Inheritance of alleles for Cgy1 and Gy4 storage protein genes in soybean

Summary A cultivar lacking the glycinin subunit A₅A₄B₃ (Raiden) was crossed with one lacking the -subunit of -conglycinin (Keburi). Analysis of F₂ and F₃ progeny indicated that the missing bands of the A₅A₄B₃ and the -subunit were each controlled by a recessive allele of two independently segregating genes. Gene symbols Gy ₄/gy ₄ and Cgy ₁/cgy ₁ were proposed for the genes which confer the presence or absence of the glycinin and conglycinin subunits, respectively.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 9675. Financial support from the American Soybean Association Research Foundation is gratefully acknowledged     

Photosynthetic glow peaks and their relationship with the free energy changes

This paper is concerned with relating thermoluminescence to the total free-energy change, G, involved in detrapping a particular electron-hole pair as a photosynthetic sample is warmed from an initial low temperature. It extends a mathematical discussion of four possible mechanisms introduced in an earlier paper [DeVault, Govindjee and Arnold, Proc Nat'l Acad Sci USA 80: 983–987 (1983)]; here, particular attention is paid to the dependence of the absolute temperature of the maximum of a glow-peak, T _m , on the total free-energy change, G. The conclusion from the cases studied is that T _m =G/(k _B W) where G is evaluated at T _m , W is a complicated function of temperature and of thermodynamic parameters in the steps of the mechanism, and k _B is the Boltzmann constant. If the rate limiting step in the mechanism of detrapping is not preceded by any step in which G is appreciably negative, W is likely to have a value of about 33 and T _m is approximately proportional to G. Otherwise W can become much smaller and more strongly dependent on temperature and T _m is no longer proportional to G. These conclusions are of significance in lending theoretical support to the practice of inferring redox midpoint potential changes from shifts in T _m .     

A theoretical and experimental evaluation of a novel radial-flow hollow fiber reactor for mammalian cell culture

A hollow fiber perfusion reactor constructed from pairs of concentric fibers forming a thin annular space is analyzed theoretically in terms of mass transfer resistances, and is shown experimentally to support the growth of an anchorage-dependent cell line in high-density culture. Hollow fiber perfusion reactors described in the literature typically employ a perfusion pathlength much greater than the distance that could be supported by diffusion alone, and analyses of these reactors typically incorporate the assumption of uniform perfusion throughout the cell mass despite many reported observations of inhomogeneous cell growth in perfusion reactors. The mathematical model developed for the annular reactor predicts that the metabolism of oxygen, carbon substrates, and proteins by anchorage-dependent cells can be supported by the reactor even in the absence of perfusion. The implications of nonuniform cell growth in perfusion reactors in general is discussed in terms of nutrient distribution. In the second part of the paper, the growth and metabolism of the mouse adrenal tumor line Y-1 in flask culture and in the annular reactor are compared. The reactor is shown to be a promising means for culturing anchorage-dependent cells at high density.List of Symbols c mol/dm³ substrate concentration - D _mm²/s effective diffusivity of substrate in the membrane - D _tm²/s effective diffusivity of substrate in the cell region - L _pm²s/kg hydraulic permeability of fiber - Pe _m Peclet number for membrane transport, _wR₁/D _m - Pe _t Peclet number for transport through cell mass, v _wR₂/D _t - Q mol/m³s zero-order consumption rate of substrate per unit volume of cell mass - r m radial distance from centerline of fiber lumen - R ₁, R ₂ m inner and outer radii of inner annular fiber (Fig. 1) - R ₃, ₄ m inner and outer radii of outer annular fiber (Fig. 1) - v _wm/s fluid velocity through the fiber wall at R ₁ - fraction of shell side filled with cells - dimensionless radial distance, R ₃/R₁ - dimensionless radial distance, R ₂/R ₁ - cm² hydraulic conductivity - viscosity - ², Thiele modulus - dimensionless radial distance, R ₄/R ₁     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Anaerobic digestion of palm oil mill effluent and condensation water waste: an overall kinetic model for methane production and substrate utilization

