Growth and laccase production kinetics of <Emphasis Type="Italic">Trametes versicolor</Emphasis> in a stirred tank reactor

White rot fungi are a promising option to treat recalcitrant organic molecules, such as lignin, polycyclic aromatic hydrocarbons, and textile dyes, because of the lignin-modifying enzymes (LMEs) they secrete. Because knowledge of the kinetic parameters is important to better design and operate bioreactors to cultivate these fungi for degradation and/or to produce LME(s), these parameters were determined using Trametes versicolor ATCC 20869 (ATCC, American Type Culture Collection) in a magnetic stir bar reactor. A complete set of kinetic data has not been previously published for this culture. Higher than previously reported growth rates with high laccase production of up to 1,385 U l⁻¹ occurred during growth without or glucose limitation. The maximum specific growth rate averaged 0.94 ± 0.23 day⁻¹, whereas the maximum specific substrate consumption rates for glucose and ammonium were 3.37 ± 1.16 and 0.15 ± 0.04 day⁻¹, respectively. The maximum specific oxygen consumption rate was 1.63 ± 0.36 day⁻¹.     

Epipelic and pelagic primary production in Alaskan Arctic lakes of varying depth

We compared on eight dates during the ice-free period physicochemical properties and rates of phytoplankton and epipelic primary production in six arctic lakes dominated by soft bottom substrate. Lakes were classified as shallow ( < 2.5 m), intermediate in depth (2.5 m <  < 4.5 m), and deep ( > 4.5 m), with each depth category represented by two lakes. Although shallow lakes circulated freely and intermediate and deep lakes stratified thermally for the entire summer, dissolved oxygen concentrations were always >70% of saturation values. Soluble reactive phosphorus and dissolved inorganic nitrogen (DIN = NO₃ ⁻–N + NH₄ ⁺–N) were consistently below the detection limit (0.05 μmol l⁻¹) in five lakes. However, one lake shallow lake (GTH 99) periodically showed elevated values of DIN (17 μmol l⁻¹), total-P (0.29 μmol l⁻¹), and total-N (33 μmol l⁻¹), suggesting wind-generated sediment resuspension. Due to increased nutrient availability or entrainment of microphytobenthos, GTH 99 showed the highest average volume-based values of phytoplankton chlorophyll a (chl a) and primary production, which for the six lakes ranged from 1.0 to 2.9 μg l⁻¹ and 0.7–3.8 μmol C l⁻¹ day⁻¹. Overall, however, increased resulted in increased area-based values of phytoplankton chl a and primary production, with mean values for the three lake classes ranging from 3.6 to 6.1 mg chl a m⁻² and 3.2–5.8 mmol C m⁻² day⁻¹. Average values of epipelic chl a ranged from 131 to 549 mg m⁻² for the three depth classes, but levels were not significantly different due to high spatial variability. However, average epipelic primary production was significantly higher in shallow lakes (12.2 mmol C m⁻² day⁻¹) than in intermediate and deep lakes (3.4 and 2.4 mmol C m⁻² day⁻¹). Total primary production (6.7–15.4 mmol C m⁻² day⁻¹) and percent contribution of the epipelon (31–66%) were inversely related to mean depth, such that values for both variables were significantly higher in shallow lakes than in intermediate or deep lakes. Handling editor: L. Naselli-Flores     

Bacterial growth and substrate degradation by BTX-oxidizing culture in response to salt stress

Interactions between microbial growth and substrate degradation are important in determining the performance of trickle-bed bioreactors (TBB), especially when salt is added to reduce biomass formation in order to alleviate media clogging. This study was aimed at quantifying salinity effects on bacterial growth and substrate degradation, and at acquiring kinetic information in order to improve the design and operation of TBB. Experiment works began by cultivating a mixed culture in a chemostat reactor receiving artificial influent containing a mixture of benzene, toluene, and xylene (BTX), followed by using the enrichment culture to degrade the individual BTX substrates under a particular salinity, which ranged 0–50 g l⁻¹ in batch mode. Then, the measured concentrations of biomass and residual substrate versus time were analyzed with the microbial kinetics; moreover, the obtained microbial kinetic constants under various salinities were modeled using noncompetitive inhibition kinetics. For the three substrates the observed bacterial yields appeared to be decreased from 0.51–0.74 to 0.20–0.22 mg mg⁻¹ and the maximum specific rate of substrate utilization, declined from 0.25–0.42 to 0.07–0.11 h⁻¹, as the salinity increased from 0 to 50 NaCl g l⁻¹. The NaCl acted as noncompetitive inhibitor, where the modeling inhibitions of the coefficients, K _T(S), were 22.7–29.7 g l⁻¹ for substrate degradation and K _T(μ), 13.0–19.0 g l⁻¹, for biomass formation. The calculated ratios for the bacterial maintenance rate, m _S, to further indicated that the percentage energy spent on maintenance increased from 19–24 to 86–91% as salinity level increased from 0 to 50 g l⁻¹. These results revealed that the bacterial growth was more inhibited than substrate degradation by the BTX oxidizers under the tested salinity levels. The findings from this study demonstrate the potential of applying NaCl salt to control excessive biomass formation in biotrickling filters.     

