Colonization dynamics of biofilm-associated ciliate morphotypes at different flow velocities

The impact of flow velocity on initial ciliate colonization dynamics on surfaces were studied in the third order Ilm stream (Thuringia, Germany) at a slow flowing site (0.09 m s^?1) and two faster flowing sites (0.31 m s^?1) and in flow channels at 0.05, 0.4, and 0.8 m s^?1. At the slow flowing stream site, surfaces were rapidly colonized by ciliates with up to 60 cells cm^?2 after 24 h. In flow channels, the majority of suspended ciliates and inorganic matter accumulated at the surface within 4.5 h at 0.05 m s^?1. At 0.4 m s^?1 the increase in ciliate abundance in the biofilm was highest between 72 and 168 h at about 3 cells cm^?2 h^?1. Faster flow velocities were tolerated by vagile flattened ciliates that live in close contact to the surface. Vagile flattened and round filter feeders preferred biofilms at slow flow velocities. Addition of inorganic particles (0, 0.6, and 7.3 mg cm^?2) did not affect ciliate abundance in flow channel biofilms, but small ciliate species dominated and number of species was lowest (16 species cm^?2) in biofilms at high sediment content. Although different morphotypes dominated the communities at contrasting flow velocities, all functional groups contributed to initial biofilm communities implementing all trophic links within the microbial loop.     

Kinematics of cross-country sit skiing during a Paralympic race

The study had three purposes: to verify a hypothesized speed decrease during the 15 km cross-country sit skiing (CCSS) race; documenting this possible fatigue effect (speed decrease), to evaluate changes among the four laps in kinematics parameters (cycle speed, cycle duration, cycle length, duty cycle (percentage ratio between pushing and total cycle duration), pole inclination, trunk inclination and shoulder–hand distance); to compare the kinematics parameters in cross-country sit skiers of different level. Video recordings were carried out during the 2006 Turin Winter Paralympic Games with two conventional digital video-cameras positioned on a flat and an uphill (8.3°) track, respectively. Better performing skiers (G1) had significantly higher speeds than worse performers (G2) both in the flat (6.54 ± 0.13 vs. 5.89 ± 0.50 m s^?1 and 5.55 ± 0.14 vs. 4.62 ± 0.22 m s^?1 in the first and last lap, respectively) and in the uphill track (3.67 ± 0.45 vs. 3.05 ± 0.59 m s^?1 and 3.20 ± 0.36 vs. 2.26 ± 0.36 m s^?1 in the first and last lap, respectively). The G1 athletes were able to maintain the high-speed better than the G2 over the entire race. Significant differences in cycle length and duty cycle between groups would be justified by the higher physical fitness of G1 skiers.     

The harmful alga Aureococcus anophagefferens utilizes 19′-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities

Aureococcus anophagefferens is a picoplanktonic microalga that is very well adapted to growth at low nutrient and low light levels, causing devastating blooms (“brown tides”) in estuarine waters. To study the factors involved in long-term acclimation to different light intensities, cells were acclimated for a number of generations to growth under low light (20 μmol photons m^? 2 s^? 1), medium light (60 or 90 μmol photons m^? 2 s^? 1) and high light (200 μmol photons m^? 2 s^? 1), and were analyzed for their contents of xanthophyll cycle carotenoids (the D pool), fucoxanthin and its derivatives (the F pool), Chls c₂ and c₃, and fucoxanthin Chl a/c polypeptides (FCPs). Higher growth light intensities resulted in increased steady state levels of both diadinoxanthin and diatoxanthin. However, it also resulted in the conversion of a significant fraction of fucoxanthin to 19′-butanoyloxyfucoxanthin without a change in the total F pool. The increase in 19′-butanoyloxyfucoxanthin was paralleled by a decrease in the effective antenna size, determined from the slope of the change in F₀ as a function of increasing light intensity. Transfer of acclimated cultures to a higher light intensity showed that the conversion of fucoxanthin to its derivative was a relatively slow process (time-frame of hours). We suggest the replacement of fucoxanthin with the bulkier 19′-butanoyloxyfucoxanthin results in a decrease in the light-harvesting efficiency of the FCP antenna and is part of the long-term acclimative response to growth at higher light intensities.     

