Genetic stability of wood density and diameter in <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don plantation estate across Australia

Genetic variation for wood quality traits and diameter growth for radiata pine (Pinus radiata D. Don) at age 20/21 years was estimated from eight trials in Australia. The traits studied were wood density, acoustic time-of-flight (an indirect measure of stiffness) and diameter at breast height (DBH). Wood density and DBH exhibited significant additive genetic variation whereas non-additive effects were not significantly different from zero. Time of flight was also not significantly different from zero for both additive and non-additive effects, respectively. Average single-site heritability estimates (±SE) for wood density and DBH were 0.38 ± 0.10 and 0.16 ± 0.08, respectively. Pooled-site heritability estimates for wood density and DBH were 0.38 ± 0.10 and 0.08 ± 0.10, respectively. For density, there was little evidence of genotype-by-environment interaction (GEI) across the eight trials at the additive level (type B additive genetic correlation; r _BADD = 0.73 ± 0.08) and type B genetic correlation for full-sib families (r _BFS = 0.64 ± 0.08). In contrast, the type B additive genetic correlation for DBH was lower, (r _BADD = 0.51 ± 0.14), suggesting evidence of GEI. However, type B genetic correlation for full-sib families was moderate (0.63 ± 0.11) for DBH, suggesting that there may be some stable full-sib families. On the basis of the results of this study, GEI should be considered in order to optimise deployment of improved germplasm in Australia.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

A candidate gene for lignin composition in Eucalyptus: cinnamoyl-CoA reductase (CCR)

Lignin content and composition are considered as mandatory traits of eucalyptus breeding programs, especially for pulp, paper, and bioenergy production. In this article, we used 33 Eucalyptus urophylla full-sib families of an 8 × 8 factorial design to provide estimates of genetic parameters for lignin- and growth-related traits. Secondly, from the sequencing of the 16 unrelated founders, we described the nucleotide and haplotype variability of cinnamoyl-CoA reductase (CCR), a candidate gene for lignin-related traits encoding the cinnamoyl-CoA reductase. Finally, we tested the association between CCR polymorphisms and trait variation using a mixed linear model. A high value of narrow sense heritability was obtained for lignin content (h2 = 0.85) and S/G ratio (h2 = 0.62) indicating that these traits are under strong genetic control. High levels of nucleotide (θ_π = 0.0131) and haplotype (Hd = 0.958) diversity were detected for CCR. From an initial set of 152 biallelic single nucleotide polymorphisms (SNPs), a subset of 65 nonredundant loci was selected. Three intronic SNPs were found to be associated to the variation of S/G ratio after multiple testing correction. In the line of what has been obtained in forest trees, these SNPs explained between 2.45% and 2.87% of the genetic variance of the trait. This study demonstrates the interest of the candidate gene approach for quantitative trait nucleotide detection in Eucalyptus and paves the way to gene assisted selection of lignin composition in E. urophylla.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Genetic variation in basic density and modulus of elasticity of coastal Douglas-fir

Douglas-fir trees from 39 open-pollinated families at four test locations were assessed to estimate heritability of modulus of elasticity (MOE) and basic density. After trees were felled, sound velocity was measured on 4-m logs with the Director HM200. Disks were taken to estimate dry and green wood density; dynamic MOE was estimated as green density × (sound velocity)². Heritability estimates of MOE (across-site h ²=0.55) were larger than those for total height (0.15) and diameter at breast height (DBH; 0.29), and similar to those for density (0.59). Negative genetic correlations were found for MOE with height (r _A=−0.30) and DBH (r _A=−0.51), and were similar to those found for density with height (r _A=−0.52) and DBH (r _A=−0.57). The partial correlations of height with MOE and density, while holding DBH constant, were positive, implying that the observed negative correlations between height and the wood properties were a function of the high positive correlation between height and DBH and the strong negative correlations between DBH and the wood properties. Taper [DBH/(height−1.4)] was found to be negatively associated with MOE. Selection for MOE may produce greater gains than selection for density because MOE had a larger coefficient of additive variation (9.6%) than density (5.1%). Conversely, selection for growth may have a more negative impact on MOE than density because of the greater genetic variation associated with MOE. Family mean correlations of the wood quality traits with stem form and crown health were mostly nonsignificant.     