The effects of suspended sediment concentration on turbulence in an annular flume

The effect of suspended sediment concentrations (SSC) on fluid turbulence in an annular flume was investigated. Flow speed was held constant at 0.57 m s⁻¹, and the resulting turbulent conditions were recorded using a 3-D Acoustic Doppler Velocimeter (ADV) at height (z) of 8.5 cm above the bed. The suspended material was composed of a natural glacial clay made up of particles smaller than 6 μm. The SSC in the flume were increased from clear water to 4800 mg l⁻¹ in nine discrete increments; temporal variations of SSC were monitored using three optical backscatter sensors (OBS) mounted in the flume wall at heights of 0.03, 0.10 and 0.20 m above the flume base. The results showed that turbulent intensity ( ) and energy dissipation rate (ɛ) did not change significantly between clear water and 200 mg l⁻¹, but decreased by nearly 30% in the SSC range between 200 and 2400 mg l⁻¹. Above 2400 mg l⁻¹, no further decrease was observed. Analyses of the velocity variances over narrow frequency bands (0.2 Hz wide) from 0 to 12.5 Hz showed that most of the flow turbulent energy (~70–80%) was contained within the lower frequencies i.e. larger eddies, and that these eddies experienced the greatest decrease in energy due to turbidity. It is proposed that these patterns are the consequence of the increase in suspended sediment concentrations and of the vertical stratification of sediments for SSC >200 mg l⁻¹.     

Leaching losses of inorganic N and DOC following repeated drying and wetting of a spruce forest soil

Forest soils are frequently subjected to dry–wet cycles, but little is known about the effects of repeated drying and wetting and wetting intensity on fluxes of , and DOC. Here, undisturbed soil columns consisting of organic horizons (O columns) and organic horizons plus mineral soil (O + M columns) from a mature Norway spruce stand at the Fichtelgebirge; Germany, were repeatedly desiccated and subsequently wetted by applying different amounts of water (8, 20 and 50 mm day⁻¹) during the initial wetting phase. The constantly moist controls were not desiccated and received 4 mm day⁻¹ during the entire wetting periods. Cumulative inorganic N fluxes of the control were 12.4 g N m⁻² (O columns) and 11.4 g N m⁻² (O + M columns) over 225 days. Repeated drying and wetting reduced cumulative and fluxes of the O columns by 47–60 and 76–85%, respectively. Increasing (0.6–1.1 g N m⁻²) and decreasing fluxes (7.6–9.6 g N m⁻²) indicate a reduction in net nitrification in the O + M columns. The negative effect of dry–wet cycles was attributed to reduced net N mineralisation during both the desiccation and wetting periods. The soils subjected to dry–wet cycles were considerably drier at the final wetting period, suggesting that hydrophobicity of soil organic matter may persist for weeks or even months. Based on results from this study and from the literature we hypothesise that N mineralisation is mostly constrained by hydrophobicity in spruce forests during the growing season. Wetting intensity did mostly not alter N and DOC concentrations and fluxes. Mean DOC concentrations increased by the treatment from 45 mg l⁻¹ to 61–77 mg l⁻¹ in the O tlsbba columns and from 12 mg l⁻¹ to 21–25 mg l⁻¹ in the O + M columns. Spectroscopic properties of DOC from the O columns markedly differed within each wetting period, pointing to enhanced release of rather easily decomposable substrates in the initial wetting phases and the release of more hardly decomposable substrates in the final wetting phases. Our results suggest a small additional DOC input from organic horizons to the mineral soil owing to drying and wetting.     

