Magnetically treated water for removal of surface contamination by Malathion on Chinese Kale (Brassica oleracea L.).

Malathion® is a persistent organophosphate pesticide used against biting and chewing insects on vegetables. It is a difficult-to-remove surface contaminant of vegetables and contaminates surface and ground water and soils. Malathion® is only partially water soluble, but use of detergent carriers makes adhering Malathion® residues difficult to subsequently remove. Magnetically treated water (MTW) successfully removed Malathion® from Chinese Kale (Brassica oleracea L.), meeting Maximum Residue Load (MRL) standards. Samples were soaked in MTW for 30 min prior to detection with GC/MS/MS, 98.5±3.02% of Malathion® was removed after washing by MTW. Removal by simple washing was only ≈42±1.2% which was not nearly sufficient to meet MRL criteria.

Using dielectrophoretic spectra to identify and separate viable yeast cells.

Immotile yeast cells were transiently moved in nonuniform sinusoidal electric fields using multiple pairs of micro-parallel cylindrical electrodes equipped with a sequential signal generator (SSG) to analyze cell viability at a clinical scale for the brewery/fermentation industry. Living yeast cells of Saccharomyces cerevisiae during the exponential-stationary phase, with a cell density of 1.15 × 105 cells mL-1 were suspended in sucrose medium. The conductivity (σs) was adjusted to 0.01 S m-1 with added KCl. Cells exposed in electric field strengths ranging from 32.89 to 40.98 kV m-1, exhibited positive dielectrophoresis (pDEP) with the lower critical frequencies (LCF) at 85.72 ± 3.59 kHz. The optimized value of LCF was shifted upwards to 780.00 ± 83.67 kHz when σswas increased to 0.10 S m-1. Dielectrophoretic and LCF spectra (translational speed of cells vs. electric field frequencies) of yeast suspensions during positive dielectrophoresis were analyzed in terms of the dielectric properties of the cell membrane and cytoplasm which reflect yeast cell viability and metabolic health status. The dielectrophoretic collection yield of cells using positive dielectrophoresis was reported on the monitor of sequential signal generator software to evaluate the number of living and dead cells through a real-time image processing analyzer. The spectra of both positive dielectrophoresis of the living and dead cells had distinguishable dielectric properties. The conductivity of the yeast cytoplasm (σc) of the dead cells was significantly less (≈ ≤ 0.05 S m-1) than that of the living yeast cells which typically had a cytoplasmic conductivity of ≈ 0.2 S m-1. This difference between viable and non-viable cells is sufficient for cell separation procedures. KEY POINTS: • Dielectrophoresis can be used to separate viable and non-viable yeast cells, • Cellular dielectric properties can be derived from the analysis of their dielectric spectra, • Cytoplasmic conductivity of viable cells is ≈ 0.2 S m-1 while that of non-viable cells ≈ ≤ 0.05 S m-1.

Influence of Na+ disorder on cytoplasmic conductivity and cellular electromagnetic (EM) energy absorption of human erythrocytes (PONE-D-21-36089).

Cytoplasmic conductivity of human erythrocytes may be significantly disturbed by the composition of the external suspending media. Effects of external NaCl on cytoplasmic conductivity of human erythrocyte (Human Red Blood Cells, HRBC) were investigated in a simple NaCl system. Using thermodynamic theory cytoplasmic conductivities could be calculated from internal [K+], [Na+], [Cl-] and [HCO3-]. Effect of cell volume and cell water changes were introduced and allowed for using the Debye-Hückel-Onsager relation and Walden's rule of viscosity. Cell volume and cell water change of HRBCs were measured in suspending isotonic solutions with conductivities from 0.50 S m-1 up to hypertonic solutions of conductivity of 2.02 S m-1 at selected temperatures of 25°C (standard benchmark temperature) and 37°C (physiological temperature). In isotonic solutions, cytoplasmic conductivity of human erythrocyte decreases with rise in the external media ionic concentration and vice versa for hypertonic solutions. The HRBC is capable of rapidly regulating its volume (and shape) over quite a wide range of osmolality. Specific Absorption Rate (SAR, 900 MHz) values (W kg-1) of electromagnetic radiation are below safe limits at non-physiological 25°C but above legal limits at 37°C [National Council on Radiation Protection and Measurements, NCRP]. However, at 37°C under both hypertonic [Na+] and isotonic but low [Na+], SAR increases further beyond legal limits.

