0× strength of NH₄NO₃. The highest production of bacoside A content was also recorded in the medium of 2.0× NH₄NO₃, which produced 17.935 mg g⁻¹ DW. The number of adventitious shoot biomass and bacoside A content were optimum when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum number of shoots (70.00 shoots explant⁻¹), biomass (fresh weight 1.137 g and dry weight 0.080 g) and also bacoside A content (27.106 mg g⁻¹ DW) were obtained at NH₄ ⁺/NO₃ ⁻ ratio of 14.38/37.60 mM. Overall, MS medium supplemented with 2.0× NH₄NO₃ is recommended for most efficient bacoside A production.     

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a NH₄ ⁺/NO₃ ^? balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture (28.5 μg/g DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM NH₄ ⁺/NO₃ ^? and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with 10.74 μM naphthaleneacetic acid and 0.93 μM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and 8.2 μg/g DW), respectively.     

Effect of plant growth regulators and medium composition on cell growth and saponin production during cell-suspension culture of mountain ginseng (Panax ginseng C. A. mayer)

We have established cell-suspension cultures of mountain ginseng (Panax ginseng G A. Mayer), and have attempted to increase the yield of saponin by manipulating our processing method and culturing factors (e.g., media strengths; the presence of plant growth regulators or sucrose; ratios of NO⁺ ₃/ NH^- ₄). Maximum biomass yield was obtained in media containing 2,4-D. However, saponin productivity was much higher in a medium comprising either IBA or NAA; 7.0 mg/L IBA was optimal for promoting both cell growth (10.0 g/L dry weight) and saponin production (7.29 mg/g DW total ginsenoside). Although the addition of cytokinins (BA and kinetin) did not affect cell growth, the level of saponin (particularly in the Rb group) was enhanced when the media were supplemented with either 0.5 mg/L BA or 0.5 mg/L kinetin. Half- and full-strength MS media were equally suitable for inducing both biomass as well as saponin production. We also investigated the effect of various concentrations of sucrose and nitrogen, and found that 30 g/L sucrose enhanced biomass yield as well as saponin content However, further increases (i.e., up to 70 g/L) led to a decrease in saponin accumulation and biomass production. Maximum growth and saponin productivity were reported from treatments with an initial nitrogen concentration of 30 mM. In general, the amount of saponin increased when the test media had high NO⁺ ₃/ NH^- ₄ ratios; in fact, saponin production was greatest when nitrate was the sole nitrogen source.     

The effect of total inorganic nitrogen and the balance between its ionic forms on adventitious bud formation and callus growth of ‘Węgierka Zwykła’ plum (<Emphasis Type="Italic">Prunus domestica</Emphasis> L.)

The effect of different ammonium NH ₄ ⁺ and nitrate NO ₃ ^? ratios (4:1, 2:1, 1:1, 1:2, 1:4, 1:6) on organogenesis of ‘W?gierka Zwyk?a’ leaf explants cultivated on media with nitrogen levels equalling full- or half-MS was investigated. On media with total nitrogen equal to ½ MS, explant regeneration increased significantly and was highest on media with 1:2 or 1:4 NH ₄ ⁺ :NO ₃ ^? ratio. An excess of ammonium versus nitrate ions had a negative effect on both regeneration and biomass. Addition of potassium to the medium increased the fresh weight of explants and the number of adventitious buds.     

Adventitious root suspension cultures of <Emphasis Type="BoldItalic">Hypericum perforatum</Emphasis>: effect of nitrogen source on production of biomass and secondary metabolites

The present study investigated the effect of nitrogen source (NH₄⁺; NO₃⁻) at different concentrations on the accumulation of biomass and secondary metabolites in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium with B5 vitamins, 1.0 mg l⁻¹ indole-3-butyric acid, 0.1 mg l⁻¹ kinetin, 3% (w/v) sucrose, and different ratios of ammonium and nitrate (0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM, using NH₄Cl and KNO₃). The cultures were maintained in darkness. The medium supplemented with 5:25 (mM) NH₄⁺/NO₃⁻ resulted in the optimum accumulation of biomass and total phenols and flavonoids. The antioxidant potential of a methanolic extract, measured as the 1, 1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities, of H. perforatum adventitious roots showed that antioxidant activity was high from root extracts that were grown on higher concentrations of NO₃⁻ nitrogen (15, 20, and 25 mM). Further, assessment of hydrogen peroxide (H₂O₂) and malondialdehyde content of the root extracts revealed that cultures supplemented with higher levels of NO₃⁻ nitrogen (15–30 mM) were under oxidative stress, which boosted the levels of secondary metabolites in the adventitious roots. These results suggest that optimal adventitious root biomass could be achieved with the supplementation of cultures with 5:25 ratios of MS nitrogen sources.     

Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives

In an attempt to scale-up of adventitious root cultures of Echinacea angustifolia for the production of biomass and caffeic acid derivatives, i.e. echinacoside, chlorogenic acid, cichoric acid, caftaric acid, and cynarin, the effects of Murashige and Skoog (MS) medium dilutions, and initial sucrose concentrations were investigated in a 5-L airlift bioreactor. In addition, the kinetics of adventitious root growth and accumulation of secondary metabolites were also studied. The greatest root dry weight (6.50 g L^?l) and accumulation of total phenolics [22.06 mg g^?1 DW (dry weight)], total flavonoids (5.77 mg g^?1 DW) and total caffeic acid derivatives (10.63 mg g^?1 DW) were obtained at quarter-strength MS medium. Of the various gradients of sucrose tested, 5 % sucrose supplementation was regarded as an optimal concentration for enhancing productivity of biomass and bioactive compounds. Neither higher salt strength (3/4–2 MS) nor sucrose concentrations (7 and 9 %) showed promotive effect on root growth and metabolite production. The kinetic studies revealed that 4 weeks of culture period is the optimal time to achieve highest productivity of metabolites. Based on these results, a large-scale (20 L) and a pilot-scale (500 L) adventitious root culture system was established. In the pilot-scale bioreactor, adventitious roots were elicitor-treated with 100 μM methyl jasmonate (MJ) on day 28. After 1 week of elicitation, 1.75 kg dry root biomass was harvested containing 60.41 mg g^?1 DW of total phenolics, 16.45 mg g^?1 DW of total flavonoids, and 33.44 mg g^?1 DW of total caffeic acid derivatives. Among the caffeic acid derivatives, the accumulation of echinacoside (the major bioactive compound) in MJ-treated adventitious roots grown in the 500-L bioreactor was the highest (12.3 mg g^?1 DW), which is approximately threefold more than the non-MJ-treated roots cultured in 5- and 20-L bioreactors.     

Withanolide A production from Withania somnifera hairy root cultures with improved growth by altering the concentrations of macro elements and nitrogen source in the medium

Withania somnifera is an important medicinal plant that contains withanolides as bioactive compounds. We have investigated the effects of macroelements and nitrogen source in hairy roots of W. somnifera with the aim of optimizing the production of biomass and withanolide A content. The effects of the macroelements NH₄NO₃, KNO₃, CaCl₂, MgSO₄ and KH₂PO₄ at concentrations of 0, 0.5, 1.0, 1.5 and 2.0× strengths and of nitrogen source [NH₄ ⁺/NO₃ ^? (0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20 and 14.38/37.60 mM)] in Murashige and Skoog medium were evaluated for biomass and withanolide A production. The highest accumulation of biomass (139.42 g l^?1 FW and 13.11 g l^?1 DW) was recorded in the medium with 2.0× concentration of KH₂PO₄, and the highest production of withanolide A was recorded with 2.0× KNO₃ (15.27 mg g^?1 DW). The NH₄ ⁺/NO₃ ^? ratio also influenced root growth and withanolide A production, with both parameters being larger when the NO₃ ^? concentration was higher than that of NH₄ ⁺. Maximum biomass growth (148.17 g l^?1 FW and 14.79 g l^?1 DW) was achieved at NH₄ ⁺/NO₃ ^? ratio of 14.38/37.60 mM, while withanolide A production was greatest (14.68 mg g^?1 DW) when the NH₄ ⁺/NO₃ ^? ratio was 0.00/18.80 mM. The results are useful for the large scale cultivation of Withania hairy root culture for the production of withanolide A.     

A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum

An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated transformation and growth rate of hairy root. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, and three explants types, hypocotyls, leaves and excised shoots were examined. The highest frequency of transformation was achieved using LBA9402 strain in the excised shoots. Several inoculation and co-cultivation media and different concentration of arginine were evaluated using LBA9402 strain and the excised shoots as explant. Interestingly, a drastic increase in the frequency of transformation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH₄NO₃ KH₂PO₄, KNO₃ and CaCl₂ was used. The effect of sucrose concentration and the ratio of NH₄ ⁺: NO₃ ^? on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH₄ ⁺ to NO₃ ^? on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization.     

