Alan P. Jackman

Numerical modeling of coupled nitrification-denitrification in sediment perfusion cores from the hyporheic zone of the Shingobee River, MN

Abstract Nitrification and denitrification kinetics in sediment perfusion cores were numerically modeled and compared to experiments on cores from the Shingobee River MN, USA. The experimental design incorporated mixing groundwater discharge with stream water penetration into the cores, which provided a well-defined, one-dimensional simulation of in situ hydrologic conditions. Ammonium (NH 4 + ) and nitrate (NO 3 − ) concentration gradients suggested the upper region of the cores supported coupled nitrification–denitrification, where groundwater-derived NH 4 + was first oxidized to NO 3 − then subsequently reduced via denitrification to N 2 . Nitrification and denitrification were modeled using a Crank–Nicolson finite difference approximation to a one-dimensional advection–dispersion equation. Both processes were modeled using first-order reaction kinetics because substrate concentrations (NH 4 + and NO 3 − ) were much smaller than published Michaelis constants. Rate coefficients for nitrification and denitrification ranged from 0.2 to 15.8 h −1 and 0.02 to 8.0 h −1 , respectively. The rate constants followed an Arrhenius relationship between 7.5 and 22 °C. Activation energies for nitrification and denitrification were 162 and 97.3 kJ/mol, respectively. Seasonal NH 4 + concentration patterns in the Shingobee River were accurately simulated from the relationship between perfusion core temperature and NH 4 + flux to the overlying water. The simulations suggest that NH 4 + in groundwater discharge is controlled by sediment nitrification that, consistent with its activation energy, is strongly temperature dependent.

Ammonium sorption to channel and riparian sediments: A transient storage pool for dissolved inorganic nitrogen

Sediment (0.5 mm–2.0 mm grain size) was incubated in nylon bags (200 μm mesh) below the water table in the channel and in two transects of shallow wells perpendicular to the banks (to 18 m) of a third-order stream during August, 1987. One transect of wells drained steep old-growth forest, and the other a steep 23 year-old clear-cut partially regenerated in alder. At approximately 6-week intervals between October, 1987, and June, 1988, bags were retrieved. Total exchangeable ammonium was determined on sediment, and dissolved oxygen, nitrate and ammonium were determined in stream and well water. Exchangeable ammonium ranged from 10 μeq/100 g of sediment in the stream where nitrification potential and subsurface exchange with stream water were high, to 115 μeq/100 g sediment 18 m inland where channel water-groundwater mixing and nitrification potential were both low. Sorbed ammonium was highest during summer/autumn base flow and lowest during winter storm flow. Both channel and well water contained measurable dissolved oxygen at all times. Ammonium concentration was typically < 10 μg-N/L in channel water, increased with distance inland, but did not exceed 365 μg-N/L at any site. Nitrate concentration was typically higher in well water than channel water. Nitrate levels increased dramatically in wells at the base of the clear-cut following the onset of autumn rains. The results indicate a potential for temporary storage of ammonium on riparian sediments which may influence biotic nitrogen cycling, and alter the timing and form of dissolved inorganic nitrogen transport from the watershed.

Modeling biotic uptake by periphyton and transient hyporrheic storage of nitrate in a natural stream

To a convection-dispersion hydrologic transport model we coupled a transient storage submodel (Bencala, 1984) and a biotic uptake submodel based on Michaelis-Menten kinetics (Kim et al., 1990). Our purpose was threefold: (1) to simulate nitrate retention in response to change in load in a third-order stream, (2) to differentiate biotic versus hydrologie factors in nitrate retention, and (3) to produce a research tool whose properties are consistent with laboratory and field observations. Hydrodynamic parameters were fitted from chloride concentration during a 20-day chloride-nitrate coinjection (Bencala, 1984), and biotic uptake kinetics were based on flume studies by Kim et al. (1990) and Triska et al. (1983). Nitrate concentration from the 20-day coinjection experiment served as a base for model validation. The complete transport retention model reasonably predicted the observed nitrate concentration. However, simulations which lacked either the transient storage submodel or the biotic uptake submodel poorly predicted the observed nitrate concentration. Model simulations indicated that transient storage in channel and hyporrheic interstices dominated nitrate retention within the first 24 hours, whereas biotic uptake dominated thereafter. A sawtooth function for Vmax ranging from 0.10 to 0.17 μg NO3-N s−1 gAFDM−1 (grams ash free dry mass) slightly underpredicted nitrate retention in simulations of 2–7 days. This result was reasonable since uptake by other nitrate-demanding processes were not included. The model demonstrated how ecosystem retention is an interaction between physical and biotic processes and supports the validity of coupling separate hydrodynamic and reactive submodels to established solute transport models in biological studies of fluvial ecosystems.