Photosynthetic electron transport in pitcher plants (Nepenthes mirabilis).

Pitcher plants (Nepenthes sp.) are insectivorous angiosperm plants with modified leaves known as pitchers best known as acting as traps for insects. Pitcher plants are typically found under boggy conditions under both forest cover and open areas with very poor nutrient status, particularly N-status. The pitchers have low photosynthetic activity. The Chl a content of the pitcher tissue of both Nepenthes mirabilis (green and red) varieties was very low. Chl b/a ratios of the green variety phyllodes (lamina) and pitchers were ≈ 0.24 to 0.29. In the red variety, the mature phyllodes had a Chl b/a ratio ≈ 0.28 but both the pitchers and the young phyllodes had Chl b/a ratios of nearly 0.5. Photosynthetic electron transport (ETR) was measured using PAM technology. Phyllodes of both varieties showed photoinhibition at supra-optimal irradiances [Nepenthes mirabilis (green variety), Eopt ≈ 200-250 µmol photon m-2 s-1; red variety, Eopt ≈ 100-150 µmol photon m-2 s-1]. Pitchers had low optimum irradiances (Eopt ≈ 40-90 µmol photon m-2 s-1). Maximum ETR (ETRmax) of phyllodes of both varieties was low (ETRmax ≈ 50 µmol e- g-1 Chl a s-1); ETRmax was higher for pitchers on a Chl a basis (ETRmax ≈ 80-100 µmol e- g-1 Chl a s-1); a consequence of their low Chl a content on a surface area basis. ETRmax of cut disks of phyllodes did not respond strongly to incubation in NH4+, glutamate or aspartate as N-sources but did respond positively to added urea.

Griess-doped polyvinyl alcohol thin film for on-site simultaneous sample preparation and nitrite determination of processed meat products.

To facilitate on-site nitrite determination for processed meat products, Griess-doped polyvinyl alcohol film was synthesized in the bottom of a plastic tube for in-tube determination. The tube's aperture was used to control the sample dimensions. Each sample, cut into eight sectors, was subjected to nitrite extraction by water. Use of tap water or commercial drinking water vs. ultrapure water was associated with < 2% differences in nitrite levels. The use of film and digital image colorimetry showed a low limit of detection (12.6 ± 0.5 µg L-1), good precision (1.0%RSD, n = 5 days), and good accuracy (93.2 ± 3.5 to 108.5 ± 1.8%recovery). Using these methods, sodium nitrite concentrations in 700 processed meat products for sale in Phuket, Thailand, were found to range from 6.8 ± 0.2 to 113.6 ± 1.3 mg kg-1. These results showed no significant differences with the HPLC standard method (p > 0.05, n = 45).

Energy absorption of human red blood cells and conductivity of the cytoplasm influenced by temperature.

The energy absorbed into tissues is known as the specific energy absorption (SAR) which is dependent on conductivity of the tissue. We calculated cytoplasmic conductivity of human red blood cell (HRBC) using the intracellular ionic concentrations and the Debye-Hückel-Onsager relation. The overall concentration is determined by cell volume and cell water content. The calculated HRBC conductivity at 25 o C was σc,25 = 0.5566 ± 0.0146 S m-1, ±SE). It is exponentially related to temperature: Q10 ≈ 1.866. At 37 o C, the calculated SAR value is 1.6 W kg-1 using a linear temperature compensation of conductivity. However, if using a biologically realistic non-linear temperature compensated conductivity, the SAR is ≈ 2.62 ± 0.05 W kg-1. The relationship between SAR and temperature increase is not straightforward. Since there is a wide variance in cellular ionic and water perfusion rates more tissue-specific SAR limits which consider temperature-related factors would be valuable.

