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.     

Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml^?1 Rf, and 10.6 U ml^?1 enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l^?1 h^?1 after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l^?1 h^?1 after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml^?1 APPT from 2.29 mg ml^?1 PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l^?1 h^?1, and 0.62 mg ml^?1 APPT from 1.35 mg ml^?1 PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l^?1 h^?1. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.     

The role of gross and net N transformation processes and NH4 + and NO3 − immobilization in controlling the mineral N pool of a temperate mixed deciduous forest soil

In many forests of Europe and north-eastern North America elevated N deposition has opened the forest N cycle, resulting in NO₃ ^? leaching. On the other hand, despite this elevated N deposition, the dominant fate of NO₃ ^? and NH₄ ⁺ in some of these forests is biotic or abiotic immobilization in the soil organic matter pool, preventing N losses. The environmental properties controlling mineral N immobilization and the variation and extent of mineral N immobilization in forest soils are not yet fully understood. In this study we investigated a temperate mixed deciduous forest, which is subjected to an average N deposition of 36.5 kg N ha^?1 yr^?1, but at the same time shows low NO₃ ^? concentrations in the groundwater. The aim of this study was to investigate whether the turnover rate of the mineral N pool could explain these low N leaching losses. A laboratory ¹⁵N pool dilution experiment was conducted to study gross and net N mineralization and nitrification and mineral N immobilization in the organic and uppermost (0–10 cm) mineral layer of the forest soil. Two locations, one at the forest edge (GE) and another one 145 m inside the forest (GF1), were selected. In the organic layers of GE and GF1, the gross N mineralization averaged 10.9 and 11.1 mg N kg^?1 d^?1, the net N mineralization averaged 6.1 and 6.8 mg N kg^?1 d^?1 and NH₄ ⁺ immobilization rates averaged 3.8 and 3.6 mg N kg^?1 d^?1. In the organic layer of GE and GF1, the average gross nitrification was 3.8 and 4.6 mg N kg^?1 d^?1, the average net nitrification was ?25.2 and ?31.3 mg N kg^?1 d^?1 and the NO₃ ^? immobilization rates averaged 29.0 and 35.9 mg N kg^?1 d^?1. For the mineral (0–10 cm) layer the same trend could be observed, but the N transformation rates were much lower for the NH₄ ⁺ pool and not significantly different from zero for the NO₃ ^? pool. Except for the turnover of the NH₄ ⁺ pool in the mineral layer, no significant differences were observed between location GE and GF1. The ratio of NH₄ ⁺ immobilization to gross N mineralization, gross N mineralization to gross nitrification, and NO₃ ^? immobilisation to gross nitrification led to the following observations. The NH₄ ⁺ pool of the forest soil was controlled by N mineralization and NO₃ ^? immobilization was importantly controlling the forest NO₃ ^? pool. Therefore it was concluded that this process is most probably responsible for the limited NO₃ ^? leaching from the forest ecosystem, despite the chronically high N deposition rates.     

Net sediment N2 fluxes in a southern New England estuary: variations in space and time

Over the past three decades, Narragansett Bay has undergone various ecological changes, including significant decreases in water column chlorophyll a concentrations, benthic oxygen uptake, and benthic nutrient regeneration rates. To add to this portrait of change, we measured the net flux of N₂ across the sediment–water interface over an annual cycle using the N₂/Ar technique at seven sites in the bay for comparison with measurements made decades ago. Net denitrification rates ranged from about 10–90 μmol N₂–N m^?2 h^?1 over the year. Denitrification rates were not significantly different among sites and had no clear correlation with temperature. Net nitrogen fixation (?5 to ?650 μmol N₂–N m^?2 h^?1) was measured at three sites and only observed in summer (June–August). Neither denitrification nor nitrogen fixation exhibited a consistent relationship with sediment oxygen demand or with fluxes of nitrite, nitrate, ammonium, total dissolved inorganic nitrogen, or dissolved inorganic phosphate across all stations. In contrast to the mid-bay historical site where denitrification rates have declined, denitrification rates in the Providence River Estuary have not changed significantly over the past 30 years.     