The process of anaerobic digestion is viewed as a series of reactions which can be described kinetically both in terms of substrate utilization and methane production. It is considered that the rate limiting factor in the digestion of complex wastewaters is hydrolysis and this cannot be adequately described using a Monod equation. In contrast readily assimilable wastewaters conform well to this approach. A generalized equation has thus been derived, based on both the Monod and Contois equations, which serves extreme cases. The model was verified experimentally using continuous feed anaerobic digesters treating palm oil mill effluent (POME) and condensation water from a thermal concentration process. POME represents a complex substrate comprising of unhydrolyzed materials whereas the condensation water is predominantly short chain volatile fatty acids. Substrate removal and methane production in both cases could be predicted accurately using the generalized equation presented.List of Symbols A (=K_skY/K_h) Kinetic parameter - B Specific methane yield, 1 of CH₄/g of substrate added B₀ Maximum specific methane yield, 1 of CH₄/g of substrate added at infinity - C Empirical constant in Contois equation - F Volumetric substrate removal rate, g/l day - k Hydrolysed substrate transport rate coefficient, 1/days - K (=YC) Kinetic parameter in Chen-Hashimoto equation - K _h Substrate hydrolysis rate coefficient, 1/days - K _s Half-saturation constant for hydrolysed substrate, g/l - M _v Volumetric methane production rate, 1 of CH₄/l day - MS Mineral solids, g/l - MSS Mineral suspended soilds, g/l - POME Palm oil mill effluent - R (=S_r/S_T0) Refractory coefficient - S _h Concentration of hydrolysed substrate, g/l - S _u Intracellular concentration of hydrolysed substrate, g/l - S ₀ Input biodegradable substrate concentration, g/l - S Biodegradable substrate concentration in the effluent or in the digester, g/l - S _r Refractory feed substrate concentration, g/l - S _T0 (=S₀+S_r) Total feed substrate concentration, g/l - S _T (S+S_r) Total substrate concentration in the effluent, g/l - TS Total solids, g/l - TSS Total suspended solids, g/l - VFA Total volatile fatty acids, g/l - VS Volatile solids, g/l - VSS Volatile suspended solids, g/l - X Biomass concentration, g/l - Y Biomass yield coefficient, biomass/substrate mass - Hydraulic retention time, days. - Specific growth rate of microorganisms, l/days - _m Maximum specific growth rate of microorganisms, l/days The authors wish to express their gratitude to the Departamento de Postgrado y Especialización del CSIC and to the Consejería de Educación y Ciencia de la Junta de Andalucia for their financial support of this work.     

Chloroplast DNA-relationship in palaeoaustralLopidium concinnum (Hypopterygiaceae, Musci). An example of stenoevolution in mosses Studies in austral temperate rain forest bryophytes 2

Evolutionary relationship between disjunct populations of the palaeoaustral moss taxonLopidium concinnum (Hypopterygiaceae) from New Zealand and southern South America were studied using non-coding chloroplast DNA sequences. No or only slight changes could be observed within the sequences oftrnT_UGU —trnL_UAA 5exon intergenic spacer,trnL_UAA intron andtrnL_UAA 3exon —trnF_GAA intergenic spacer. This indicates nearly no genetic divergence between extant New Zealand and Chilean populations, i.e. no significant differing pathways of evolution within the 80–60 million years of disrupted areas with interrupted gene flow. Molecular data support the idea of an old Gondwanan relict species of stenoevolutionary character. Ecological data on short-range dispersal strengthen this assessment.     

Correlation between testcross performance of lines at early and late selfing generations