Spectrofluorometric Insights into the Application of PAM Fluorometry in Photosynthetic Research.

Although pulse amplitude modulation (PAM) fluorometry has revolutionized photosynthetic studies, Photosynthetic Electron Transport Rate (ETR) cannot be measured using PAM technology in some organisms. We compare in vivo absorbance information on a selection of photosynthetic organisms using an integrating sphere spectrophotometry on a variety of oxygenic and nonoxygenic photo-organisms and provide fluorescence data to help in understanding why PAM technology is unsuccessful on some organisms, particularly cyanobacteria. The study includes anoxygenic photosynthetic bacteria: Afifella marina, Rhodopseudomonas palustris and Thermochromatium which are all RC-2 type photosynthetic bacteria (Bacteriochlorophyll a or BChl a) which are known to have measureable delayed fluorescence, Yield and hence measureable ETR. The common unicellular green alga, Chlorella sp (Chl a + b) uses the same primary photosynthetic pigments as vascular plants. Comparisons are made to some other representative oxygenic unicellular organisms: Trebouxia (Chlorophyta, Chl a + b), Chaetoceros (a diatom, Chl a + c1 c2 ) and the unusual cyanobacterium Acaryochloris marina which has Chl d + a but uses Chl d as its primary photosynthetic pigment. Synechococcus R-2 (Cyanobacteria) has only Chl a. Its fluorescence is outside the range normally used for measuring photosynthesis using PAM technology: delayed fluorescence is not readily detectable.

Photosynthetic electron transport rate (ETR) in the littoral herb Launaea sarmentosa known as mole crab in Thailand.

Launaea sarmentosa (Willd.) Sch. Bip ex Kunze (Asteracaeae) is a littoral sand dune herb found in the Indian Ocean region, used as a folk medicine and as a savory vegetable in Thailand. It is in the transition stage from a kitchen & cottage industry to a commercial proposition. Rapid light curves to measure the photosynthetic electron transport rate (ETR) were conducted on the plants over the course of daylight from 6:00 to 18:00 using a PAM fluorometer on plants grown under 50% (nominal) green horticultural and 20% black (nominal) shade cloth and in the open. Plants grown in the open were sun plants. Eopt (µmol photon m-2 s-1) decreased slightly under shade cloth (open air: 890 > green shade cloth, 778 > green shade cloth, 713). Launaea shows limited shade adaptation. ETR decreased under shade cloth on both a surface area and Chl a basis (µmol e- g Chl a-1 s-1) [open air: 388 > (green shade cloth, 209 = black shade cloth, 263)]. Maximum non-photochemical quenching (NPQmax) was significantly decreased under black shade cloth [(open air: 1.02 = green shade cloth, 0.969) > black shade cloth, 0.694]. ETR showed midday inhibition (9:00-15:00) when irradiance exceeded the Eopt of Launaea. Daily total photosynthetic electron transport was (mmol e- m-2 d-1): open air, 1890 ± 157; green shade cloth, 1620 ± 203, black shade cloth, 1217 ± 143. Green shade cloth has no effect on total daily photosynthetic electron transport of Launaea but offers some protection from desiccation and excessive evapotranspiration. Waterlogging decreases ETR by about 40%. Launaea can be grown watered with brackish (½ seawater) water unlike many other vegetables. Launeae is a physiologically undemanding cottage industry/market garden crop suitable for sandy coastline fishing communities.

A tribute to Robert John Porra (august 7, 1931-may 16, 2019).