Biogeochemical processes at the sediment–water interface, Bombah Broadwater, Myall Lakes

Myall Lakes has experienced algal blooms in recent years which threaten water quality. Biomarkers, benthic fluxes measured with chambers, and pore water metabolites were used to identify the nature and reactivity of organic matter (OM) in the sediments of Bombah Broadwater (BB), and the processes controlling sediment-nutrient release into the overlying waters. The OM in the sediments was principally from algal sources although terrestrial OM was found near the Myall River. Terrestrial faecal matter was identified in muddy sediments and was probably sourced via runoff from farm lands. The reactive OM which released nutrients into the overlying waters was from diatoms, dinoflagellates and probably cyanobacteria. Microcystis filaments were observed in surface sediments. OM degradation rates varied between 5.3 and 47.1 mmol m^?2 day^?1 (64–565 mg m^?2 day^?1), were highest in the muddy sediments and sulphate reduction rates accounted for 20–40% of the OM degraded. Diatoms, being heavy sink rapidly, and are an important vector to transport catchment N and P to sites of denitrification and P-trapping in the sediments. Denitrification rates (mean ～4 mmol N m^?2 day^?1), up to 7 mmol N m^?2 day^?1 (105 mg N m^?2 day^?1) were measured, and denitrification efficiencies were highest (mean = 86 ± 4%) in the sandy sediments (～20% of the area of BB), but lower in the muddy sediments (mean = 63 ± 15%). These differences probably result from higher OM loads and anaerobic respiration in muddy sediments. Most DIP (>70%) from OM degradation was not released into overlying waters but remained trapped in surface sediments. Biophysical (advective) processes were responsible for the measured metabolite (O₂, CO₂, DSi, DIN and DIP) fluxes across the sediment–water interface.     

A low molecular mass cutinase of Thielavia terrestris efficiently hydrolyzes poly(esters)

A low molecular mass cutinase (designated TtcutA) from Thielavia terrestris was purified and biochemically characterized. The thermophilic fungus T. terrestris CAU709 secreted a highly active cutinase (90.4 U ml^?1) in fermentation broth containing wheat bran as the carbon source. The cutinase was purified 19-fold with a recovery yield of 4.8 %. The molecular mass of the purified TtcutA was determined as 25.3 and 22.8 kDa using SDS-PAGE and gel filtration, respectively. TtcutA displayed optimal activity at pH 4.0 and 50 °C. It was highly stable up to 65 °C and in the broad pH range 2.5–10.5. Extreme stability in high concentrations (80 %, v/v) of solvents such as methanol, ethanol, acetone, acetonitrile, isopropanol, and dimethyl sulfoxide was observed for the enzyme. The K _m values for this enzyme towards p-nitrophenyl (pNP) acetate, pNP butyrate, and pNP caproate were 7.7, 1.0, and 0.52 mM, respectively. TtcutA was able to efficiently degrade various ester polymers, including cutin, polyethylene terephthalate (PET), polycaprolactone (PCL), and poly(butylene succinate) (PBS) at hydrolytic rates of 3 μmol h^?1 mg^?1 protein, 1.1 mg h^?1 mg^?1 protein, 203.6 mg h^?1 mg^?1 protein, and 56.4 mg h^?1 mg^?1 protein, respectively. Because of these unique biochemical properties, TtcutA of T. terrestris may be useful in various industrial applications in the future.     

Biodegradation of 4-nitrotoluene with biosurfactant production by Rhodococcus pyridinivorans NT2: metabolic pathway, cell surface properties and toxicological characterization