Influence of sucrose concentration and phosphate source on biomass and metabolite accumulation in adventitious roots of Pseudostellaria heterophylla

In this study, we investigated the induction of Pseudostellaria heterophylla adventitious root and the effects of sucrose concentration and phosphate source on biomass increase and metabolites accumulation. These roots were initially cultured in Murashige and Skoog medium for 4 weeks. IBA 3.0 mg L^?1 proved to be the best auxin for inducing adventitious roots and the frequency of adventitious roots induced from roots (100 %) was higher than that from leaves (78 %) and stems (27 %). The medium with 4 % sucrose resulted in the optimum biomass i.e. 1.04 g/flask DW, and the content of saponin and polysaccharides reached the peak i.e. 0.676 and 24.4 %, respectively. With regards to phosphate source, 1.25 mM phosphate concentration was more favorable for biomass of roots (0.87 g/flask of DW), whereas the optimum saponin (0.74 %) and polysaccharides (22.09 %) were achieved with 2.5 mM phosphate. However, the saponin content at 2.5 mM phosphate did not show significant difference from the saponin content at 0.625 mM (0.69 %) or 3.75 mM phosphate (0.69 %).     

Effect of NO<Subscript>3</Subscript><Superscript>−</Superscript>:NH<Subscript>4</Subscript><Superscript>+</Superscript> ratios on growth,root morphology and leaf metabolism of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) seedlings

The effect of NO₃ ^?:NH₄ ⁺ ratio (14:1, 9:6, 7.5:7.5, 1:14, total 15 mmol/L N) in the nutrient solution on biomass, root morphology, and C and N metabolism parameter in hydroponically grown oilseed rape (Brassica napus L.) was evaluated. The dry weights of leaves and roots were significantly largest at the equal NO₃ ^?:NH₄ ⁺ ratio (7.5:7.5) compared with those of high NO₃ ^?:NH₄ ⁺ ratio (14:1) or low NO₃ ^?:NH₄ ⁺ ratio (1:14). Additionally, low NO₃ ^?:NH₄ ⁺ ratio (1:14) reduced total root length and root surface area compared with the equal NO₃ ^?:NH₄ ⁺ ratio (7.5:7.5), while high NO₃ ^?:NH₄ ⁺ ratio (14:1) did not show any significant effect on root morphology except average diameter. The maximum of chlorophyll a, chlorophyll b and carotenoid were obtained under 7.5:7.5 treatment, whereas the maximum of the leaf net photosynthetic (P _n), stomatal conductance (G _s) and transpiration rate (T _r) were increased with increase in NH₄ ⁺ concentration in the nutrient solution. The activity of nitrate reductase (NR) showed a significant difference at different NO₃ ^?:NH₄ ⁺ ratios and ranged 9:6 > 7.5:7.5 > 14:1 > 1:14, whereas the range of soluble sugar and soluble protein was 7.5:7.5 > 1:14 > 9:6 > 14:1. Our study reveals that oilseed rape growth is greater under 7.5:7.5 treatment than that under three other treatments. Oilseed rape growth at high or low NO₃ ^?:NH₄ ⁺ ratios was inhibited by decreased pigments, NR activity, soluble sugar, and soluble protein, whereas subdued root growth should be apprehended considerate under high NH₄ ⁺ condition.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Ammonia oxidizing bacteria and archaea in horizontal flow biofilm reactors treating ammonia-contaminated air at 10 °C

The objective of this study was to demonstrate the feasibility of novel, Horizontal Flow Biofilm Reactor (HFBR) technology for the treatment of ammonia (NH₃)-contaminated airstreams. Three laboratory-scale HFBRs were used for remediation of an NH₃-containing airstream at 10 °C during a 90-d trial to test the efficacy of low-temperature treatment. Average ammonia removal efficiencies of 99.7 % were achieved at maximum loading rates of 4.8 g NH₃ m³ h^?1. Biological nitrification of ammonia to nitrite (NO₂ ^?) and nitrate (NO₃ ^?) was mediated by nitrifying bacterial and archaeal biofilm populations. Ammonia-oxidising bacteria (AOB) were significantly more abundant than ammonia-oxidising archaea (AOA) vertically at each of seven sampling zones along the vertical HFBRs. Nitrosomonas and Nitrosospira, were the two most dominant bacterial genera detected in the HFBRs, while an uncultured archaeal clone dominated the AOA community. The bacterial community composition across the three HFBRs was highly conserved, although variations occurred between HFBR zones and were driven by physicochemical variables. The study demonstrates the feasibility of HFBRs for the treatment of ammonia-contaminated airstreams at low temperatures; identifies key nitrifying microorganisms driving the removal process; and provides insights for process optimisation and control. The findings are significant for industrial applications of gas oxidation technology in temperate climates.     