Robert John Porra (7.8.1931-16.5.2019) is probably best known for his substantial practical contributions to plant physiology and photosynthesis by addressing the problems of both the accurate spectroscopic estimation and the extractability of chlorophylls in many organisms. Physiological data and global productivity estimates, in particular of marine primary productivity, are often quoted on a chlorophyll basis. He also made his impact by work on all stages of tetrapyrrole biosynthesis: he proved the C5 pathway to chlorophylls, detected an alternative route to protoporphyrin in anaerobes and the different origin of the oxygen atoms in anaerobes and aerobes. A brief review of his work is supplemented by personal memories of the authors.

Photosynthesis in a Vanda sp orchid with Photosynthetic Roots.

PAM fluorometry showed that the orchid Vanda sp (Gaud ex Pfitzers, Vandeae) had photosynthetic electron transport yields in leaves reaching ≈ 0.617 ± 0.262 at midday. Yield decayed exponentially as irradiance increased (Y½ = 128 ± 12.4 µmol photon m-2 s-1). Optimum irradiance (Eopt) for ETR (Photosynthetic Electron Transport Rate) was ≈ 369 ± 23.3 µmol photon m-2 s-1; the maximum photosynthetic ETR (ETRmax) (on a Chl a basis) ≈ 97.6 ± 3.76 µmol e-g-1 Chl a s-1. Rapid light curves exhibited classic photoinhibition at high irradiances: Vanda sp is a shade plant. Photosynthetic kinetics was strongly diurnal with minimal Eopt and ETRmax in the early morning, reaching a maximum at midday and decreasing in the afternoon. The aerial roots were normally photosynthetically dormant but rapidly activated when wet (homiochlorophyllous) then becoming dormant again after drying. Wet roots deliberately incubated under moist conditions had photosynthetic light curves comparable to leaves (Ymax ≈0.332, Y½ = ≈ 78.3 ± 27.8 µmol photons m-2 s-1, Eopt ≈ 278 µmol photons m-2 s-1 and ETRmax ≈ 317 ± 86.9 µmol e-g-1 Chl a s-1): wetting for only 15 min activated photosynthesis. Leaves showed a small degree of diurnal cycling of titratable acid but acid was accumulated in the early morning, not at night, this is a type of CAM-cycling. Titratable acid was low at sunrise (≈ 54.1 µmol H+g-1 FW), but increased until about 9 a.m. (≈ 137 µmol H+g-1 FW) and then gradually decreased over the course of the day.

Masking Phosphate with Rare-Earth Elements Enables Selective Detection of Arsenate by Dipycolylamine-ZnII Chemosensor.

Functional reassessment of the phosphate-specific chemosensors revealed their potential as arsenate detectors. A series of dipicolylamine (Dpa)-ZnII chemosensors were screened, among which acridine Dpa-ZnII chemosensor showed the highest capability in sensing arsenate. The presence of excess ZnII improved sensitivity and strengthened the binding between acridine Dpa-ZnII complex to arsenate as well as phosphate. However, due to their response to phosphate, these sensors are not suited for arsenate detection when phosphate is also present. This study demonstrated for the first time that rare-earth elements could effectively mask phosphate, allowing the specific fluorescence detection of arsenate in phosphate-arsenate coexisting systems. In addition, detection of arsenate contamination in the real river water samples and soil samples was performed to prove its practical use. This sensor was further employed for the visualization of arsenate and phosphate uptake in vegetables and flowering plants for the first time, as well as in the evaluation of a potent inhibitor of arsenate/phosphate uptake.

Bioaccumulation and physiological responses of the Coontail, Ceratophyllum demersum exposed to copper, zinc and in combination.