A FRET-based assay for the discovery of West Nile Virus NS2B-NS3 protease inhibitors

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K_m of 3.35 ± 0.31 μM, a k_cat of 0.0717 ± 0.0016 s^?1 and a k_cat/K_m of 21,400 ± 2000 M^?1 s^?1.     

Conduction velocity of the human phrenic nerve in the neck

PurposeTo measure phrenic nerve conduction velocity in the neck in humans.ScopeWe studied 15 healthy subjects (9 men, 32.4 ± 6.7). We performed bipolar electrical phrenic stimulation in the neck, from a distal and a proximal stimulation site, and recorded diaphragm electromyographic responses on the surface of the chest. The ratio of the between-site distance to the latency difference provided phrenic velocities. Ulnar motor velocity was assessed similarly. In addition, five homogeneous patients with Charcot-Marie-Tooth disease type 1A (CMT1A) were studied for validation purposes. We obtained diaphragmatic responses from the two stimulation sites in all cases. The distal latencies (anterior axillary line recording) were 6.51 ± 0.63 ms (right) and 6.13 ± 0.64 ms (left). The minimal between site distance was 39 mm. Phrenic motor velocity was 55.2 ± 6.3 m s^?1 (right) and 56.3 ± 7.2 m s^?1 (left). In CMT1A, phrenic velocities were 17.1 ± 8.1 m s^?1 (from 7 to 32 m s^?1) and were similar to ulnar and median velocities.ConclusionsPhrenic nerve velocities can be estimated in humans and compare with upper limb motor conduction velocities. This should refine the investigation of phrenic function in peripheral neuropathies.     

Effect of light quality on Bacillus amyloliquefaciens JBC36 and its biocontrol efficacy

Light is one of the most important environmental signals regulating physiological processes of many microorganisms. However, very few studies have been reported on the qualitative or quantitative effects of light on control of postharvest spoilage using antagonistic bacteria. In this study, we investigated the effects of white, red, green, and blue light at photon flux densities of 40, 240, and 360 μmol m^?2 s^?1 on Bacillus amyloliquefaciens JBC36 (JBC36), which has been reported as a promising candidate for biocontrol of green and blue mold on mandarin fruit. With the exception of blue light at 240 and 360 μmol m^?2 s^?1, light generally stimulated growth of JBC36 compared to the controls grown in the dark. Red light increased swarming motility irrespective of intensity and significantly enhanced biofilm formation at 240 μmol m^?2 s^?1. Production of antifungal metabolites and antifungal activity on Penicillium digitatum was also affected by light quality. Interestingly, antifungal activity was significantly increased when JBC36 and P. digitatum was co-incubated under red and green light at an intensity of 240 μmol m^?2 s^?1. We also demonstrated that the quality of light resulted in changes in colonization of JBC36 on mandarin fruit and control of green mold. In particular, red light increased the population level on mandarin fruit and biocontrol efficacy against green mold. These results represent the first report on the effect of light quality on an antagonistic bacterium for the control of postharvest spoilage. We believe that an improved understanding of the JBC36 response to light quality may help in the development of strategies to increase biocontrol efficacy of postharvest spoilage.     

Using chitosan-modified clays to control black-bloom-induced black suspended matter in Taihu Lake: Deposition and resuspension of black matter/clay flocs