Summary In hybrid breeding programs, testcross evaluation of lines can be done during the early stages of selfing (early testing) or delayed until the lines are near-homozygous. To evaluate the usefulness of early testing, the expected genetic and phenotypic correlations between testcross performance at different selfing generations were examined. The genetic correlation (r _GnGn ) between testcross performance of S _n and S _n , (n>n) individuals or lines is equal to the square root of the ratio of their testcross genetic variances, and it is a function of the inbreeding coefficients (F) at the two selfing generations, i.e., r _GnGn=[(1+F _n )/(1+F _n )]^0.5. The genetic correlation between testcross performance of lines and their directly descended homozygous (n=) lines is 0.71 for S₁; 0.87 for S₂, 0.93 for S₃, 0.97 for S₄, 0.98 for S₅, and 0.99 for S₅ lines. The effectiveness of early testing is limited mainly by nongenetic effects. The square root of testcross heritability at generation n sets the upper limit on the correlation between phenotypic value at generation n and genotypic value at homozygosity. The probabilities of correctly retaining S _n individuals or lines that have superior testcross performance at homozygosity (n=) indicate that early testing should be effective in identifying lines with above- and below-average combining ability. However, the risk of losing lines with superior combining ability is high if strong (best 10%) selection pressure is applied during early testing. If only a small proportion of lines is retained based on testcross performance and/or if the heritability of the trait is low, selfing for two or three generations prior to testcrossing may be desirable to increase the likelihood of retaining lines that perform well at homozygosity. The theoretical results in this study support the testcross evaluation procedures for grain yield used by most maize (Zea mays L.) breeders.A contribution from Limagrain Genetics, a Groupe Limagrain company     

An investigation of the role of circulating catecholamines in the control of ventilation during acute moderate hypoxia in rainbow trout (Oncorhynchus mykiss)

Summary Gill ventilation volume ( w), arterial blood oxygen tension (PaO₂) and arterial blood oxygen content (CaO₂) of rainbow trout (Oncorhyncus mykiss) were monitored during normoxia [waterPO₂ (PwO₂) 155 Torr], hypoxia (PwO₂=72±5.8 Torr), or hyperoxia (PwO₂=643±32 Torr). Fish hyperventilated during acute (30 min) hypoxia and hypoventilagted during acute hyperoxia. Plasma catecholamine levels were unchanged after 30 min of hypoxia or hyperoxia. In addition, selective adrenoceptor blockade with either propranolol (-adrenoceptor antagonist) or phentolamine (-adrenoceptor antagonist) did not modify the hyperventilatory response during hypoxia. These results indicate that circulating catecholamines are not involved in the control of ventilation in moderately hypoxic rainbow trout. In the summer, intra-arterial infusion of catecholamine in normoxic trout caused transient (adrenaline) or persistent (noradrenaline) hypoventilation. These observations also do not support a role for catecholamines in the stimulation of ventilation.During hypoxia,PwO₂,PaO₂ andCaO₂ were depressed whereas during hyperoxia, onlyPwO₂ andPaO₂ was elevated significantly. Thus, it is suggested that the hypoventilatory response to hyperoxia is mediated by a direct effect of elevatedPwO₂/PaO₂, whereas the hyperventilatory response to hypoxia is mediated by changes inPwO₂/PaO₂, and/orCaO₂.     

Structure and activity of chloroplasts of sunflower leaves having various water potentials

Summary Changes in membrane integrity, conformation and configuration, and in photosystem II (PS II) activity (measured as dichloroindophenol photoreduction) of sunflower (Helianthus annuus L.) chloroplasts were studied after leaf tissue had been desiccated to various water potentials ( _w ). Fixatives for electron microscopy were adjusted osmotically to within 1 bar of the _w of the tissue to prevent rehydration during fixation. PS II activity decreased to 50% of the control activity at a _w of-26 bar. At this _w , leaf viability was being lost but there was virtually no loss of integrity of the thylakoid lamellar system. Even at extreme _w (below-100 bar), thylakoids retained much structural detail but were less stained. At-26 bar, intrathylakoid spacing (configuration) and lamellar thickness (conformation) were decreased in vivo. Upon isolation of the plastids, the differences in configuration disappeared but the differences in conformation remained. The decreases in membrane conformation and PS II activity both, in vivo and in vitro suggest that alterations in conformation may cause decreases in chloroplast activity at _w as low as-26 bar. Since structural detail was maintained, however, previous observations of altered membrane integrity, which involved tissue fixed without osmotic support, may have been affected by tissue rehydration during fixation.Abbreviations DCIP sodium 2,6-dichloroindophenol - PS II photosystem II - _w leaf water potential     