A novel 4-nitrotoluene-degrading bacterial strain was isolated from pesticides contaminated effluent-sediment and identified as Rhodococcus pyridinivorans NT2 based on morphological and biochemical properties and 16S rDNA sequencing. The strain NT2 degraded 4-NT (400 mg l^?1) with rapid growth at the end of 120 h, reduced surface tension of the media from 71 to 29 mN m^?1 and produced glycolipidic biosurfactants (45 mg l^?1). The biosurfactant was purified and characterized as trehalose lipids. The biosurfactant was stable in high salinity (10 % w/v NaCl), elevated temperatures (120 °C for 15 min) and a wide pH range (2.0–10.0). The noticeable changes during biodegradation were decreased hydrophobicity; an increase in degree of fatty acid saturation, saturated/unsaturated ratio and cyclopropane fatty acid. Biodegradation of 4-NT was accompanied by the accumulation of ammonium (NH₄ ⁺) and negligible amount of nitrite ion (NO₂ ^?). Product stoichiometry showed a carbon (C) and nitrogen (N) mass balance of 37 and 35 %, respectively. Biodegradation of 4-NT proceeded by oxidation at the methyl group to form 4-nitrobenzoate, followed by reduction and hydrolytic deamination yielding protocatechuate, which was metabolized through β-ketoadipate pathway. In vitro and in vivo acute toxicity assays in adult rat (Rattus norvegicus) showed sequential detoxification and the order of toxicity was 4-NT >4-nitrobenzyl alcohol >4-nitrobenzaldehyde >4-nitrobenzoate >> protocatechuate. Taken together, the strain NT2 could be used as a potential bioaugmentation candidate for the bioremediation of contaminated sites.     

Biodegradation of para-nitrophenol by Citricoccus nitrophenolicus strain PNP1T at high pH

A gram-positive bacterium Citricoccus nitrophenolicus (strain PNP1^T, DSM 23311^T, CCUG 59571^T) isolated from a waste water treatment plant was capable of effectively degrading p-nitrophenol (pNP) as a source of carbon, nitrogen and energy for growth. Degradation of pNP required oxygen and resulted in the stoichiometric release of nitrite. Strain PNP1^T also degraded 4-chlorophenol, phenol and salicylate. pNP was degraded at pH values between 6.8 and 10.0 and at temperatures between 15–32 °C. pNP at concentrations up to 150 mg L^?1 were degraded during growth in media at pH ≤ 10, whereas 200 mg L^?1 was completely inhibitory to growth. When incubated in an NH₄Cl-free medium (pH 10) containing both pNP and acetate, pNP is degraded with concomitant release of nitrite which was subsequently assimilated during acetate degradation. Intact cells of strain PNP1^T suspended in NaHCO₃/Na₂CO₃ buffer were able to continuously degrade 200 mg L^?1 pNP over a 40 day period at pH 10.     

Heterotrophic nitrification and aerobic denitrification by a novel Halomonas campisalis

A novel halophilic strain that could carry out heterotrophic nitrification and aerobic denitrification was isolated and named as Halomonas campisalis ha3. It removed inorganic nitrogen compounds (e.g. NO₃ ^?, NO₂ ^? and NH₄ ⁺) simultaneously, and grew well in the medium containing up to 20 % (w/v) NaCl. PCR revealed four genes in the genome of ha3 related to aerobic denitrification: napA, nirS, norB and nosZ. The optimal conditions for aerobic denitrification were pH 9.0, at 37 °C, with 4 % (w/v) NaCl and sodium succinate as carbon source. The nitrogen removal rate was 87.5 mg NO₃ ^?–N l^?1 h^?1. Therefore, this strain is a potential aerobic denitrifier for the treatment of saline wastewater.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

Nitrification and denitrification in a midwestern stream containing high nitrate: in situ assessment using tracers in dome-shaped incubation chambers