A cyanobacteria-induced black bloom in Taihu Lake, 2007, subjected nearly one million Wuxi City residents to a drinking water crisis. This black bloom attracted wide attention in China and the rest of the world. However, black bloom is a highly weather-dependent event, and its rapid movement in lakes makes it difficult to predict where it will occur. Therefore, jar-tests and simulated flow experiments were performed to investigate the flocculation, deposition, and resuspension of black-bloom-induced black matter. At a dosage of 0.2 g L⁻¹ chitosan + 1 g L⁻¹ diatomite, 90% of the turbidity was removed within 1 h in jar-tests and in low flow speed simulated experiments. However, the black matter/clay flocs did not fully sink, and stratification of turbidity apparently occurred in the lower part of the tank during the simulated flow experiments. The resuspension experiments under simulated flow speeds showed that at a wind speed of 1–6 m s⁻¹ in Taihu Lake produced currents that did not greatly affect floc resuspension, but a wind speed of 3–4 m s⁻¹ produced waves that could induce floc resuspension. Quartz sand was sprayed over the flocs to verify its effect on inhibiting resuspension. A shear stress of 0.37 N m⁻² did not induce floc resuspension, which indicated that a wind speed of 6 m s⁻¹ would not induce floc resuspension in Taihu Lake.     

Nitrification rates of algal–bacterial biofilms in wastewater stabilization ponds under light and dark conditions

The objective of this study was to investigate nitrification rates in algal–bacterial biofilms of waste stabilization ponds (WSP) under different conditions of light, oxygen and pH. Biofilms were grown on wooden plates of 6.0 cm by 8.0 cm by 0.4 cm in a PVC tray continuously fed with synthetic wastewater with initial NH₄-N and Chemical Oxygen Demand (COD) concentrations of 40 mg l^?1 and 100 mg l^?1, respectively, under light intensity of 85–95 μE m^?2 s^?1. Batch activity tests were carried out by exposure of the plates to light conditions as above (to simulate day time), dim light of 1.8–2.2 μE m^?2 s^?1 (to simulate reduced light as in deeper locations in WSP) and dark conditions (to simulate night time). Dissolved oxygen (DO) concentration and pH were controlled. At some experiments, both parameters were kept constant, and at others they were left to vary as in WSP. Results show biofilm nitrification rates of 945–1817 mg-N m^?2 d^?1 and 1124–1615 mg-N m^?2 d^?1 for light and dark experiments. When the minimum DO was 4.1 mg l^?1, the biofilm nitrification rates under light and dark conditions did not differ significantly at 95% confidence. When the minimum DO in the dim light experiment was 3.2 mg l^?1, the nitrification rates under light and dim light conditions were 945 mg-N m^?2 d^?1 and 563 mg-N m^?2 d^?1 and these significantly differed. Further decrease of DO to 1.1 mg l^?1 under dark conditions resulted in more decrease of the nitrification rates to 156 mg-N m^?2 d^?1. It therefore seems that under these experimental conditions, biofilm nitrification rates are significantly reduced at a certain point when bulk water DO is between 3.2 mg l^?1 and 4.1 mg l^?1. As long as bulk water DO under dark is high, light is not important in influencing the process of nitrification.     

Proteomics analysis of proteins in green alga Haematococcus lacustris (Chlorophyceae) expressed under combined stress of nitrogen starvation and high irradiance

This study is aimed at identifying the proteins that are up-regulated during astaxanthin accumulation in Haematococcus lacustris. For this H. lacustris cells were cultivated in photobioreactors under normal light irradiance of 40 μE m^?2 s^?1 for 6 days and then induced to accumulate astaxanthin for 3 days further by exposure to continuous high irradiance of 200 μE m^?2 s^?1 with fluorescent lamps as light source after the cells reached the stationary phase in a nitrogen-depleted condition. Under this condition, the average astaxanthin content per cell increased from 91 mg/l up to 406 mg/l after 3 days of induction. The proteomics data from a two-dimensional electrophoretic comparison demonstrated that a combination of nitrogen source depletion and 1 h high light have significantly changed the pattern of protein expression in H. lacustris. A total of 49 protein spots were picked after 1 h of stress induction. They consisted of 13 down-regulated proteins and 36 up-regulated proteins. Fifteen proteins which had highly up-regulated expression were further analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results will point toward interesting proteins that can be pursued for further analysis of astaxanthin biosynthesis pathway.     

Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 °C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (k_cat/K_m = 18.5 s^?1 μM^?1) and 2,6-dimethoxyphenol (k_cat/K_m = 13.9 s^?1 μM^?1) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield.     