Ceratophyllum demersum is a submerged aquatic angiosperm which is fast growing in contaminated water. This plant has no roots and so takes up nutrients from the water column without the complicating factor of differential shoot/root uptake of nutrients. This study aimed to compare the bioaccumulative capacities of Cu, Zn and their combination by C. demersum and physiological responses (growth, chlorophyll content, and photosynthetic rate) of C. demersum to Cu and Zn. Additionally, pulse amplitude modulation (PAM) technology and integrating sphere spectrometer were applied to detect copper and zinc toxicity effects on the light reactions of photosynthesis C. demersum is an aquatic plant that could be a good accumulator of Cu and Zn in actual solution in the water column. Additionally, RGR (relative growth rate) and chlorophyll content of C. demersum show that toxic effects of Cu or Zn increased over time. Cu and Zn effects manifested themselves more slowly than expected: at least 5 to 10 d were needed for noticeable effects both macroscopically (physical appearance), biochemical (chlorophyll content) and from measurements of photosynthesis using Pulse Amplitude Modulation (PAM) fluorometry. Moreover, the combination of Cu and Zn caused more toxic effect than either Cu or Zn separately. Whole plant scans using an integrating sphere spectrophotometer showed that Cu, Zn and Zn + Cu toxicity effects could be identified from spectral scans but were not specific enough for Cu, Zn and Zn + Cu toxicity to be distinguished from one another.

Arsenic toxicity in the water weed Wolffia arrhiza measured using Pulse Amplitude Modulation Fluorometry (PAM) measurements of photosynthesis.

Accumulation of arsenic in plants is a serious South-east Asian environmental problem. Photosynthesis in the small aquatic angiosperm Wolffia arrhiza is very sensitive to arsenic toxicity, particularly in water below pH 7 where arsenite (As (OH)3) (AsIII) is the dominant form; at pH >7 AsO4(2-) (As(V) predominates). A blue-diode PAM (Pulse Amplitude Fluorometer) machine was used to monitor photosynthesis in Wolffia. Maximum gross photosynthesis (Pgmax) and not maximum yield (Ymax) is the most reliable indicator of arsenic toxicity. The toxicity of arsenite As(III) and arsenate (H2AsO4(2-)) As(V) vary with pH. As(V) was less toxic than As(III) at both pH 5 and pH 8 but both forms of arsenic were toxic (>90% inhibition) at below 0.1molm(-3) when incubated in arsenic for 24h. Arsenite toxicity was apparent after 1h based on Pgmax and gradually increased over 7h but there was no apparent effect on Ymax or photosynthetic efficiency (α0).

Measurement of photosynthesis using PAM technology in a purple sulfur bacterium Thermochromatium tepidum (Chromatiaceae).

We demonstrate that Blue-diode-based pulse amplitude modulation (PAM) technology can be used to measure the photosynthetic electron transport rate (ETR) of purple sulfur bacteria (Thermochromatium tepidum, Chromatiaceae). Previous studies showed that PAM technology could be used to estimate photosynthesis in purple nonsulfur bacteria and so PAM technology can be used to estimate photosynthesis of both kinds of purple photosynthetic bacteria. The absorptance of Thermochromatium films on glass fiber disks was measured and used to calculate actual ETR. ETR vs Irradiance (P vs E) curves fitted the waiting-in-line model (ETR = (ETRmax × E/Eopt) × exp (1−E/Eopt)). Yield (Y) was only ≈ 0.3­0.4. Thermochromatium saturates at 325 ± 13.8 µmol photons m(−2) s(−1) or ≈15% sunlight and shows photoinhibition at high irradiances. A pond of Thermochromatium would exhibit classic surface inhibition. Photosynthesis is extremely low in the absence of an electron source: ETR increases in the presence of acetate (5 mol m(−3)) provided as an organic carbon source and also increases in the presence of sulfite (3 mol m(−3)) but not sulfide and is only marginally increased by the presence of Fe(2+). Nonphotochemical quenching does occur in Thermochromatium but at very low levels compared to oxygenic photo-organisms or Rhodopseudomonads.

Anaerobic digestion of food waste using yeast.