Interaction of benthic microalgae and macrofauna in the control of benthic metabolism, nutrient fluxes and denitrification in a shallow sub-tropical coastal embayment (western Moreton Bay, Australia)

Benthic biogeochemistry and macrofauna were investigated six times over 1 year in a shallow sub-tropical embayment. Benthic fluxes of oxygen (annual mean ?918 μmol O₂ m^?2 h^?1), ammonium (NH₄ ⁺), nitrate (NO₃ ^?), dissolved organic nitrogen, dinitrogen gas (N₂), and dissolved inorganic phosphorus were positively related to OM supply (N mineralisation) and inversely related to benthic light (N assimilation). Ammonium (NH₄ ⁺), NO₃ ^? and N₂ fluxes (annual means +14.6, +15.9 and 44.6 μmol N m^?2 h^?1) accounted for 14, 16 and 53 % of the annual benthic N remineralisation respectively. Denitrification was dominated by coupled nitrification–denitrification throughout the study. Potential assimilation of nitrogen by benthic microalgae (BMA) accounted for between 1 and 30 % of remineralised N, and was greatest during winter when bottom light was higher. Macrofauna biomass tended to be highest at intermediate benthic respiration rates (?1,000 μmol O₂ m^?2 h^?1), and appeared to become limited as respiration increased above this point. While bioturbation did not significantly affect net fluxes, macrofauna biomass was correlated with increased light rates of NH₄ ⁺ flux which may have masked reductions in NH₄ ⁺ flux associated with BMA assimilation during the light. Peaks in net N₂ fluxes at intermediate respiration rates are suggested to be associated with the stimulation of potential denitrification sites due to bioturbation by burrowing macrofauna. NO₃ ^? fluxes suggest that nitrification was not significantly limited within respiration range measured during this study, however comparisons with other parts of Moreton Bay suggest that limitation of coupled nitrification–denitrification may occur in sub-tropical systems at respiration rates exceeding ?1,500 μmol O₂ m^?2 h^?1.     

Production of Biomass and Ginsenosides from Adventitious Roots of Panax ginseng in Bioreactor Cultures

Organic nutrients play a central role during Panax ginseng adventitious root culture in bioreactor systems. To understand how the nutrient elements were uptaken during the adventitious root growth as well as the production of biomass and natural ginsenosides, a biotechnological approach to identifying the nutritional physiology of ginseng in a commercial‐scale bioreactor was necessary. Normal MS medium nutrient in the bioreactor culture of adventitious roots resulted in slow growth, low biomass, and Rg and Rb ginsenoside contents. When the ginsenoside production increased to higher levels, a group of regulatory nutritional elements that have the potential to interact with biomass was identified. The effects of the salt strength of the medium, of macroelements, metal elements, the ammonia/nitrate ratio, sucrose concentration, and osmotic agents on the growth, the formation of biomass and the production of ginsenosides from adventitious roots were investigated. Appropriate conditions allowed for a maximum ginsenoide production of up to 12.42 [mg/g DW] to be obtained after 5 weeks of culture. The results demonstrated that the key organic nutrients can be regulated to improve the biomass and growth, and increase the ginsenoside yield in bioreactor cultures of P. ginseng adventitious roots.     

Production of biomass and bioactive compounds by adventitious root suspension cultures of Morinda citrifolia (L.) in a liquid-phase airlift balloon-type bioreactor