Thermoimaging as a tool for studying light-induced heating of leaves: Correlation of heat dissipation with the efficiency of photosystem II photochemistry and non-photochemical quenching

Thermoimaging – a highly sensitive and non-invasive method of temperature measurement – was applied to explore the role of changing photosynthetic efficiency in light-induced heating of tobacco (Nicotiana tabacum cv. Samsun) leaves. In the absence of evaporative cooling through the stomata, which was achieved by covering leaves with Vaseline, illumination with 50–1400 μM photons m^?2 s^?1 intensity of photosynthetically active radiation resulted in ≈1–5 °C leaf temperature increase in about 2 min. The heating effect showed a non-linear correlation with the extent of non-photochemical quenching (NPQ) resulting in higher leaf temperatures at higher NPQ values. When leaves were adapted to excessive irradiance (1300 μM photons m^?2 s^?1 for 6 h), which resulted in reduction of photosynthetic efficiency and amplification of NPQ the light-induced heating effect was enhanced. The experimental results have been explained on the basis of a simple theoretical model characterizing the balance of energy fluxes in leaves in relation to the efficiency of photosystem II photochemistry and non-photochemical quenching. The role of alternative energy dissipation pathways outside of PSII in the phenomenon of light-induced leaf heating is also discussed.     

Solids hydrolysis and accumulation in a hybrid anaerobic digester-constructed wetlands system

The properties and behaviour of solids retained in a pilot plant constituted of an up-flow anaerobic sludge blanket (UASB) reactor and two constructed wetlands (CWs) were monitored over a 3-year period. The UASB (25.5 m³) was fed with raw municipal wastewater at a flow rate of 61–112 m³ d^?1 and a volumetric loading rate (VLR) of 0.75–1.70 kg TCOD m^?3 d^?1. The CWs (75 m² each) were operated in series and received a fraction (17–20 m³ d^?1) of the UASB effluent. The applied surface loading rates (SLR) were in the range of 3800–8700 g TCOD m^?2 d^?1 (UASB) and 11–15 g BOD₅ m^?2 d^?1 (CWs). The overall system removed 95% TSS, 85% TCOD and 87% BOD₅ on average. For influent VSS, the UASB removed 72.1% and gave a hydrolysis of 63.5%, while the average surplus sludge generation was 8.7%. Over the 3-year period, TSS and VSS accumulated in the CWs at rates of 1.07 and 0.56 kg m^?2 year^?1, respectively. The aerobic biodegradability of the accumulated solids ranged from 23 to 92 mg O₂ g VSS^?1 d^?1 and increased downstream in the CWs. About 59% of the VSS that entered the CWs was removed by hydrolysis, while 24% accumulated on granular media. These low solids accumulation rates were especially remarkable considering the high COD and BOD₅ loading rates applied. The system lay-out appear to be promising in terms of preventing clogging.     

Nitrate removal and hydraulic performance of organic carbon for use in denitrification beds

Denitrification beds are a cost-effective technology for removing nitrate from point source discharge. To date, field trials and operational beds have primarily used wood media as the carbon source; however, the use of alternative more labile carbon media could provide for increased removal rate, lower installation costs and reduced bed size. While previous laboratory experiments have investigated the potential of alternative carbon sources, these studies were typically of short duration and small scale and did not necessarily provide reliable information for denitrification bed design purposes. To address this issue, we compared nitrate removal, hydraulic and nutrient leaching characteristics of nine different carbon substrates in 0.2 m³ barrels, at 14 and 23.5 °C over a 23-month period. Mean nitrate removal rates for the period 10–23 months were 19.8 and 15 g N m^?3 d^?1 (maize cobs), 7.8 and 10.5 g N m^?3 d^?1 (green waste), 5.8 and 7.8 g N m^?3 d^?1 (wheat straw), 3.0 and 4.9 g N m^?3 d^?1 (softwood), and 3.3 and 4.4 g N m^?3 d^?1 (hardwood) for the 14 and 23.5 °C treatments, respectively. Maize cobs provided a 3–6.5-fold increase in nitrate removal over wood media, without prohibitive decrease in hydraulic conductivity, but had higher rates of nutrient leaching at start-up. Significant difference in removal rate occurred between the 14 and 23.5 °C treatments, with the mean Q₁₀ temperature coefficient = 1.6 for all media types in the period 10–23 months.     