Fermentative breakdown of food waste seems a plausible alternative to feeding food waste to pigs, incineration or garbage disposal in tourist areas. We determined the optimal conditions for the fermentative breakdown of food waste using yeast (Saccharomyces cerevisiae) in incubations up to 30days. Yeast efficiently broke down food waste with food waste loadings as high as 700g FW/l. The optimum inoculation was ≈46×10(6)cells/l of culture with a 40°C optimum (25-40°C). COD and BOD were reduced by ≈30-50%. Yeast used practically all the available sugars and reduced proteins and lipids by ≈50%. Yeast was able to metabolize lipids much better than expected. Starch was mobilized after very long term incubations (>20days). Yeast was effective in breaking down the organic components of food waste but CO2 gas and ethanol production (≈1.5%) were only significant during the first 7days of incubations.

Characterization of exopolymeric substances from selected Rhodopseudomonas palustris strains and their ability to adsorb sodium ions.

Removal of Na(+) by binding with exopolymeric substances (EPS) from Rhodopseudomonas palustris TN114 and PP803 was investigated. The moderate negative correlation pairs (rp) between remaining Alcian blue and amount of Na(+) adsorbed on EPS from strains TN114 and PP803 were -0.652 and -0.609. Both strains showed positive relationships between the amounts of EPS produced and bacterial growth. EPS from strain PP803 had a higher efficiency in removing Na(+) than the EPS from strain TN114 based on their EC50 values (1.79 and 1.49 mg/mL for TN114 and PP803, respectively). The principal component from EPS of strain PP803 which was responsible for salt removal was purified and it was identified as a polysaccharide (≈18 kDa) mainly composed of galacturonic acid. Overall results suggested that EPS is a key factor that our strains used to bind Na(+) allowing their survival in high NaCl concentrations.

Probabilistic risk assessment of diuron and prometryn in the Gwydir River catchment, Australia, with the input of a novel bioassay based on algal growth.

A probabilistic risk assessment of the selected herbicides (diuron and prometryn) in the Gwydir River catchment was conducted, with the input of the EC50 values derived from both literature and a novel bioassay. Laboratory test based on growth of algae exposed to herbicides assayed with a microplate reader was used to examine the toxicity of diuron and prometryn on the growth of Chlorella vulgaris. Both herbicides showed concentration dependent toxicity in inhibiting the growth of Chlorella during the exposure period of 18-72 h. Diuron caused more toxicity as judged by growth rates than prometryn. Thalaba Creek at Merrywinebone was identified as the 'hotspot' for diuron and prometryn risk in the Gwydir catchment. The use of microplate assays coupled with probabilistic risk assessment is recommended for rapid assessment of ecotoxicity of indigenous species, allowing identification of locations in river catchments requiring environmental management.

Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes

Background: Rice is globally one of the most important food crops, and NaCl stress is a key factor reducing rice yield. Amelioration of NaCl stress was assessed by determining the growth of rice seedlings treated with culture supernatants containing 5-aminolevulinic acid (ALA) secreted by strains of Rhodopseudomonas palustris (TN114 and PP803) and compared to the effects of synthetic ALA (positive control) and no ALA content (negative control). Results: The relative root growth of rice seedlings was determined under NaCl stress (50 mM NaCl), after 21 d of pretreatment. Pretreatments with 1 μM commercial ALA and 10X diluted culture supernatant of strain TN114 (2.57 μM ALA) gave significantly better growth than 10X diluted PP803 supernatant (2.11 μM ALA). Rice growth measured by dry weight under NaCl stress ordered the pretreatments as: commercial ALA N TN114 N PP803 N negative control. NaCl stress strongly decreased total chlorophyll of the plants that correlated with non-photochemical quenching of fluorescence (NPQ). The salt stress also strongly increased hydrogen peroxide (H2O2) concentration in NaCl-stressed plants. The pretreatments were ordered by reduction in H2O2 content under NaCl stress as: commercial ALA N TN114 N PP803 N negative control. The ALA pretreatments incurred remarkable increases of total chlorophyll and antioxidative activities of catalase (CAT), ascorbate peroxide (APx), glutathione reductase (GR) and superoxide dismutase (SOD); under NaCl stress commercial ALA and TN114 had generally stronger effects than PP803. Conclusions: The strain TN114 has potential as a plant growth stimulating bacterium that might enhance rice growth in saline paddy fields at a lower cost than commercial ALA.