A Novel Approach for Bioremediation of a Coastal Marine Wastewater Effluent Based on Artificial Microbial Mats

A novel alternative for wastewater effluent bioremediation was developed using constructed microbial mats on low-density polyester. This biotechnology showed high removal efficiencies for nitrogen and phosphorous in a short retention time (48 h): 94% for orthophosphate (7.78 g m³ d⁻¹), 79% for ammonium (11.30 g m⁻³ d⁻¹), 78% for nitrite (7.46 g m⁻³ d⁻¹), and 83% for nitrate (8.55 g m⁻³ d⁻¹). The microbial mats were dominated by Cyanobacteria genera such as Chroococcus sp., Lyngbya sp., and bacteria of the subclass Proteobacteria representative of the Eubacteria Domain. Nitzschia sp. was the dominant Eukaryote Domain. Various N and P substrates in the wastewater permit the growth of self-forming and self-sustaining bacterial, microalgal, and cyanobacterial communities on a polyester support. The result is the continuous, self-sufficient growth of microbial mats. This is an innovative, economical, and environmentally safe alternative for the treatment of wastewater effluents in coastal marine environments.     

Utilizing pigment-producing fungi to add commercial value to American beech (<Emphasis Type="Italic">Fagus grandifolia</Emphasis>)

American beech (Fagus grandifolia) is an abundant, underutilized tree in certain areas of North America, and methods to increase its market value are of considerable interest. This research utilized pigment-producing fungi to induce color in American beech to potentially establish its use as a decorative wood. Wood samples were inoculated with Trametes versicolor, Xylaria polymorpha, Inonotus hispidus, and Arthrographis cuboidea to induce fungal pigmentation. Black pigmentation (T. versicolor, X. polymorpha, I. hispidus) was sporadic, occurred primarily on the surfaces of the heartwood, but not internally. Pink pigmentation (A. cuboidea) occurred throughout all of the tested beech samples, but was difficult to see in the heartwood due to the darker color of the wood. To increase the visibility of the pink stain, beech blocks were pretreated with T. versicolor for 4 weeks before being inoculated with A. cuboidea. This method significantly increased the saturation of the pink stain on both beech heartwood and sapwood, creating coloration similar to that found on sugar maple. This value-adding process should be particularly effective for small-scale wood pigmentation, and should help establish a market for this currently underutilized wood species.     

Isolation and characterization of a fungus <Emphasis Type="Italic">Aspergillus</Emphasis> sp. strain F-3 capable of degrading alkali lignin

A fungus strain F-3 was selected from fungal strains isolated from forest soil in Dalian of China. It was identified as one Aspergillus sp. stain F-3 with its morphologic, cultural characteristics and high homology to the genus of rDNA sequence. The budges or thickened node-like structures are peculiar structures of hyphae of the strain. The fungus degraded 65% of alkali lignin (2,000 mg l⁻¹) after day 8 of incubation at 30°C at pH 7. The removal of colority was up to 100% at 8 days. The biodegradation of lignin by Aspergillus sp. F-3 favored initial pH 7.0. Excess acid or alkali conditions were not propitious to lignin decomposing. Addition of ammonium l-tartrate or glucose delayed or repressed biodegradation activities. During lignin degradation, manganese peroxidase (28.2 U l⁻¹) and laccase (3.5 U l⁻¹)activities were detected after day 7 of incubation. GC-MS analysis of biodegraded products showed strain F-3 could convert alkali lignin into small molecules or other utilizable products. Strain F-3 may co-culture with white rot fungus and decompose alkali lignin effectively.     

A preliminary study of the bioremediation potential of Codium fragile applied to seaweed integrated multi-trophic aquaculture (IMTA) during the summer

In integrated multi-trophic aquaculture (IMTA), seaweeds have the capacity to reduce the environmental impact of nitrogen-rich effluents in coastal ecosystems. To establish such bioremediation systems, selection of suitable seaweed species is important. The distribution and productivity of seaweeds vary seasonally based on water temperature and photoperiod. In Korea, candidate genera such as Pophyra, Laminaria, and Undaria grow from autumn to spring. In contrast, Codium grows well at relatively high water temperatures in summer. Thus, aquaculture systems potentially could capitalize on Codium’s capacity for rapid growth in the warm temperatures of late summer and early fall. In this study, we investigated ammonium uptake and removal efficiency by Codium fragile. In laboratory experiments, we grew C. fragile under various water temperatures (10, 15, 20, and 25°C), irradiances (dark, 10, and 100 μmol photons m⁻² s⁻¹), and initial ammonium concentrations (150 and 300 μM); in all cases, C. fragile exhausted the ammonium supply for 6 h. At 150 μM of , ammonium removal efficiency was greatest (99.5 ± 2.6%) when C. fragile was incubated at 20°C under 100 μmol photons m⁻² s⁻¹. At 300 μM of , removal efficiency was greatest (86.3 ± 2.1%) at 25°C under 100 μmol photons m⁻² s⁻¹. Ammonium removal efficiency was significantly greater at 20 and 25°C under irradiance of 100 μmol photons m⁻² s⁻¹ than under other conditions tested.     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Decolorization of 1:2 metal complex dye Acid blue 193 by a newly isolated fungus, Cladosporium cladosporioides