Three-dimensional lumbar segment kinetics of fast bowling in cricket

Cricket fast bowlers have a high incidence of serious lumbar injuries, such as lesions in the pars interarticularis. Kinematic studies have shown that bowling actions with large shoulder counter-rotation are associated with significantly higher incidences of lumbar injury. However, in bowling, there has been no calculation of the spinal loads, which are the causal mechanisms of such injuries. In this study, 21 fast bowlers (22.4±3.9 years) of premier grade level and above were tested using a three-dimensional (3D) motion analysis system. The mean ball release speed was 31.9±2.8 m s^?1 and ranged from 27.0 to 35.6 m s^?1. Kinematics and kinetics were calculated for lumbar spine lateral bending, rotation, and flexion during the delivery and power phases of bowling. Power calculations were used to define the actuation of lumbar spine motion as either active or controlled. The actuation of the lumbar spine was complex, involving multiple sequences of active and controlled motion. In addition, lumbar spine loads were largest during the power phase when the ground reaction forces were highest. In conclusion, the dynamic loads and the cyclical nature of their application when the spine is positioned near its end range of motion may be significant factors of injury to this region. In addition, the lumbar spine in bowling has to vigorously flex, laterally bend and rotate simultaneously in a complex interdependent sequence of actuation patterns. Therefore, any technical change to reduce injury susceptibility needs to consider the mechanics of whole body coordination and timing.     

Influence of the Włocławek Reservoir on hydrochemistry and plankton of a large,lowland river (the Lower Vistula River,Poland)

The research on the W?oc?awek Reservoir (WR), situated on the Lower Vistula River (Poland), was carried out during two periods: between 1986 and 2002, hydrochemistry was studied, and between 1994 and 2000 – plankton. WR is 70 km² in area, its capacity is 370 × 10⁶ m³ and the retention time with the average flow rate (Q) amounts to 930 m³ s^?1 – only 5 days (!). Until 2002, due to cyclic work of a hydroelectric power plant, considerable variability in the daily flow rate was recorded (from 600 to 1800 m³ s^?1). Its specific hydrology and fluvial character mean that WR functions in a completely different way compared to dam reservoirs described in textbooks, with significantly longer retention time.WR significantly improves most of the water quality variables in the river. It reduces the concentration of suspended matter (on average by more than 50%), P_tot (by 16%), BOD₅ (by 40%) and chlorophyll “a” (by 50–60%) (unpublished data).Phytoplankton is rich in species – altogether 441 taxa, with the count of 7.4 × 10⁶ individuals per dm^?3 and biomass of 16 mg dm^?3. Throughout the research period, a significant reduction in the abundance of phytoplankton was recorded in WR, sometimes more than 60%. The total number of zooplankton taxa at the site upstream from WR and in the reservoir was almost identical (68 and 67), however in the reservoir itself, a small decrease in the number of Rotifera species and an increase in the number of Crustacea species was observed, as well as a significant, fourfold increase in the total zooplankton biomass. The aforementioned reduction of phytoplankton and enrichment of the river with Crustacea are one of the major biocenotic consequences of the functioning of the W?oc?awek Reservoir.     

Response of ecosystem CO2 exchange to biomass productivity in a high yield grassland

We measured the biomass production and ecosystem carbon CO₂ exchange in a high yield grassland dominated by Miscanthus sinensis. The experimental grassland is managed by mowing once a year in winter every year and the harvested biomass on the ground is left to become the humus. The maximum aboveground and belowground biomasses were 1117 and 2803 g d.w. m^?2 in our grassland. Although the high potential of our grassland for biomass production led to higher carbon uptake than with other types of grassland, the large biomass contributed to a higher respired carbon loss. Biomass increase led to a linear increase in ecosystem respiration. Over the 3 years, RE₁₀ increased with increasing aboveground biomass. The potential gross primary production at a photosynthetic photon flux density of 2000 μmol m² s^?1 logarithmic increased with LAI. These responses of CO₂ exchange to biomass production suggest this grassland behaved as weak CO₂ sink or near carbon neutral (?78 and 17 g C m^?2 year^?1) in current management.     