The use of solar radiation by the photosynthetic bacterium, Rhodopseudomonas palustris: model simulation of conditions found in a shallow pond or a flatbed reactor.

Photosynthetic bacteria are attractive for biotechnology because they produce no oxygen and so H2 -production is not inhibited by oxygen as occurs in oxygenic photoorganisms. Rhodopseudomonas palustris and Afifella marina containing BChl a can use irradiances from violet near-UV (VNUV) to orange (350-650 nm) light and near-infrared (NIR) light (762-870 nm). Blue diode-based pulse amplitude modulation technology was used to measure their photosynthetic electron transport rate (ETR). ETR vs Irradiance curves fitted the waiting-in-line model--ETR = (ETRmax × E/Eopt) × exp (1 - E/Eopt). The equation was integrated over pond depth to calculate ETR of Afifella and Rhodopseudomonas in a pond up to 30 cm deep (A376, 1 cm = 0.1). Afifella saturates at low irradiances and so photoinhibition results in very low photosynthesis in a pond. Rhodopseudomonas saturates at ≈15% sunlight and shows photoinhibition in the surface layers of the pond. Total ETR is ≈335 µmol (e(-)) m(-2) s(-1) in NUV + photosynthetically active radiation light (350-700 nm). Daily ETR curves saturate at low irradiances and have a square-wave shape: ≈11-13 mol (e(-)) m(-2) day(-1) (350-700 nm). Up to 20-24% of daily 350-700 nm irradiance can be converted into ETR. NIR is absorbed by water and so competes with the bacterial RC-2 photosystem for photons.

The ammonia transport, retention and futile cycling problem in cyanobacteria.

Ammonia is the preferred nitrogen source for many algae including the cyanobacterium Synechococcus elongatis (Synechococcus R-2; PCC 7942). Modelling ammonia uptake by cells is not straightforward because it exists in solution as NH(3) and NH (4) (+) . NH(3) is readily diffusible not only via the lipid bilayer but also through aquaporins and other more specific porins. On the other hand, NH (4) (+) requires cationic transporters to cross a membrane. Significant intracellular ammonia pools (≈1-10 mol m(-3)) are essential for the synthesis of amino acids from ammonia. The most common model envisaged for how cells take up ammonia and use it as a nitrogen source is the "pump-leak model" where uptake occurs through a simple diffusion of NH(3) or through an energy-driven NH (4) (+) pump balancing a leak of NH(3) out of the cell. The flaw in such models is that cells maintain intracellular pools of ammonia much higher than predicted by such models. With caution, [(14)C]-methylamine can be used as an analogue tracer for ammonia and has been used to test various models of ammonia transport and metabolism. In this study, simple "proton trapping" accumulation by the diffusion of uncharged CH(3)NH(2) has been compared to systems where CH(3)NH (3) (+) is taken up through channels, driven by the membrane potential (ΔU (i,o)) or the electrochemical potential for Na(+) (ΔµNa (i,o) (+) ). No model can be reconciled with experimental data unless the permeability of CH(3)NH(2) across the cell membrane is asymmetric: permeability into the cell is very high through gated porins, whereas permeability out of the cell is very low (≈40 nm s(-1)) and independent of the extracellular pH. The best model is a Na (in) (+) /CH(3)NH (3) (+) (in) co-porter driven by ΔµNa (i,o) (+) balancing synthesis of methylglutamine and a slow leak governed by Ficks law, and so there is significant futile cycling of methylamine across the cell membrane to maintain intracellular methylamine pools high enough for fixation by glutamine synthetase. The modified pump-leak model with asymmetric permeability of the uncharged form is a viable model for understanding ammonia uptake and retention in plants, free-living microbes and organisms in symbiotic relationships.