To improve root growth and production of bioactive compounds such as anthraquinones (AQ), phenolics, and flavonoids by adventitious root cultures of Morinda citrifolia, the effects of aeration rate, inoculum density, and Murashige and Skoog (MS) medium salt strengths were investigated using a balloon-type bubble bioreactor. The possible mechanisms underlying changes in activities of enzymic (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase) and nonenzymic (vitamin E) antioxidants, phenylalanine ammonia lyase, and stress levels (accumulation of hydrogen peroxide and proline, peroxidation of lipids) were also studied. Low aeration rate (0.05 vvm [air volume/culture volume/min]) accelerated accumulation of root fresh weight and dry weight (DW). High aeration rates (0.1 to 0.3 vvm) stimulated accumulation of AQ, phenolics, and flavonoids and reduced root growth. Low inoculum densities (5 and 10 g l^–1) increased accumulation of those metabolites but inhibited root growth. Culture of adventitious roots with high concentrations of MS salts (1× and 1.5× MS) resulted in induction of oxidative stress that strongly inhibited root growth. Overall, an aeration rate of 0.05 vvm, 15 g l^–1 inoculum density, and half-strength (0.5×) MS medium were optimal for enhancing accumulation of root dry biomass (4.38 g l^–1), AQ (103.08 mg g^–1 DW), phenolics (54.81 mg g^–1 DW), and flavonoids (49.27 mg g^–1 DW).     

Nitrate reduction with biotic and abiotic cathodes at various cell voltages in bioelectrochemical denitrification system

Electrochemical treatment of nitrate ions was attempted using different catalysts on the cathode in bioelectrochemical denitrification systems. The carbon cathode coated by biofilm (biocathode) could remove 91 % of nitrate ions at 1.0 V, which was almost same as the Pt-coated electrode (90 %). The exchange current density of biocathode was 0.0083 A/m², which was almost 22 times higher than with an abiotic plain carbon cathode. The formation of intermediate products in nitrate reduction varied depending on the cell voltage. At 0.5 V, a large portion of nitrate was converted to ammonia, but at more increased cell voltage (0.7 and 1 V) a high amount of nitrite ions was found with little ammonia formation in cathodic solution. The maximum nitrate removal rate was 0.204 mg NO₃-N/cm²d by biocathode, while plain carbon paper showed only 0.176 mg NO₃-N/cm²d. Electrochemical analysis of chronoamperometry showed a higher stable current generation for biocathode (3.1 mA) and Pt-coated cathode (2.8 mA) as compared to plain carbon (0.6 mA) at 0.7 V of poised voltage.     

Influence of bioturbation on denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in freshwater sediments

Intensive agriculture leads to increased nitrogen fluxes (mostly as nitrate, NO₃ ^?) to aquatic ecosystems, which in turn creates ecological problems, including eutrophication and associated harmful algal blooms. These problems have focused scientific attention on understanding the controls on nitrate reduction processes such as denitrification and dissimilatory nitrate reduction to ammonium (DNRA). Our objective was to determine the effects of nutrient-tolerant bioturbating invertebrates (tubificid oligochaetes) on nitrogen cycling processes, specifically coupled nitrification–denitrification, net denitrification, DNRA, and biogeochemical fluxes (O₂, NO₃ ^?, NH₄ ⁺, CO₂, N₂O, and CH₄) in freshwater sediments. A mesocosm experiment determined how tubificid density and increasing NO₃ ^? concentrations (using N¹⁵ isotope tracing) interact to affect N cycling processes. At the lowest NO₃ ^? concentration and in the absence of bioturbation, the relative importance of denitrification to DNRA was similar (i.e., 49.6 and 50.4 ± 8.1 %, respectively). Increasing NO₃ ^? concentrations in the control cores (without fauna) stimulated denitrification, but did not enhance DNRA, which significantly altered the relative importance of denitrification compared to DNRA (94.6 vs. 5.4 ± 0.9 %, respectively). The presence of tubificid oligochaetes enhanced O₂, NO₃ ^?, NH₄ ⁺ fluxes, greenhouse gas production, and N cycling processes. The relative importance of denitrification to DNRA shifted towards favoring denitrification with both the increase in NO₃ ^? concentrations and the increase of bioturbation activity. Our study highlights that understanding the interactions between nutrient-tolerant bioturbating species and nitrate contamination is important for determining the nitrogen removal capacity of eutrophic freshwater ecosystems.     

NH4 : NO3 nutrition influence on biomass productivity and root respiration of poplar and willow clones

