Further reading in geomicrobiology

Microbial volatilization of selenium (Se) may be an effective bioremediation technique to remove Se from dewatered sediments. In this laboratory study, soil management parameters (wetting and drying cycles, aeration, mixing, aggregate size, and water quality) were assessed for their influence upon Se volatilization. Selenium volatilization rates were higher under continuously moist conditions (—33 kPa) compared with wetting and drying cycles. After 6 months of incubation, a continuously moist seleniferous soil had lost approximately 21% of the Se inventory, whereas the same soil incubated under wetting and drying cycles had dissipated 7% of the total Se. Incubation under anoxia (N₂ atmosphere) increased evolution of dimethyl selenide (DMSe) 1.4‐fold compared with aerated conditions. When soil samples were incubated under static versus continuously mixed conditions, the latter treatment enhanced volatilization 1.8‐fold. This was attributed to increased availability of the Se to the methylating soil microbiota. The optimum aggregate size to promote volatilization of Se was 0.53 mm when compared to 0.15, 1, and 2 mm. The application of saline well water (7.5 dS m^‐1) over 6 months, compared with deionized water, had little effect on volatilization rates of Se from a highly saline (22 dS mr^‐1) seleniferous dewatered sediment. Each of these parameters should be considered in promoting volatilization of Se as a bioremediation approach in the cleanup of seleniferous sediments.     

Effect of fertilization on selenium content of tea and the nutritional function of Se-enriched tea in rats

The present study examined the effect of fertilization with sodium selenite on the selenium content of tea and the nutritional function of Se-enriched tea. Selenium content of tea leaves was increased up to 0.36 g g^–1 by the application of sodium selenite to soil at 0.5 and 1.0 kg Se ha^–1. Application by a Se-enriched organic manure at a rate of 0.5 kg Se ha^–1 provided a higher biological availability of selenium for plant uptake compared with a similar amount of sodium selenite. Foliar spray of sodium selenite at 50–100 g Se ha^–1 increased the selenium content to 0.32–1.45 g g^–1 in tea leaves sampled at the 8–26 days after spraying. Selenium content in the blood and liver, glutathione peroxidase activity in blood of rats were significantly enhanced by feeding of an extracted solution of Se-enriched tea leaves and sodium selenite. Glutathione peroxidase activity in liver of rats fed with Se-enriched tea was higher than that fed with sodium selenite, indicating that the selenium in Se-enriched tea leaves is a more effective Se source than sodium selenite. Increasing the Se level in food products through the application of a selenium fertilizer is a safe, effective and feasible means of increasing the selenium intake of human and animals in low selenium areas of China.     

Diurnal changes in nitric oxide emissions from conventional tillage and pasture sites in the Amazon Basin: influence of soil temperature

We have investigated a subset of restoration practices applied to a degraded pasture at Fazenda Nova Vida, a 22000 ha cattle ranch in Rond^onia, Brazil. Nitric oxide (NO) and carbon dioxide (CO₂) emissions from soils were measured in conventional tillage and current pasture sites to assess N and C losses. Mean daily NO emissions from tilled plots were at least twice those from the pasture. Nitric oxide emissions from the tilled sites showed a strong diurnal pattern, while those from the pasture sites did not. Mean daytime NO emissions from the tilled sites were 9.7 g NO-N m^–2 h^–1, while mean nighttime emissions were 29.7 g NO-N m^–2 h^–1. In the pasture sites, NO emissions were 7.6 g NO-N m^–2 h^–1 during the day, and 7.7 g NO-N m^–2 h^–1 at night. Surface soil temperature was a good inverse predictor (r ²=0.75) of NO emissions from the tilled sites. Carbon dioxide emissions from the tilled sites were generally larger than CO₂ emissions from the pasture sites. The mean CO₂ emission rate from the tilled sites was 179 mg C m^–2 h^–1, while it was 123 mg C m^–2 h^–1 from the pasture sites. There was no distinct diurnal pattern for CO₂ emissions. We found that the very high temperatures measured at the soil surface in the tillage plots, in the range of 40–45°C, reduced the rate of NO emission. The reduction in NO emissions may be because of the sensitivity of autotrophic nitrifiers to high temperatures. This study provides insights on how land-use change may alter regional NO fluxes by exposing certain microbial communities to extreme environmental conditions. Future studies of NO emissions in tropical agricultural systems where soils are bare for extend periods need to make diurnal measurements or the daily fluxes will be substantially underestimated.     