Summary A fungus Cladosporium cladosporioides isolated from coal sample as a decolorizing microorganism. It decolorized five different azo and triphenylmethane dyes like acid blue 193, acid black 210, crystal violet, reactive black B(S) and reactive black BL/LPR both on solid and in liquid broth medium. Culture broth of this fungus decolorized completely 100 mg of acid blue 193 l⁻¹ in 8 days. The extracellular enzyme of Cladosporium cladosporioides decolorized acid blue 193 on repeated addition to a total (out of 700 mg l⁻¹) concentration of 564 mg l⁻¹ within 168 h without significant decline in the activity, showing the resistant property of Cladosporium cladosporioides to a high concentration of the dye. The optimal temperature 40 °C, pH 5.6 and sugar concentration of 4% required for decolorization of acid blue 193. Cladosporium cladosporioides showed manganese peroxidase activity with 41 U l⁻¹, laccase activity with 1413 U l⁻¹ and lignin peroxidase activity was negligible after day 8 of incubation.     

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l⁻¹). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l⁻¹. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn²⁺) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.     

Soil Respiration in European Grasslands in Relation to Climate and Assimilate Supply

Soil respiration constitutes the second largest flux of carbon (C) between terrestrial ecosystems and the atmosphere. This study provides a synthesis of soil respiration (R _s) in 20 European grasslands across a climatic transect, including ten meadows, eight pastures and two unmanaged grasslands. Maximum rates of R _s ( ), R _s at a reference soil temperature (10°C; ) and annual R _s (estimated for 13 sites) ranged from 1.9 to 15.9 μmol CO₂ m⁻² s⁻¹, 0.3 to 5.5 μmol CO₂ m⁻² s⁻¹ and 58 to 1988 g C m⁻² y⁻¹, respectively. Values obtained for Central European mountain meadows are amongst the highest so far reported for any type of ecosystem. Across all sites was closely related to . Assimilate supply affected R _s at timescales from daily (but not necessarily diurnal) to annual. Reductions of assimilate supply by removal of aboveground biomass through grazing and cutting resulted in a rapid and a significant decrease of R _s. Temperature-independent seasonal fluctuations of R _s of an intensively managed pasture were closely related to changes in leaf area index (LAI). Across sites increased with mean annual soil temperature (MAT), LAI and gross primary productivity (GPP), indicating that assimilate supply overrides potential acclimation to prevailing temperatures. Also annual R _s was closely related to LAI and GPP. Because the latter two parameters were coupled to MAT, temperature was a suitable surrogate for deriving estimates of annual R _s across the grasslands studied. These findings contribute to our understanding of regional patterns of soil C fluxes and highlight the importance of assimilate supply for soil CO₂ emissions at various timescales.     

Seasonality and controls of phytoplankton productivity in the middle Cape Fear River, USA

Our 1 year study was aimed at assessing seasonal patterns and controls on phytoplankton primary production (PPR) and biomass (chlorophyll a) in a fourth order section of the middle Cape Fear River in North Carolina, USA, and to determine the impact of three low-head lock and dam (LD) structures on these variables within the 70 km study reach of this coastal river. Mean concentrations of NO₃ ⁻–N, NH₄ ⁺–N and soluble reactive phosphorus (SRP) averaged 52.9, 6.0, and 3.6 μmol l⁻¹ in monthly sampling, while the average light attenuation coefficient was 2.4 m⁻¹. The average euphotic depth was 2.1 m. Nutrient concentrations and attenuation coefficients were not significantly different above versus below each LD, or along the entire study reach. Significantly higher concentrations of dissolved O₂ below versus above each LD were attributed to re-aeration during spillway transit. No seasonal pattern in physicochemical properties was apparent. Phytoplankton chlorophyll a concentrations ranged from <1 to 36 μg l⁻¹, while rates of primary production ranged from 18 to 2,580 mg C m⁻² day⁻¹, with values for both variables peaking in the spring and early summer. Chlorophyll a and primary productivity values were consistently higher above versus below each LD in May and June suggesting a seasonal effect, but values were otherwise similar such that overall means were not significantly different. Several factors point to light as the primary control on phytoplankton in the middle Cape Fear River: high nutrient concentrations; a low ratio of euphotic : mixing depth (0.46); progressive increases in chlorophyll a and radiocarbon uptake in all treatments in quarterly nutrient enrichment bioassays conducted at levels of irradiance elevated relative to in situ river values; and consistently low quarterly values of (maximum rate of chlorophyll-normalized C uptake; ≤3.7 mg C mg chl a⁻¹ h⁻¹) and I _k (light saturation parameter; ≤104 μmol photons m⁻² s⁻¹) for photosynthetic light–response (P–I) curves. Handling editor: L. Naselli-Flores     