Biomass Expansion Factors of Natural Japanese Red Pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) Forests in Korea

Biomass expansion factors, which convert the timber volume (or dry weight) to biomass, are used to estimate the forest biomass and account for the carbon budget at the national or regional level. This study estimated the biomass conversion and expansion factors (BCEF), root to shoot ratio (R), biomass expansion factors (BEF) of natural Japanese Red Pine (Pinus densiflora Sieb. et Zucc.) forests based on direct field measurements and publications in Korea. This study attempted to fit the non-linear relationships between the biomass expansion factors (BCEF and BEF) and main stand factors [stand age, tree height, and diameter at breast height (DBH)]. The relationship between BEF and each main stand factor was expressed as a simple logarithmical equation. The BCEF was also expressed as a logarithmical equation of the tree height, DBH, and stand volume, whereas there was no significant relationship between BCEF and stand age. The mean value for BCEF, BEF, and R was 0.5821 Mg m⁻³ (n = 22, SD = 0.1196), 1.4465 (n = 22, SD = 0.2905), and 0.2220 (n = 17, SD = 0.0687), respectively. The values of the biomass expansion factors in this study may indicate much representativeness to estimate forest biomass in natural Japanese Red Pine forests of Korea than the default values given by the IPCC (2003, 2006).     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at a productivity of 0.63–1.09 g PHA l⁻¹ h⁻¹ with final PHA content ranging from 42.6 to 55.8% in single-stage, carbon-limited, fed-batch fermentations of Pseudomonas putida KT2440. A mixture of nonanoic acid (NA) and 10-undecenoic acid (UDA⁼) was fed exponentially to control growth rate. Varying the specific growth rate (0.14 h⁻¹ vs. 0.23 h⁻¹) at similar substrate feed ratios (NA:UDA⁼ = 5:1) had little effect on the final PHA content and relative composition. However, decreasing the NA:UDA⁼ ratio decreased the final amount of PHA produced from 56% with NA:UDA⁼ = 5.07:1 to only 42% at NA:UDA⁼ = 2.47:1. The molar fraction of all 3-hydroxyalkanoate monomers in the PHA product was relatively constant throughout each fermentation, indicating that the final product was homogeneous rather than a mixture of different copolymers. A linear relationship between unsaturation of the PHA produced and unsaturation of the carbon feed was found, which demonstrates the feasibility of producing unsaturated MCL-PHAs with controlled polymeric composition in a fed-batch process.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Density of tiger and leopard in a tropical deciduous forest of Mudumalai Tiger Reserve,southern India,as estimated using photographic capture–recapture sampling

Density of tiger Panthera tigris and leopard Panthera pardus was estimated using photographic capture–recapture sampling in a tropical deciduous forest of Mudumalai Tiger Reserve, southern India, from November 2008 to February 2009. A total of 2,000 camera trap nights for 100 days yielded 19 tigers and 29 leopards within an intensive sampling area of 107 km². Population size of tiger from closed population estimator model M_b Zippin was 19 tigers (SE = ±0.9) and for leopards M_h Jackknife estimated 53 (SE = ±11) individuals. Spatially explicit maximum likelihood and Bayesian model estimates were 8.31 (SE = ±2.73) and 8.9 (SE = ±2.56) per 100 km² for tigers and 13.17 (SE = ±3.15) and 13.01 (SE = ±2.31) per 100 km² for leopards, respectively. Tiger density for MMDM models ranged from 6.07 (SE = ±1.74) to 9.72 (SE = ±2.94) per 100 km² and leopard density ranged from 13.41 (SE = ±2.67) to 28.91 (SE = ±7.22) per 100 km². Spatially explicit models were more appropriate as they handle information at capture locations in a more specific manner than some generalizations assumed in the classical approach. Results revealed high density of tiger and leopard in Mudumalai which is unusual for other high density tiger areas. The tiger population in Mudumalai is a part of the largest population at present in India and a source for the surrounding Reserved Forest.     