Proximity of the CTLA-1 serine esterase and Tcr α loci in mouse and man

The serine esterase CTLA-1 gene was shown by in situ hybridization to map to the D segment of mouse chromosome 14, the same localization as a member of the immunoglobulin super family, Tcr . To further demonstrate the proximity of CTLA-1 and Tcr , genetic linkage was tested in mouse using restriction fragment length polymorphisms and a backcross progeny, and no recombination was observed in the 100 backcross products studied. Recombination events between Tcr /CTLA-1 and the markers Gdh-X and NP-1 show that the most probable order of these loci in the mouse 14D region is NP-1-Tcr /Ctla-1-Gdh-X. In man, the human homologue of CTLA-1 was shown by in situ hybridization to map on chromosome 14, at 14q11-q12, where Tcr also maps. Using the human cell line SUP-Tl, bearing the inversion inv(14)(q11;q32), we further demonstrated the loci order in man to be centromere-NP-1-Tcr -CTLA-1. To complement the cytogenetic and genetic mapping data, we tried to determine the physical distance between the two genes by pulsed field gel electrophoresis (PFGE). DNA prepared from various cell types, both mouse and human, were digested with a panel of rare cutter enzymes and hybridized first with CTLA-1, then with Tcr probes. None of the bands identified hybridized with both Tcr and CTLA-1 probes for either mouse or human cells. Although the physical mapping by PFGE is inconclusive, the cytogenetic and genetic data support close linkage of the Tcr and CTLA-1 genes in both mouse and man, suggesting homology between the D region of mouse chromosome 14 and the q11–q12 region of human chromosome 14, encompassing the Tcr and CTLA-1 loci. These findings also provide another example of proximity of genes coding for a member of the Ig super-family and a serine esterase.     

Short-term changes in carbon-isotope discrimination identify transitions between C3 and C4 carboxylation during Crassulacean acid metabolism

Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO₂ collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO₂ exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO₂ (Phase I), discrimination () ranged from 4.4 to 6.6, equivalent to an effective carbon-isotope ratio (¹³C) of –12.3 to –14.5 versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO₂ uptake and refixation of respiratory CO₂, characteristic of CAM in the Bromeliaceae. When for the relative proportion of external (p _a ) and internal (p _i) CO₂ is taken into account, calculated p _i/p _a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of during Phase II, early in the light period, showed the transition between C₄ and C₃ pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as increased from 10.5 to 21.2 During decarboxylation in the light period (Phase III), CO₂ leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of calculated from on-line measurements (64.4) showed that the CO₂ lost was considerably enriched in ¹³C, and this was confirmed by direct analysis of the CO₂ diffusing out into a CO₂-free atmosphere ( ¹³C = + 51.6 versus PDB). Instantaneous discrimination was characteristic of the C₃ pathway during Phase IV (late in the light period), but a reduction in showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that double carboxylation involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.Abbreviations and Symbols CAM Crassulacean acid metabolism - H⁺ (dawn-dusk) variation in titratable acidity - ¹³C carbonisotope ratio of plant organic material, relative to Pee Dee Belemnite (vs. PDB) - discrimination against ¹³CO₂, - p _i, p _a internal, external partial pressures of CO₂ - Rubisco ribulose1,5-bisphosphate carboxylase - PAR photosynthetically active radiation - PEPCase phosphoenolpyruvate carboxylase We are grateful for financial support in respect of research grants (GR3/5360, GR3/6419) and a studentship awarded by the Natural Environment Research Council, UK.     

Skeletal muscle pH dynamics during arousal from hibernation measured by31P NMR spectroscopy