The extent to which in-stream processes alter or remove nutrient loads in agriculturally impacted streams is critically important to watershed function and the delivery of those loads to coastal waters. In this study, patch-scale rates of in-stream benthic processes were determined using large volume, open-bottom benthic incubation chambers in a nitrate-rich, first to third order stream draining an area dominated by tile-drained row-crop fields. The chambers were fitted with sampling/mixing ports, a volume compensation bladder, and porewater samplers. Incubations were conducted with added tracers (NaBr and either ¹⁵N[NO₃ ^?], ¹⁵N[NO₂ ^?], or ¹⁵N[NH₄ ⁺]) for 24–44 h intervals and reaction rates were determined from changes in concentrations and isotopic compositions of nitrate, nitrite, ammonium and nitrogen gas. Overall, nitrate loss rates (220–3,560 μmol N m^?2 h^?1) greatly exceeded corresponding denitrification rates (34–212 μmol N m^?2 h^?1) and both of these rates were correlated with nitrate concentrations (90–1,330 μM), which could be readily manipulated with addition experiments. Chamber estimates closely matched whole-stream rates of denitrification and nitrate loss using ¹⁵N. Chamber incubations with acetylene indicated that coupled nitrification/denitrification was not a major source of N₂ production at ambient nitrate concentrations (175 μM), but acetylene was not effective for assessing denitrification at higher nitrate concentrations (1,330 μM). Ammonium uptake rates greatly exceeded nitrification rates, which were relatively low even with added ammonium (3.5 μmol N m^?2 h^?1), though incubations with nitrite demonstrated that oxidation to nitrate exceeded reduction to nitrogen gas in the surface sediments by fivefold to tenfold. The chamber results confirmed earlier studies that denitrification was a substantial nitrate sink in this stream, but they also indicated that dissolved inorganic nitrogen (DIN) turnover rates greatly exceeded the rates of permanent nitrogen removal via denitrification.     

Study the biocatalyzing effect and mechanism of cellulose acetate immobilized redox mediators technology (CE-RM) on nitrite denitrification

The biocatalyzing effect of a novel cellulose acetate immobilized redox mediators technology (CE-RM) on nitrite denitrification process was studied with anthraquinone, 1,8-dichloroanthraquinone, 1,5-dichloroanthraquinone and 1,4,5,8-tetrachloroanthraquinone. The results showed that the immobilized 1,4,5,8-tetrachloroanthraquinone presented the best biocatalyzed effect which increased nitrite denitrification rate to 2.3-fold with 12 mmol/L 1,4,5,8-tetrachloroanthraquinone. The unequal biocatalyzing effect was due to the quantity and position of –Cl substituent in anthraquinone-structure. Moreover, the nitrite denitrification rate was increased with the oxidation reduction potential (ORP) values becoming more negative during the biocatalyzing process. The stabilized ORP value with 12 mmol/L immobilized 1,4,5,8-tetrachloroanthraquinone were 81 mV lower than the control. At the same time, the more OH^? was produced with the higher nitrite removal rate achieved in the nitrite denitrification process. In addition, a positive linear correlation was found between the nitrite removal reaction constants k [gNO₂ ^?–N/(gVSS d)] and immobilized 1,4,5,8-tetrachloroanthraquinone concentration (C _{1,4,5,8-tetrachloroanthraquinone}), which was k = 1.8443 C _{1,4,5,8-tetrachloroanthraquinone} + 33.75(R ² = 0.9411). The initial nitrite concentration of 179 mgNO₂ ^?–N/L resulted in the maximum nitrite removal rate, which was 6.526[gNO₂ ^?–N/(gVSS d)]. These results show that the application of cellulose acetate immobilized redox mediators (CE-RM) can be valuable for increasing nitrite denitrification rate.     

Nutrient Biogeochemistry During the Early Stages of Delta Development in the Mississippi River Deltaic Plain

Nutrient biogeochemistry associated with the early stages of soil development in deltaic floodplains has not been well defined. Such a model should follow classic patterns of soil nutrient pools described for alluvial ecosystems that are dominated by mineral matter high in phosphorus and low in carbon and nitrogen. A contrast with classic models of soil development is the anthropogenically enriched high nitrate conditions due to agricultural fertilization in upstream watersheds. Here we determine if short-term patterns of soil chemistry and dissolved inorganic nutrient fluxes along the emerging Wax Lake delta (WLD) chronosequence are consistent with conceptual models of long-term nutrient availability described for other ecosystems. We add a low nitrate treatment more typical of historic delta development to evaluate the role of nitrate enrichment in determining the net dinitrogen (N₂) flux. Throughout the 35-year chronosequence, soil nitrogen and organic matter content significantly increased by an order of magnitude, whereas phosphorus exhibited a less pronounced increase. Under ambient nitrate concentrations (>60 μM), mean net N₂ fluxes (157.5 μmol N m^?2 h^?1) indicated greater rates of gross denitrification than gross nitrogen fixation; however, under low nitrate concentrations (<2 μM), soils switched from net denitrification to net nitrogen fixation (?74.5 μmol N m^?2 h^?1). As soils in the WLD aged, the subsequent increase in organic matter stimulated net N₂, oxygen, nitrate, and nitrite fluxes producing greater fluxes in more mature soils. In conclusion, soil nitrogen and carbon accumulation along an emerging delta chronosequence largely coincide with classic patterns of soil development described for alluvial floodplains, and substrate age together with ambient nitrogen availability can be used to predict net N₂ fluxes during early delta evolution.     