Water influx and efflux in free-flying pigeons

We used tritium-labeled water to measure total body water, water influx (which approximated oxidative water production) and water efflux in free-flying tippler pigeons (Columba livia) during flights that lasted on average 4.2 h. At experimental air temperatures ranging from 18 to 27 °C, mean water efflux by evaporation and excretion [6.3 ± 1.3 (SD) ml · h⁻¹, n = 14] exceeded water influx from oxidative water and inspired air (1.4 ± 0.7 ml · h⁻¹, n = 14), and the birds dehydrated at 4.9 ± 0.9 ml · h⁻¹. This was not significantly different from gravimetrically measured mass loss of 6.2 ± 2.1 g · h⁻¹ (t = 1.902, n = 14, P>0.05). This flight-induced dehydration resulted in an increase in plasma osmolality of 4.3 ± 3.0 mosmol · kg⁻¹ · h⁻¹ during flights of 3–4 h. At 27 °C, the increase in plasma osmolality above pre-flight levels (ΔP _osm = 7.6±4.29 mosmol · kg⁻¹ · h⁻¹, n = 6) was significantly higher than that at 18 °C (ΔP _osm = 0.83±2.23 mosmol · kg⁻¹ · h⁻¹, (t = 3.43, n = 6, P < 0.05). Post-flight haematocrit values were on average 1.1% lower than pre-flight levels, suggesting plasma expansion. Water efflux values during free flight were within 9% of those in the one published field study (Gessaman et al. 1991), and within the range of values for net water loss determined from mass balance during wind tunnel experiments (Biesel and Nachtigall 1987). Our net water loss rates were substantially higher than those estimated by a simulation model (Carmi et al. 1992) suggesting some re-evaluation of the model assumptions is required. Accepted: 8 April 1997     

Genetic parameters of morphological and physiological characteristics of containerized white spruce (Picea glauca [Moench.] Voss) seedlings

The root systems of containerized seedlings must be sufficiently developed and have adequate root plug cohesion to permit handling and the planting of the seedlings with minimal root damage. Genetic variability in morphological and physiological seedling characteristics of 75 open-pollinated white spruce families was estimated to determine whether genetic selection for improved seedling root systems is possible. Seedlings were grown for 2 years under standard cultural practices in a forest nursery. Gas exchange measurements and seedling morphological characteristics (height, diameter, shoot and root dry mass, root to shoot ratio) were measured at the end of the two growing seasons whereas seedling mineral (N, P, and K) status was assessed at the end of the first growing season. Genetic parameters (heritabilities—h ²—and genetic correlations) were estimated for every seedling characteristic and a strong genetic control associated with a large genetic variation was observed at both family (0.20 ≤ h_f² h_f^2  ≤ 0.88) and individual (0.21 ≤ h_i² h_i^2  ≤ 0.97) levels. A single, late-season measurement of physiological characteristics did not reveal physiological basis for family variability in seedling root growth. Nevertheless, the family variation was large enough to permit genetic improvement of 2-year-old seedling juvenile morphological characteristics. Strong, positive genetic correlations enable us to foresee using root collar diameter as an effective method for indirectly selecting white spruce families with heavier root systems.     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems

The change of dilution rate (D) on both Methylophilus methylotrophus NCIMB11348 and Methylobacterium sp. RXM CCMI908 growing in trimethylamine (TMA) chemostat cultures was studied in order to assess their ability to remove odours in fish processing plants. M. methylotrophus NCIMB11348 was grown at dilution rates of 0.012–0.084 h⁻¹ and the biomass level slightly increased up to values of D around 0.07 h⁻¹. The maximum cell production rate was obtained at 0.07 h⁻¹ corresponding to a maximum conversion of carbon into cell mass (35%). The highest rate of TMA consumption was 3.04 mM h⁻¹ occurring at D=0.076 h⁻¹. Methylobacterium sp. RXM CCMI908 was grown under similar conditions. The biomass increased in a more steep manner up to values of D around 0.06 h⁻¹. The maximum cell production rate (0.058 g l⁻¹h⁻¹) was obtained in the region close to 0.06 h⁻¹ where a maximum conversion of the carbon into cell mass (40%) was observed. The maximum TMA consumption was 2.33 mM h⁻¹ at D=0.075 h⁻¹. The flux of carbon from TMA towards cell synthesis and carbon dioxide in both strains indicates that the cell is not excreting products but directing most of the carbon source to growth. Carbon recovery levels of approximately 100% show that the cultures are carbon-limited. Values for theoretical maximum yields and maintenance coefficients are presented along with a kinetic assessment based on the determination of the substrate saturation constant and maximum growth rate for each organism. Received: 25 February 1999 / Received revision: 14 May 1999 / Accepted: 17 May 1999     

Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Changes in Morphology and Elemental Composition of <Emphasis Type="Italic">Vibrio splendidus</Emphasis> Along a Gradient from Carbon-limited to Phosphate-limited Growth

We examined morphology, elemental composition (C, N, P), and orthophosphate-uptake efficiency in the marine heterotrophic bacterium Vibrio splendidus grown in continuous cultures. Eight chemostats were arranged along a gradient of increasing glucose concentrations in the reservoirs, shifting the limiting factor from glucose to phosphate. The content of carbon, nitrogen, and phosphorus was measured in individual cells by x-ray microanalysis using a transmission electron microscope (TEM). Cell volumes (V) were estimated from length and width measurements of unfixed, air-dried cells in TEM. There was a transition from coccoid cells in C-limited cultures toward rod-shaped cells in P-limited cultures. Cells in P-limited cultures with free glucose in the media were significantly larger than cells in glucose-depleted cultures (P < 0.0001). We found functional allometry between cellular C-, N-, and P content (in femtograms) and V (in cubic micrometers) in V. splendidus (C = 224 × V ^0.89, N = 52.5 × V ^0.80, P = 2 × V ^0.65); i.e., larger bacteria had less elemental C, N, and P per V than smaller cells, and also less P relative to C. Biomass-specific affinity for orthophosphate uptake in large P-limited V. splendidus approached theoretical maxima predicted for uptake limited by molecular diffusion toward the cells. Comparing these theoretical values to respective values for the smaller, coccoid, C-limited V. splendidus indicated, contrary to the traditional view, that large size did not represent a trade-off when competing for the non-C-limiting nutrients.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.     

Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.

Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl (DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg²⁺. Kinetic data for chorismate in the forward reaction (K _m = 807 μM, V _max = 6.2 pkat · mg⁻¹) and for isochorismate in the reverse reaction (K _m = 675 μM, V _max = 5.9 pkat · mg⁻¹) were determined. Received: 18 November 1996 / Accepted: 28 December 1996     

Mouse to human comparative genetics reveals a novel immunoglobulin E-controlling locus on Hsa8q12

Atopy is a predisposition to hyperproduction of immunoglobulin E (IgE) against common environmental allergens. It is often associated with development of allergic diseases such as asthma, rhinitis, and dermatitis. Production of IgE is influenced by genetic and environmental factors. In spite of progress in the study of heredity of atopy, the genetic mechanisms of IgE regulation have not yet been completely elucidated. The analysis of complex traits can benefit considerably from integration of human and mouse genetics. Previously, we mapped a mouse IgE-controlling locus Lmr9 on chromosome 4 to a segment of <9 Mb. In this study, we tested levels of total IgE and 25 specific IgEs against inhalant and food allergens in 67 Czech atopic families. In the position homologous to Lmr9 on chromosome 8q12 marked by D8S285, we demonstrated a novel human IgE-controlling locus exhibiting suggestive linkage to composite inhalant allergic sensitization (limit of detection, LOD = 2.11, P = 0.0009) and to nine specific IgEs, with maximum LOD (LOD = 2.42, P = 0.0004) to plantain. We also tested 16 markers at previously reported chromosomal regions of atopy. Linkage to plant allergens exceeding the LOD > 2.0 was detected at 5q33 (D5S1507, LOD = 2.11, P = 0.0009) and 13q14 (D13S165, LOD = 2.74, P = 0.0002). The significant association with plant allergens (quantitative and discrete traits) was found at 7p14 (D7S2250, corrected P = 0.026) and 12q13 (D12S1298, corrected P = 0.043). Thus, the finding of linkage on chromosome 8q12 shows precision and predictive power of mouse models in the investigation of complex traits in humans. Our results also confirm the role of loci at 5q33, 7p14, 12q14, and 13q13 in control of IgE. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.