Soil algal communities inhabiting zinc and lead mine spoils

Algal communities inhabiting four calamine mine spoils differing in time since cessation of exploitation and loaded with high concentrations of zinc (20,284–61,599 μg g⁻¹ soil DW), lead (2,620–3,885 μg g⁻¹ DW) and cadmium (104–232 μg g⁻¹ DW) were studied. In dump soils of slightly alkaline pH (7.28–7.52) and low nutrient (, , ) concentrations, chlorophyll a content ranged from 0.41 to 2.27 μg g⁻¹ soil DW. In total, 23 algal species were recorded. Chlorophyta were the dominant taxonomic group (42–55% of all identified species) followed by Cyanobacteria (28–36%) and Heterokontophyta (13–21%). The highest species richness (18) was observed in the oldest dump (120 years old) with natural succession, while in younger dumps it was lower (11–15). Total algal abundance ranged between 5.5 and 19.1 × 10² ind. g⁻¹ soil DW, and values of Margalef’s diversity indices (1.59–2.25) were low. These results may suggest that both high concentrations of heavy metals and low nutrient content influenced the algal communities in all the dumps studied. The differences in algal microflora observed between tailing dumps may indicate that habitat quality improved with time and that algae isolated from Zn/Pb-loaded soils may be Zn/Pb-resistant ecotypes of ubiquitous species.     

Bifurcation Analysis of Genetically Engineered Pacemaking in Mammalian Heart

Genetically engineered pacemaking in ventricular cells has been achieved by down-regulation of the time independent inward rectifying current (I _K1), or insertion of the hyperpolarisation-activated funny current (I _f). We analyse the membrane system (i.e. ionic concentrations clamped) of an epicardial Luo-Rudy dynamic cell model using continuation algorithms with the maximum conductance () of I _K1 and I _f as bifurcation parameters. Pacemaker activity can be induced either via Hopf or homoclinic bifurcations. As _K1 is decreased by ≈74%, autorhythmicity emerged via a homoclinic bifurcation, i.e., the periodicity first appear with infinitely large periods. In contrast, the insertion of _f induced periodicity via a subcritical Hopf bifurcation at _f≈ 0.25 mSμF⁻¹. Stable autorhythmic action potentials occurred at _f > 0.329 mSμF⁻¹.     

Can biological nitrification inhibition (BNI) genes from perennial Leymus racemosus (Triticeae) combat nitrification in wheat farming?

Using a recombinant luminescent Nitrosomonas europaea assay to quantify biological nitrification inhibition (BNI), we found that a wild relative of wheat (Leymus racemosus (Lam.) Tzvelev) had a high BNI capacity and releases about 20 times more BNI compounds (about 30 ATU g⁻¹ root dry weight 24 h⁻¹) than Triticum aestivum L. (cultivated wheat). The root exudate from cultivated wheat has no inhibitory effect on nitrification when applied to soil; however, the root exudate from L. racemous suppressed formation and kept more than 90% of the soil’s inorganic-N in the -form for 60 days. The high-BNI capacity of L. racemosus is mostly associated with chromosome Lr#n. Two other chromosomes Lr#J, and Lr#I also have an influence on BNI production. Tolerance of L. racemosus to is controlled by chromosome 7Lr#1-1. Sustained release of BNI compounds occurred only in the presence of in the root environment. Given the level of BNI production expressed in DALr#n and assuming normal plant growth, we estimated that nearly 87,500,000 ATU of BNI activity ha⁻¹ day⁻¹ could be released in a field of vigorously growing wheat; this amounts to the equivalent of the inhibitory effect from the application of 52.5 g of the synthetic nitrification inhibitor nitrapyrin (one AT unit of BNI activity is equivalent to 0.6 μg of nitrapyrin). At this rate of BNI production it would take only 19 days for a BNI-enabled wheat crop to produce the inhibitory power of a standard commercial application of nitrapyrin, 1 kg ha⁻¹. The synthetic nitrification inhibitor, dicyandiamide, blocked specifically the AMO (ammonia monooxygenase) pathway, while the BNI from L. racemosus blocked the HAO (hydroxylamine oxidoreductase) pathway in Nitrosomonas. Here we report the first finding of high production of BNI in a wild relative of any cereal and its successful introduction and expression in cultivated wheat. These results demonstrate the potential for empowering the new generation of wheat cultivars with high-BNI capacity to control nitrification in wheat-production systems. Responsible Editor: Hans Lambers.     