Production of Human α-1-Antitrypsin from Transgenic Rice Cell Culture in a Membrane Bioreactor

Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two‐compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant α‐1‐antitrypsin (rAAT). Expression of rAAT is controlled by the rice α‐amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactorapos;s relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100–247 mg/L (4–10% of the total extracellular protein) in the cell compartment at 5–6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.

Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures.

Alfalfa (Medicago sativa L.) cells were grown in 500 ml, aerated and stirred batch bioreactors using Schenk and Hildebrant medium. For cultures in which the pH was allowed to vary, we observed two fairly distinct growth phases. Evidence is presented which indicates that the two-phase growth is most likely a result of the two nitrogen sources in the medium. The ammonium present in the medium is directly utilized during the first growth phase and ammonium resulting from intracellular nitrate reduction is utilized during the second phase. During the first growth phase, sucrose is completely hydrolyzed to glucose and fructose with some glucose and fructose consumption. In the second growth phase glucose is consumed preferentially over fructose. Attempts at maintaining the pH at 5.5 using 1N NaOH as the base titrant resulted in very little cell growth compared with cultures for which the pH was allowed to vary.

Bioreactor Production of Human α1‐Antitrypsin Using Metabolically Regulated Plant Cell Cultures

Transgenic rice cell cultures, capable of producing recombinant human α1‐antitrypsin (rAAT), were scaled up from shake flasks to a 5‐L bioreactor. The maximum specific growth rates (μmax) observed from two bioreactor runs were 0.40 day−1 (doubling time of 1.7 days) and 0.47 day−1 (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O2/(g dw h). Using a metabolically regulated rice α‐amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar‐free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar‐free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots.

Transport and concentration controls for chloride, strontium, potassium and lead in Uvas Creek, a small cobble-bed stream in Santa Clara County, California, U.S.A.

Abstract Three models describing solute transport of conservative ion species and another describing transport of species which adsorb linearly and reversibly on bed sediments are developed and tested. The conservative models are based on three different conceptual models of the transient storage of solute in the bed. One model assumes the bed to be a well-mixed zone with flux of solute into the bed proportional to the difference between stream concentration and bed concentration. The second model assumes solute in the bed is transported by a vertical diffusion process described by Ficks law. The third model assumes that convection occurs in a selected portion of the bed while the mechanism of the first model functions everywhere. The model for adsorbing species assumes that the bed consists of particles of uniform size with the rate of uptake controlled by an intraparticle diffusion process. All models are tested using data collected before, during and after a 24-hr. pulse injection of chloride, strontium, potassium and lead ions into Uvas Creek near Morgan Hill, California, U.S.A. All three conservative models accurately predict chloride ion concentrations in the stream. The model employing the diffusion mechanism for bed transport predicts better than the others. The adsorption model predicts both strontium and potassium ion concentrations well during the injection of the pulse but somewhat overestimates the observed concentrations after the injection ceases. The overestimation may be due to the convection of solute deep into the bed where it is retained longer than the 3-week post-injection observation period. The model, when calibrated for strontium, predicts potassium equally well when the adsorption equilibrium constant for strontium is replaced by that for potassium.

Characterization of plant suspension cultures using the focused beam reflectance technique

Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R2>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.

A cyclical semicontinuous process for production of human α1-antitrypsin using metabolically induced plant cell suspension cultures

Transgenic rice suspension cultures were utilized to produce a human therapeutic protein, recombinant α1‐antitrypsin (rAAT), in a cyclical, semicontinuous operation. Recombinant protein production was induced by removing the carbon source from the cell culture medium. The transgenic rice cells secreted the rAAT into the medium, and therefore medium exchanges could be performed for consecutive growth and protein expression phases. The process consisted of three cycles over a 25–28 day period, with growth phases lasting 4–6 days each and protein expression phases lasting 2.5–5 days each. Biomass and sugar concentrations, oxygen uptake rate, cell viability, culture pH, total extracellular protein, and active rAAT were measured throughout the cyclical process. The data profiles were reproducible between separate cyclical runs where, following each induction period, cell growth and viability could be reestablished once sucrose was added back to the culture. Volumetric productivities ranged from 3 to 12 mg active rAAT/(L day) for individual cycles with overall volumetric productivities of 4.5 and 7.7 mg active rAAT/(L day).

Expression of recombinant trichosanthin, a ribosome-inactivating protein, in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.