Methane and nitrous oxide exchange in differently fertilised grassland in southern Germany

We examined the effect of fertilisation (200 kg cattle slurry-N ha^–1 year^–1) on the exchange of N₂O and CH₄ in the soil–plant system of meadow agroecosystems in southern Germany. From 1996 to 1998, we regularly determined the gas fluxes (closed chamber method) and associated environmental parameters. N₂O and CH₄ fluxes were not significantly affected by fertilisation. N₂O fluxes at the unfertilised and fertilised plots were small, generally between 50 and –20 g N m^–2 h^–1. We identified some incidents of N₂O uptake. CH₄-C fluxes ranged from 1.3 to –0.2 mg m^–2 h^–1 and were not significantly different from 0 at both plots. We budgeted an annual net emission of 15.5 and 29.6 mg m^–2 N₂O-N and an annual CH₄-C net emission of 184.2 and 122.7 mg m^–2 at the unfertilised and fertilised plots, respectively. Apparently, rapid N mineralization and uptake in the densely rooted topsoil prevents N losses and the inhibition of CH₄ oxidation.     

Phytoextraction of selenium from soils irrigated with selenium-laden effluent

This two-part study compared the efficacy of different plant species to extract Se from soils irrigated with Se-laden effluent. The species used were: Brassica napus L. (canola), Brassica juncea Czern L. and Coss (Indian mustard), and Hordeum vulgare L. (barley). In Study 1 we irrigated the plants with a saline effluent containing 0.150 mg Se L^–1, while in Study 2, the same species were planted in a saline soil selenized with 2 mg Se L^–1. Plants were simultaneously harvested 120 days after planting. In Study 1, there were only slight effects of treatment on dry matter (DM) yield. Plant Se concentrations averaged 21 g Se g^–1DM for the Brassica species, and 4.0 g Se g^–1 DM for barley. Total Se added to soils via effluent decreased by 40% for Brassica species and by 20% for barley. In Study 2, total DM decreased for all species grown in saline soils containing Se. Plant Se concentrations averaged 75 g g^–1 DM for Brassica species and 12 g Se g^–1 DM for barley. Total Se added to soils prior to planting decreased by 40% for Brassica species and up to 12% for barley. In both studies, plant accumulation of Se accounted for at least 50% of the Se removed in soils planted to Brassica and up to 20% in soils planted to barley. Results show that although the tested Brassica species led to a significant reduction in Se added to soil via use of Se-laden effluent, additional plantings are necessary to further decrease Se content in the soil.     

Pioneer and late stage tropical rainforest tree species (French Guiana) growing under common conditions differ in leaf gas exchange regulation,carbon isotope discrimination and leaf water potential

Leaf gas exchange rates, predawn _wp and daily minimum _wm leaf water potentials were measured during a wet-to-dry season transition in pioneer (Jacaranda copaia, Goupia glabra andCarapa guianensis) and late stage rainforest tree species (Dicorynia guianensis andEperua falcata) growing in common conditions in artificial stands in French Guiana. Carbon isotope discrimination () was assessed by measuring the stable carbon isotope composition of the cellulose fraction of wood cores. The values were 2.7 higher in the pioneer species than in the late stage species. The calculated time integratedC _i values derived from the values averaged 281 mol mol^–1 in the pioneers and 240 mol mol^–1 in the late stage species. The corresponding time-integrated values of intrinsinc water-use efficiency [ratio CO₂ assimilation rate (A)/leaf conductance (g)] ranged from 37 to 47 mmol mol^–1 in the pioneers and the values were 64 and 74 mmol mol^–1 for the two late stage species. The high values were associated—at least inJ. copaia—with high maximumg values and with high plant intrinsinc specific hydraulic conductance [Cg/(_wm–_wp], which could reflect a high competitive ability for water and nutrient uptake in the absence of soil drought in the pioneers. A further clear discriminating trait of the pioneer species was the very sensitive stomatal response to drought in the soil, which might be associated with a high vulnerability to cavitation in these species. From a methodological point of view, the results show the relevance of for distinguishing ecophysiological functional types among rainforest trees.     

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD₅₀ (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of ¹O₂. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN₃, a ¹O₂ scavenger. Based on a quenching study, the rate constant of the reaction of ¹O₂ with NaN₃ was determined to be 1.5–3.5 M^–1 s^–1, which is close to the reported value (3.8×10⁸ M^–1 s^–1). The intensity of the ¹O₂-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 s) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 s), coproporphyrin I (50 s), or coproporphyrin III (36 s). These results demonstrate the photodynamic generation of ¹O₂ by Zincphyrin.     