Effects of methanol on expression of an anticoagulant hirudin in recombinant Hansenula polymorpha

A series of batch, fed-batch, and continuous cultures was carried out to analyze the effects of methanol on the fermentation characteristics of recombinant Hansenula polymorpha for the production of hirudin, an anticoagulant. Hirudin expression efficiencies were greatly influenced by the methanol concentrations in continuous and fed-batch culture modes. At a steady state of continuous culture, an optimum methanol concentration of 1.7 g l⁻¹ was determined at a dilution rate of 0.18 h⁻¹ with 1.8 mg l⁻¹ h⁻¹ hirudin productivity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 58–61. Received 21 September 2000/ Accepted in revised form 10 June 2001     

Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the γ-aminobutyric acid (GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number of transporters in the plasma membrane. The two-electrode voltage-clamp method was used to measure the transporter-mediated macroscopic current evoked by GABA ( ), macroscopic charge movements (Q _NaCl) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct correlation between Q _NaCl and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 ± 1 elementary charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 ± 1 elementary charges per GAT1 monomer. This information and the relationship between and Q _NaCl were used to estimate a GAT1 unitary turnover rate of 15 ± 2 s⁻¹ (21°C, −50 mV). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79–93 s⁻¹ (37°C, −50 to −90 mV).     

An evolutionary analytical model of a complementary circular code simulating the protein coding genes, the 5′ and 3′ regions

The self-complementary subset ∪{AAA,TTT} with = {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} of 22 trinucleotides has a preferential occurrence in the frame 0 (reading frame established by the ATG start trinucleotide) of protein (coding) genes of both prokaryotes and eukaryotes. The subsets ∪{CCC} and ∪{GGG} of 21 trinucleotides have a preferential occurrence in the shifted frames 1 and 2 respectively (frame 0 shifted by one and two nucleotides respectively in the 5′-3′ direction). and are complementary to each other. The subset contains the subset which has the rarity property (6 × 10⁻⁸) to be a complementary maximal circular code with two permutated maximal circular codes and in the frames 1 and 2 respectively. is called a C³ code. A quantitative study of these three subsets in the three frames 0, 1, 2 of protein genes, and the 5′ and 3′ regions of eukaryotes, shows that their occurrence frequencies are constant functions of the trinucleotide positions in the sequences. The frequencies of in the frame 0 of protein genes are 49, 28.5 and 22.5% respectively. In contrast, the frequencies of in the 5′ and 3′ regions of eukaryotes, are independent of the frame. Indeed, the frequency of in the three frames of 5′ (respectively 3′) regions is equal to 35.5% (respectively 38%) and is greater than the frequencies and , both equal to 32.25% (respectively 31%) in the three frames. Several frequency asymmetries unexpectedly observed (e.g. the frequency difference between and in the frame 0), are related to a new property of the subset involving substitutions. An evolutionary analytical model at three parameters (p, q, t) based on an independent mixing of the 22 codons (trinucleotides in frame 0) of with equiprobability (1/22) followed by t ≈ 4 substitutions per codon according to the proportions p ≈ 0.1; q ≈ 0.1 and r = 1 − p − q ≈ 0.8 in the three codon sites respectively, retrieves the frequencies of observed in the three frames of protein genes and explains these asymmetries. Furthermore, the same model (0.1, 0.1, t) after t ≈ 22 substitutions per codon, retrieves the statistical properties observed in the three frames of the 5′ and 3′ regions. The complex behaviour of these analytical curves is totally unexpected and a priori difficult to imagine.