Effects of shade treatments on the photosynthetic capacity,chlorophyll fluorescence,and chlorophyll content of Tetrastigma hemsleyanum Diels et Gilg

Tetrastigma hemsleyanum Diels et Gilg was grown under full sunlight and moderate and high levels of shade for one month to evaluate its photosynthetic and chlorophyll fluorescence response to different light conditions. The results showed that T. hemsleyanum attained greatest leaf size and P_n when cultivated with 67% shade. Leaves of seedlings grown with 90% shade were the smallest. Leaf color of plants grown under full sunlight and 50% shade was yellowish-green. The P_n value increased rapidly as PPFD increased to 200 μmol m^?2 s^?1 and then increased slowly to a maximum, followed by a slow decrease as PPFD was increased to 1000 μmol m^?2 s^?1. P_n was highest for the 67% shade treatment and the LSP for this shade treatment was 600 μmol m^?2 s^?1. Full sunlight and 50% shade treatments resulted in significant reduction of ETR and qP and increased NPQ. Chl a, Chl b and total chlorophyll content increased and Chl a/b values decreased with increased shading. Results showed that light intensity greater than that of 50% shade depressed photosynthetic activity and T. hemsleyanum growth. Irradiance less than that of 75% shade limited carbon assimilation and led to decreased plant growth. Approximately 67% shade is suggested to be the optimum light irradiance condition for T. hemsleyanum cultivation.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ～456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T_1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol^?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg²⁺ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) for sodium phytate were 83 nmol mg^?1 s^?1 and 0.156 mM, respectively. The catalytic turnover number (K_cat) and catalytic efficiency (K_cat/K_m) of phytase were 37.8 s^?1 and 2.4 × 10⁵ M^?1 s^?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.     

Nitrate removal and greenhouse gas production in a stream-bed denitrifying bioreactor

Denitrifying bioreactors are currently being tested as an option for treating nitrate (NO₃^?) contamination in groundwater and surface waters. However, a possible side effect of this technology is the production of greenhouse gases (GHG) including nitrous oxide (N₂O) and methane (CH₄). This study examines NO₃^? removal and GHG production in a stream-bed denitrifying bioreactor currently operating in Southern Ontario, Canada. The reactor contains organic carbon material (pine woodchips) intended to promote denitrification. Over a 1 year period, monthly averaged removal of influent (stream water) NO₃^? ranged from 18 to 100% (0.3–2.5 mg N L^?1). Concomitantly, reactor dissolved N₂O and CH₄ production, averaged 6.4 μg N L^?1 (2.4 mg N m^?2 d^?1), and 974 μg C L^?1 (297 mg C m^?2 d^?1) respectively, where production is calculated as the difference between inflow and effluent concentrations. Gas bubbles entrapped in sediments overlying the reactor had a composition ranging from 19 to 64% CH₄, 1 to 6% CO₂, and 0.5 to 2 ppmv N₂O; however, gas bubble emission rates were not quantified in this study. Dissolved N₂O production rates from the bioreactor were similar to emission rates reported for some agricultural croplands (e.g. 0.1–15 mg N m^?2 d^?1) and remained less than the highest rates observed in some N-polluted streams and rivers (e.g. 110 mg N m^?2 d^?1, Grand R., ON). Dissolved N₂O production represented only a small fraction (0.6%) of the observed NO₃^? removal over the monitoring period. Dissolved CH₄ production during summer months (up to 1236 mg C m^?2 d^?1), was higher than reported for some rivers and reservoirs (e.g. 6–66 mg C m^?2 d^?1) but remained lower than rates reported for some wastewater treatment facilities (e.g. sewage treatment plants and constructed wetlands, 19,500–38,000 mg C m^?2 d^?1).