Nitrogen fertilization often improves the yield of intensively managed, short‐rotation coppices. However, information of N nutrition form on the growth of common species and clones used for biomass production is limited. Thus, this study aims at evaluating N form effects on the growth of two Salicaceae clones. Cuttings of the poplar clone Max 4 (Populus maximovizcii × P. nigra) and the willow clone Inger (Salix triandra × S. viminialis) were fertilized in a pot experiment with four ratios of nitrate (NO₃^?) to ammonium (50%, 62.5%, 75% and 87.5% NO₃^? balanced with ammonium (NH₄⁺) to constant total N) for one growing season and under stable soil pH. Plants were harvested for analysis of biomass and morphology of leaves, stem and roots. Respiration of fine and coarse roots (RR) was determined and related to biomass growth. Salix cv. Inger accumulated more total dry matter than Populus cv. Max 4. In both Salicaceae clones, the total biomass was significantly influenced by the nitrate ratio and greatest in plants fertilized with 50% NO₃^? of the total N supply. Both clones possess a different leaf and root morphology, but no significant influence of the NO₃^? ratio on the morphology was found. Fine RR rates differed significantly between clones, with significantly greater fine RR in Max 4; 87.5% NO₃^? fertilization increased the fine RR. Fine RR and total accumulated plant biomass were closely related. Our study is the first to show the tremendous influence of fine root respiration, especially including the carbon‐intensive reduction of NO₃^? to NH₄⁺, on the aboveground growth of Salicaceae clones. Ways to improve yield in SRC are thus to lower the assimilate consumption by fine roots and to match fertilization regimes to the used clones or vice versa.     

Effect of influent substrate ratio on anammox granular sludge: performance and kinetics

Effect of influent substrate ratio on anammox process was studied in sequencing batch reactor. Operating temperature was fixed at 35 ± 1 °C. Influent pH and hydraulic retention time were 7.5 and 6 h, respectively. When influent NO₂ ^?-N/NH₄ ⁺-N was no more than 2.0, total nitrogen removal rate (TNRR) increased whereas NH₄ ⁺-N removal rate stabilized at 0.32 kg/(m³ d). ΔNO₂ ^?-N/ΔNH₄ ⁺-N increased with enhancing NO₂ ^?-N/NH₄ ⁺-N. When NO₂ ^?-N/NH₄ ⁺-N was 4.5, ΔNO₂ ^?-N/ΔNH₄ ⁺-N was 1.98, which was much higher than theoretical value (1.32). The IC₅₀ of NO₂ ^?-N was 289 mg/L and anammox activity was inhibited at high NO₂ ^?-N/NH₄ ⁺-N ratio. With regard to influent NH₄ ⁺-N/NO₂ ^?-N, the maximum NH₄ ⁺-N removal rate was 0.36 kg/(m³ d), which occurred at the ratio of 4.0. Anammox activity was inhibited when influent NH₄ ⁺-N/NO₂ ^?-N was higher than 5.0. With influent NO₃ ^?-N/NH₄ ⁺-N of 2.5–6.5, NH₄ ⁺-N removal rate and NRR were stabilized at 0.33 and 0.40 kg/(m³ d), respectively. When the ratio was higher than 6.5, nitrogen removal would be worsened. The inhibitory threshold concentration of NO₂ ^?-N was lower than NH₄ ⁺-N and NO₃ ^?-N. Anammox bacteria were more sensitive to NO₂ ^?-N than NH₄ ⁺-N and NO₃ ^?-N. TNRR would be enhanced with increasing nitrogen loading rate, but sludge floatation occurred at high nitrogen loading shock. The Han-Levenspiel could be applied to simulate nitrogen removal resulting from NO₂ ^?-N inhibition.     

Correlation of continuous ryegrass regrowth with cytokinin induced by root nitrate absorption

This study investigated the separate and combined effects of nitrate (NO₃ ^?) and cytokinin additions on continuous ryegrass regrowth after defoliation and the underlying mechanisms. Our results showed that frequent defoliation reduced the biomass of newly grown leaves and roots, the root soluble carbohydrate contents, the root vitality (an indicator of root absorption capacity), and the leaf contents of NO₃ ^?, zeatin and zeatin riboside (Z + ZR), and isopentenyl adenine and isopentenyl adenosine (IP + IPA). NO₃ ^?addition to the roots or leaves increased the biomass of newly grown leaves as well as the leaf contents of NO₃ ^?, Z + ZR, and IP + IPA without increasing the root-to-shoot delivery of endogenous cytokinin. Interestingly, cytokinin directly added to the leaves also increased the biomass of newly grown leaves and their Z + ZR and IP + IPA contents, suggesting that nitrate-induced leaf cytokinin production mediates the growth-promoting effects of nitrate. We also found that cytokinin had a direct whereas NO₃ ^? had an indirect effect on the biomass of newly grown leaves. Taken together, our results indicate that leaf cytokinin production induced by NO₃ ^? absorbed through the roots plays a key role in continuous ryegrass regrowth after defoliation.