Application of N-15 dilution for simultaneous estimation of nitrification and nitrate reduction in soil-water columns

Nitrification and denitrification rates were estimated simultaneously in soil-floodwater columns of a Crowley silt loam (Typic Albaqualfs) rice soil by an¹⁵N isotopic dilution technique. Labeled NO ₃ ^– was added to the floodwater of soil-water columns, half were treated with urea fertilizer. The (NO ₃ ^– +NO ₂ ^– )–N and (NO ₃ ^– +NO ₂ ^– )–N concentrations in the floodwater were measured over time and production and reduction rates for NO ₃ ^– calculated. Nitrate reduction in the urea amended columns averaged 515 mol N m^–2h^–1 and nitrification averaged 395 mol N m^–2h^–1 over the 35–153 d incubation. The nitrification rate for 4–19 d sampling period (1,560 mol N m^–2h^–1) in the urea amended columns was almost 9 times greater than the reduction rate (175 mol N m^–2h^–1) over the same period. Without the addition of urea the NO ₃ ^– production rate averaged 32 mol N m^–2h^–1 and reduction 101 mol N m^–2h^–1.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Concentrations of heavy metals and related trace elements in some Ethiopian rift-valley lakes and their in-flows

Concentrations of heavy metals commonly known to impact the environment and other related trace elements were quantified in the water bodies of nine Ethiopian rift-valley lakes and six rivers (their inflows) as well as in effluents from two factories. In about half of the samples the concentrations of As was 10–700 g l^–1 and Se, ranged from 10 to 28 g l^–1, were much higher than the maximum permissible level (MPL) according to international standards for drinking water. Mercury (Hg) was detected in four lakes and one river with high values ranging from 2 to 165 g l^–1. Concentrations of Mo in three soda lakes were as high as 544–2590 g l^–1. Iron (Fe) was found in high concentrations (567–4969 g l^–1) in three lakes, which are known to be discolored from inorganic colloids. Levels of Cd, Pb, and Cr ranged between 5 and 9, 12 and 20 and 104 and 121 g l^–1, respectively. The rest of the heavy metals analyzed, Ba, Cu, Mn, Ni and Zn, were either not detected in the samples or were found in much lower concentrations than the MPL for drinking water. Effluent from a tannery contained about 15, 141, 523, and 19 g l^–1 of As, Cr, Fe, and Se, respectively, and effluent from a textile factory contained high concentrations of As (10.6), Hg (3.8) and Se (20) g l^–1. Compared to more industrialized regions and other African lakes the concentrations of heavy metals in Ethiopian rift-valley lakes (with the exception of the soda lakes) and their inflows were low.     

Mercury budget of an upland-peatland watershed

Inputs, outputs, and pool sizes oftotal mercury (Hg) were measured in a forested 10 hawatershed consisting of a 7 ha hardwood-dominatedupland surrounding a 3 ha conifer-dominatedpeatland. Hydrologic inputs via throughfall andstemflow, 13±0.4 g m^–2 yr^–1over the entire watershed, were about doubleprecipitation inputs in the open and weresignificantly higher in the peatland than in theupland (19.6 vs. 9.8 g m^–2 yr^–1). Inputs of Hg via litterfall were 12.3±0.7g m^–2 yr^–1, not different in thepeatland and upland (11.7 vs. 12.5 g m^–2yr^–1). Hydrologic outputs via streamflow were2.8±0.3 g m^–2 yr^–1 and thecontribution from the peatland was higher despiteits smaller area. The sum of Hg inputs were lessthan that in the overstory trees, 33±3 gm^–2 above-ground, and much less than eitherthat in the upland soil, 5250±520 gm^–2, or in the peat, 3900±100 gm^–2 in the upper 50 cm. The annual flux of Hgmeasured in streamflow and the calculated annualaccumulation in the peatland are consistent withvalues reported by others. A sink for Hg of about20 g m^–2 yr^–1 apparently exists inthe upland, and could be due to either or bothstorage in the soil or volatilization.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

The Biogeochemical Cycle of Methane in the Coastal Zone and Littoral of the Kandalaksha Bay of the White Sea