In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana

We developed a micropropagation protocol for Cleome gynandra, a C₄ model plant with medicinal importance. Surface-sterilized nodal segments obtained from 1 to 2-month-old field grown plant were used as explants for culture establishment and plant regeneration. Multiple shoots differentiated through bud breaking on Murashige and Skoog (MS) medium with different concentrations of benzyladenine (BA) and kinetin (Kin). The optimum shoot differentiation occurred on medium with 1.5 mg l^?1 BA. Out of various concentrations and combinations of cytokinins and auxins, MS medium containing 0.5 mg l^?1 BA and 0.1 mg l^?1 IAA (indole-3-acetic acid) was found best for shoot multiplication. However, the differentiated shoots exhibited hyperhydration, leaf curling and early leaf fall during subculturing. To overcome these problems, regenerated shoots were transferred to the modified MS medium with reduced nitrates (825 mg l^?1 NH₄NO₃ and 950 mg l^?1 KNO₃) and 100 mg l^?1 (NH₄)₂SO₄. The micropropagated shoots were rooted (i) in vitro on one-fourth strength of MS salts with 0.25 mg l^?1 each of IBA (indole-3 butyric acid) and NOA (2-naphthoxyacetic acid) + 100 mg l^?1 activated charcoal, and (ii) ex vitro, by treating the shoot base(s) with 200 mg l^?1 of IBA for 3 min and transferred to soilrite moistened with one-fourth strength of MS macro salts in culture bottles. The plants were hardened in the greenhouse with 85 % survival rate. Micromorphological studies of the plants were conducted during hardening with reference to development and changes in vein spacing, glandular trichome and stomata. In comparison to leaves under in vitro condition, higher density of veins and glandular trichomes was observed in the leaves of hardened plants. In addition, stomata became functional during hardening which were non-functional under in vitro condition.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Plant growth regulators affect biosynthesis and accumulation profile of isoflavone phytoestrogens in high-productive in vitro cultures of Genista tinctoria

The influence of plant growth regulators on biomass growth and the accumulation of medicinally-relevant isoflavone phytoestrogens, derivatives of genistein and daidzein (8 compounds including aglycones, glucosides and glucoside esters) in callus cultures of Genista tinctoria (Fabaceae) was examined. The experiments included 10 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, β-naphthoxyacetic acid, picloram, 2,3,5-triiodobenzoic acid (TIBA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)] and 7 cytokinins [6-benzylaminopurine, forchlorfenuron, 1,3-diphenylurea, 2-isopentenyladenine, kinetin (KIN), thidiazuron, zeatin] applied at 0.5 and 5.0 mg l^?1, jointly with 5.0 or 0.5 mg l^?1 KIN or 2,4-D (for auxins and cytokinins, respectively—36 phytohormone combinations in total). Statistical analysis of the relationships between callus growth [expressed as growth index (Gi)] and the accumulation of isoflavones showed positive correlation in the cytokinin group (r_xy values from 0.13 to 0.61) and negative correlation within auxins (r_xy values from ?0.31 to ?0.39). Among the cytokinins tested, the highest isoflavone content (6,436.26 mg/100 g dry weight) and the fastest biomass growth (Gi = 892.46 %) were obtained for 0.5 mg l^?1 KIN used jointly with 5.0 mg l^?1 2,4-D. In the group of auxins, the combination of 0.5 mg l^?1 TIBA and 5.0 mg l^?1 KIN provided the fastest culture growth (Gi = 983.07 %) and the isoflavone concentration of 10,474.23 mg/100 g dry weight, which is so far the highest amount of these metabolites achieved in callus cultures of higher plants.     

Soil N and C trace gas fluxes and microbial soil N turnover in a sessile oak (Quercus petraea (Matt.) Liebl.) forest in Hungary

During two intensive field campaigns in summer and autumn 2004 nitrogen (N₂O, NO/NO₂) and carbon (CO₂, CH₄) trace gas exchange between soil and the atmosphere was measured in a sessile oak (Quercus petraea (Matt.) Liebl.) forest in Hungary. The climate can be described as continental temperate. Fluxes were measured with a fully automatic measuring system allowing for high temporal resolution. Mean N₂O emission rates were 1.5 μg N m⁻² h⁻¹ in summer and 3.4 μg N m⁻² h⁻¹ in autumn, respectively. Also mean NO emission rates were higher in autumn (8.4 μg N m⁻² h⁻¹) as compared to summer (6.0 μg N m⁻² h⁻¹). However, as NO₂ deposition rates continuously exceeded NO emission rates (−9.7 μg N m⁻² h⁻¹ in summer and −18.3 μg N m⁻² h⁻¹ in autumn), the forest soil always acted as a net NO _x sink. The mean value of CO₂ fluxes showed only little seasonal differences between summer (81.1 mg C m⁻² h⁻¹) and autumn (74.2 mg C m⁻² h⁻¹) measurements, likewise CH₄uptake (summer: −52.6 μg C m⁻² h⁻¹; autumn: −56.5 μg C m⁻² h⁻¹). In addition, the microbial soil processes net/gross N mineralization, net/gross nitrification and heterotrophic soil respiration as well as inorganic soil nitrogen concentrations and N₂O/CH₄ soil air concentrations in different soil depths were determined. The respiratory quotient (ΔCO_{2 resp} ΔO_{2 resp}⁻¹) for the uppermost mineral soil, which is needed for the calculation of gross nitrification via the Barometric Process Separation (BaPS) technique, was 0.8978 ± 0.008. The mean value of gross nitrification rates showed only little seasonal differences between summer (0.99 μg N kg⁻¹ SDW d⁻¹) and autumn measurements (0.89 μg N kg⁻¹ SDW d⁻¹). Gross rates of N mineralization were highest in the organic layer (20.1–137.9 μg N kg⁻¹ SDW d⁻¹) and significantly lower in the uppermost mineral layer (1.3–2.9 μg N kg⁻¹ SDW d⁻¹). Only for the organic layer seasonality in gross N mineralization rates could be demonstrated, with highest mean values in autumn, most likely caused by fresh litter decomposition. Gross mineralization rates of the organic layer were positively correlated with N₂O emissions and negatively correlated with CH₄ uptake, whereas soil CO₂ emissions were positively correlated with heterotrophic respiration in the uppermost mineral soil layer. The most important abiotic factor influencing C and N trace gas fluxes was soil moisture, while the influence of soil temperature on trace gas exchange rates was high only in autumn.     

Applicability of one-stage partial nitritation and anammox in MBBR for anaerobically pre-treated municipal wastewater

Energy consumption of municipal wastewater treatment plants can be reduced by the anaerobic pre-treatment of the main wastewater stream. After this pre-treatment, nitrogen can potentially be removed by partial nitritation and anammox (PN/A). Currently, the application of PN/A is limited to nitrogen-rich streams (>500 mg L^?1) and temperatures 25–35 °C. But, anaerobically pretreated municipal wastewater is characterized by much lower nitrogen concentrations (20–100 mg L^?1) and lower temperatures (10–25 °C). We operated PN/A under similar conditions: total ammonium nitrogen concentration 50 mg L^?1 and lab temperature (22 °C). PN/A was operated for 342 days in a 4 L moving bed biofilm reactor (MBBR). At 0.4 mg O₂ L^?1, nitrogen removal rate 33 g N m^?3 day^?1 and 80 % total nitrogen removal efficiency was achieved. The capacity of the reactor was limited by low AOB activity. We observed significant anammox activity (40 g N m^?3 day^?1) even at 12 °C, improving the applicability of PN/A for municipal wastewater treatment.     

Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose

Carbon distribution and kinetics of Candida shehatae were studied in fed-batch fermentation with xylose or glucose (separately) as the carbon source in mineral medium. The fermentations were carried out in two phases, an aerobic phase dedicated to growth followed by an oxygen limitation phase dedicated to ethanol production. Oxygen limitation was quantified with an average specific oxygen uptake rate (OUR) varying between 0.30 and 2.48 mmolO₂ g dry cell weight (DCW)^?1 h^?1, the maximum value before the aerobic shift. The relations among respiration, growth, ethanol production and polyol production were investigated. It appeared that ethanol was produced to provide energy, and polyols (arabitol, ribitol, glycerol and xylitol) were produced to reoxidize NADH from assimilatory reactions and from the co-factor imbalance of the two-first enzymatic steps of xylose uptake. Hence, to manage carbon flux to ethanol production, oxygen limitation was a major controlled parameter; an oxygen limitation corresponding to an average specific OUR of 1.19 mmolO₂ g DCW^?1 h^?1 allowed maximization of the ethanol yield over xylose (0.327 g g^?1), the average productivity (2.2 g l^?1 h^?1) and the ethanol final titer (48.81 g l^?1). For glucose fermentation, the ethanol yield over glucose was the highest (0.411 g g^?1) when the specific OUR was low, corresponding to an average specific OUR of 0.30 mmolO₂ g DCW^?1 h^?1, whereas the average ethanol productivity and ethanol final titer reached the maximum values of 1.81 g l^?1 h^?1 and 54.19 g l^?1 when the specific OUR was the highest.     

Influence of hydrological connectivity of riverine wetlands on nitrogen removal via denitrification

Wetland ecosystems in agricultural areas often become progressively more isolated from main water bodies. Stagnation favors the accumulation of organic matter as the supply of electron acceptors with water renewal is limited. In this context it is expected that nitrogen recycling prevails over nitrogen dissipation. To test this hypothesis, denitrification rates, fluxes of dissolved oxygen (SOD), inorganic carbon (DIC) and nitrogen and sediment features were measured in winter and summer 2007 on 22 shallow riverine wetlands in the Po River Plain (Northern Italy). Fluxes were determined from incubations of intact cores by measurement of concentration changes or isotope pairing in the case of denitrification. Sampled sites were eutrophic to hypertrophic; 10 were connected and 12 were isolated from the adjacent rivers, resulting in large differences in nitrate concentrations in the water column (from <5 to 1,133 μM). Benthic metabolism and denitrification rates were investigated by two overarching factors: season and hydrological connectivity. SOD and DIC fluxes resulted in respiratory quotients greater than one at most sampling sites. Sediment respiration was coupled to both ammonium efflux, which increased from winter to summer, and nitrate consumption, with higher rates in river-connected wetlands. Denitrification rates measured in river-connected wetlands (35–1,888 μmol N m^?2 h^?1) were up to two orders of magnitude higher than rates measured in isolated wetlands (2–231 μmol N m^?2 h^?1), suggesting a strong regulation of the process by nitrate availability. These rates were also significantly higher in summer (9–1,888 μmol N m^?2 h^?1) than in winter (2–365 μmol N m^?2 h^?1). Denitrification supported by water column nitrate (D_W) accounted for 60–100% of total denitrification (Dtot); denitrification coupled to nitrification (D_N) was probably controlled by limited oxygen availability within sediments. Denitrification efficiency, calculated as the ratio between N removal via denitrification and N regeneration, and the relative role of denitrification for organic matter oxidation, were high in connected wetlands but not in isolated sites. This study confirms the importance of restoring hydraulic connectivity of riverine wetlands for the maintenance of important biogeochemical functions such as nitrogen removal via denitrification.