Microbiological and biogeochemical investigations of the processes of methane production (MP) and methane oxidation (MO) in the coastal waters and littoral of the Kandalaksha Bay of the White Sea were carried out. The studies were conducted in the coastal zones and in the water areas of the Kandalaksha Preserve, Moscow State University White Sea Biological Station, and the Zoological Institute (RAS) biological station in August 1999, 2000, and 2001 and in March 2001. The rate of CO₂ assimilation in the shallow and littoral sediments was 35–27800 g C/(dm³ day) in summer and 32.8–88.9 g C/(dm³ day) in winter. The maximal rates of MP were observed in the littoral sediments in the zone of macrophyte decomposition, in local depressions, and in the estuary of a freshwater creek (up to 113 l/(dm³ day)). The maximal level of MO was observed in the shallow estuarine sediments (up to 2450 l/(dm³ day)). During the winter season, at the temperature of –0.5 to 0.5°C, the MP rate in the littoral sediments was 0.02–0.3 l/(dm³ day), while the MO rate was 0.06–0.7 l/(dm³ day). The isotopic data obtained indicate that the C_org of the mats and of the upper sediment layers is enriched with the heavy ¹³C isotope by 1–4 as compared to the C_org of the suspension. A striking difference was found between the levels of methane emission by the typical littoral microlandscapes. In fine sediments, the average emission was 675 l CH₄/(m² day); in stormy discharge stretch sediments, it was 1670 l CH₄/(m² day); and under stones and in silted pits, 1370 l CH₄/(m²day). The calculation, performed with consideration of the microlandscape areas with a high production, allowed the CH₄ production of 1 km² of the littoral to be estimated as 192–300 l CH₄/(km² day).     

Selenium modulates the activities of antioxidant enzymes,osmotic homeostasis and promotes the growth of sorrel seedlings under salt stress

Using physiological assays coupled with ultrathin tissue sections, we investigated the impacts of exogenous selenium (Se) on the growth, antioxidant enzymes, osmotic regulation and ultrastructural modifications of leaf mesophyll and root tip cells of 100 mM NaCl-stressed sorrel (Rumex patientia × R. tianshanicus) seedlings. At low concentrations (1–5 M), Se tended to stimulate the growth, the activities of superoxide dismutase and peroxidase enzymes, as well as the accumulation of water-soluble sugar in leaves of sorrel seedlings. At higher concentrations (10–30 M), Se exerted diminished beneficial effects on growth and enzyme activities. CAT activity did not change with Se addition (1–30 M). Electrolyte leakage of leaf cells declined, and K⁺ and Na⁺ ions increased in leaves with Se treatment, notably at 5 M of Se. TEM observations revealed that treatment with 5 M of Se positively promoted the integrity of membrane systems and cellular organelles, such as chloroplasts and mitochondria in leaf mesophyll and root tip cells. These results strongly suggest that an appropriate concentration of exogenous Se functions positively to promote the antioxidative and osmoregulatory capacity, and enhance the salt-resistance in sorrel seedlings.     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Phosphate uptake capacity of summer phytoplankton of the Loosdrecht Lakes in relation to phosphorus loading and irradiance

Summer populations of the phytoplankton of the Loosdrecht Lakes were enclosed in laboratory scale enclosures (LSE), supplied with 7.5 g P.l^–1.d^–1 and 105 g P.l^–1.d^–1, respectively. The maximum initial phosphate uptake rate (V_m) was related to irradiance and primary production. At phosphate uptake saturating light-irradiance V_m values up to 4 times the V_m values in the dark were measured.The phosphate uptake capacity per unit dry weight remained more or less constant throughout the experiments in the LSE receiving the lower amount of phosphorus, and declined in the LSE receiving the higher amount of phosphorus. Within the range of V_m values measured (<10 g P.mg DW^–1.h^–1 or 1,3 g P. g chla ^–1.h^–1), the growth rate of the phytoplankton was not influenced by alterations in phosphorus availability.     

Laminaria culture for reduction of dissolved inorganic nitrogen in salmon farm effluent

Finfish culture is a growing industry, and it causes a nutrient loading problem. To investigate the feasibility of an integrated culture of kelp and salmon, 15-cm long kelp (Laminaria saccharina) was grown in salmon culture effluent. The objectives were to test the effects of flow rate and kelp density on dissolved inorganic nitrogen removal (DIN), and DIN uptake and growth by the kelp. NH ₄ ⁺ , NO ₃ ^– and DIN (NH ₄ ⁺ + NO ₃ ^– ) loadings were in the ranges 6.2–25.4, 12.9–40.0, 19.7–52.7 mol 1^–1, respectively, over the experimental period.Surplus uptake of nitrogen was not evident, because the C:N ratio (10–11) was constant in all experiments. During light periods, the kelp removed from 170–339 mol 1^–1 h^–1, and approximately 26–40% of the incoming DIN. The DIN uptake rate, based on daylight sampling periods, ranged between 6.1–22.5 mol g^–1 dry mass h^–1. The highest-flow rate, lowest-density tank had the highest DIN uptake rate. Debris from the fish effluent settling on the kelp thalli in the low-flow rate tanks affected uptake. Mean DIN uptake rate based on 3 days of growth for all flow-density combinations ranged between 5.4–8.3 mol g^–1 dry mass h^–1. The kelp utilized NH ₄ ⁺ and NO ₃ ^– equally.The growth ranged between 6.5–9% d^–1. The biomass production ranged from 1–2 g per sampling period. The highest growth rate and biomass production were achieved by kelp in the highest-flow rate, lowest-density tank. Lower DIN concentrations due to higher DIN removal rates in the other tanks and light limitation due to self-shading in the high-density tanks were probably responsible for the reduced growth rate in these tanks.Author for correspondence     

Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis

In order to maintain axenic seedstock cultures axenically of thecommercially important red seaweed, Porphyra yezoensis, aprocedure was developed for axenic isolation and culture of conchocelis andmonospores. For axenic isolation of the conchocelis, contaminated microalgaewere most effectively removed by filtering contaminated samples through a100-m mesh after sonication. Removal of bacteria and otheralgaewas accomplished using a mixture of 5 agents (0.02% chitosan, 100 gml^–1 GeO₂, 10 gml^–1 ampicillin, 40 gml^–1 kanamycin and 200 gml^–1 streptomycin). Axenic single colonies wereisolatedfrom a semi-solid medium prepared from 1% transfer gel. After collectingmonospores from the 40–50% density layer on a percoll-gradient, removalofbacteria and fungi from the monospores was accomplished using a mixture of 5antibiotics (3.5 g ml^–1 nystatin, 2 mgml^–1 ampicillin, 400 gml^–1 kanamycin, 50 gml^–1 neomycin and 800 gml^–1 streptomycin). Axenic single juvenile blades wereisolated from a semi-solid medium prepared from 0.5% transfer gel.     

Compartmentation and flux characteristics of nitrate in spruce

The radiotracer¹³N was used to undertake compartmental analyses for NO ₃ ^– in intact non-mycorrhizal roots ofPicea glauca (Moench) Voss. seedlings. Three compartments were defined, with half-lives of exchange of 2.5 s, 20 s, and 7 min. These were identified as representing surface adsorption, apparent free space, and cytoplasm, respectively. Influx, efflux, and net flux as well as cytoplasmic and apparent-free-space nitrate concentrations were estimated for three different concentration regimes of external nitrate. After exposure to external NO ₃ ^– for 3 d, influx was calculated to be 0.09 mol·g^–1·h^–1 (at 10 M [NO ₃ ^– ]_o), 0.5mol·g^–1·h^–1 (at 100 M [NO _inf3 ^sup– ]_o), and 1.2 mol · g^–1· h^–1 (at 1.5 mM [NO ₃ ^– ]_o). Efflux increased with increasing [NO ₃ ^– ]_o, constituting 4% of influx at 10 M, 6% at 100 M, and 21% at 1.5 mM. Cytoplasmic [NO ₃ ^– ] was estimated to be 0.3 mM at 10 uM [NO ₃ ^– ]_o, 2mM at 100 M [NO ₃ ^– ]_o, and 4mM at 1.5 mM [NO ₃ ^– ]_o, while free-space [NO ₃ ^– ] was 16 M, 173 M, and 2.2 mM, respectively. A series of experiments was carried out to confirm the identity of the compartments resolved by efflux analysis. Pretreatment at high temperature or application of 2-chloro-ethanol, sodium dodecyl sulphate or hydrogen peroxide made it possible to distinguish the metabolic (cytoplasmic) phase from the remaining two (physical) phases. Likewise, varying [Pi] of the medium altered efflux and thereby [NO ₃ ^– ]_cyt, but did not affect [NO ₃ ^– ]_{free space}.Abbreviations and Symbols [NO ₃ ^– ]_cyt cytoplasmic NO ₃ ^– concentration - [NO ₃ ^– ]_{free space} apparent-free-space NO ₃ ^– concentration - [NO ₃ ^– ]_o concentration of NO ₃ ^– in the external solution - NO ₃ ^– flux - _co efflux from the cytoplasm - _oc influx to the cytoplasm - _net net flux - _xylem flux to the xylem - _red/vac combined flux to reduction and the vacuole The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia Campus for providing¹³NO ₃ ^– , Drs. R.D. Guy and S. Silim for providing plant material, and Dr. M.Y. Wang, Mr. J. Mehroke and Mr. P. Poon for assistance in experiments and for helpful discussions.