Summary The time-course of changes in skeletal muscle pH during arousal from hibernation in the Columbian ground squirrel was studied using³¹P NMR spectroscopy. In hibernation (T _re 7–9°C), shoulder/neck muscle pH was 7.45±0.03 and _Im was 0.60. In euthermia (T _re 37°C), muscle pH was 7.24±0.05 and _Im was 0.75. Thus the overall pH-temperature coefficient was-0.009 pH units/°C, indicating acidification of the muscle in hibernation. During the transition from hibernation to euthermia, however, the muscle shows a nonlinear pattern of pH change. In early arousal (T _sh<20–25°C,T _re<15°C) muscle pH does not change and muscle _Im increases to 0.72. In later arousal (T _sh>20–25°C,T _re>15°C) muscle pH decreases gradually toward the euthermic value and muscle _Im increases only slightly from 0.72 to 0.75. These results support the hypothesis that intracellular acidification of the muscle, present during hibernation, is reversed in early arousal. This may facilitate an increase in muscle metabolism and the contribution of maximal shivering thermogenesis to rewarming of the animal.Abbreviations _Im dissociation ratio of protein imidazole buffergroups - NST non-shivering thermogenesis - BAT brown adipose tissue - dp H/dT temperature coefficient of pH - pH _i intracellular pH - ³¹ P NMR ³¹Phosphorus nuclear magnetic resonance - P _i chemical shift of inorganic phosphate relative to PCr - PCr phosphocreatine - T _b body temperature - T _re rectal temperature - T _sh subcutaneous shoulder temperature - T _a ambient temperature     

Inheritance and biochemical analysis of four electrophoretic variants of β-conglycinin from soybean

Summary Three genes which code for variant -conglycinin subunits were identified. Alleles Cgy ₁ ^S and Cgy ₂ ^S were codominant with Cgy ₁ and Cgy ₂ and produced and subunits, respectively, with reduced electrophoretic mobility. Allele Cgy ₃ ^D increased the mobility of at least one polypeptide in the subunit family and exhibited incomplete dominance. Gene loci Cgy ₂/Cgy ₂ ^S and Cgy ₃ ^D /cgy ₃ ^D were linked, whereas Cgy ₁/Cgy ₁ ^S / cgy ₁ segregated independently of the others. Techniques developed for purification of normal -conglycinin subunits were effective in purifying the altered subunits. Deglycosylated variant proteins from seeds containing the alleles Cgy ₁ ^S , Cgy ₂ ^S , or Cgy ₃ ^D also has altered mobility relative to deglycosylated normal proteins. Therefore, the altered subunits contained changes in their amino acid sequences rather than in their carbohydrate moieties. This interpretation is consistent with the observed codominant or incompletely dominant mode of inheritance for these alleles and suggests that each contains an altered nucleotide sequence in the structural gene. A fourth variant, which exhibited doublet and a electrophoretic bands, was inherited in a recessive fashion. Deglycosylated subunit proteins from this variant were identical in electrophoretic mobility to those of the deglycosylated normal protein. This suggests that the doublet phenotype resulted from an alteration in the carbohydrate moiety of these subunits. The gene or genes which condition this variant presumably are required for normal post-translational modification of the subunit carbohydrates and as such may be useful for investigating these events.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 10,323. Financial support from the American Soybean Research Foundation is gratefully acknowledged     

A testcross procedure for selecting exotic strains to improve pure-line cultivars in predominantly self-fertilizing species

Methods for identifying germplasm carrying alleles with the potential to improve a particular single-cross hybrid have been proposed and discussed in recent years. There is a need for similar methods to be used in breeding crops for which pure-line cultivars, rather than hybrids, are the goal. The objective of this research was to develop a method to identify germplasm lines with the potential to contribute favorable alleles not present in a specified pure line or set of pure lines. Given a set of adapted pure lines (A ₁, A ₂ ..., A _m) to be improved and a set of germplasm lines (P ₁ P ₂ ..., P _f), the procedure consists of producing all f x m possible hybrids and evaluating them along with the parents. The testcross statistic T _ij is defined by T _ij=(F _ij–A _j)+(1–) (F _ij–P _i), where A _j, P _i, and F _ij represent the performance of thej ^th adapted line, the i ^th germplasm line, and their hybrid, respectively. The statistic is the mean value of T _ij over all adapted parents A _j. If =(1/2)(1+d), where d = the mean degree of dominance, then T _ij measures the potential for alleles from P _i to improve A _j and measures the potential for alleles from P _i to improve the set A ₁, A ₂ ..., A _m. Use of data on soybean and peanut hybrids published by other researchers suggests that the value assumed for d has little effect on the P _i chosen. The ability of the T _ij and statistics to identify germplasm strains carrying rare favorable alleles should be assessed in empirical studies.Joint contribution: OARDC (Journal Articale No. 161-94), USDAARS, Iowa Agriculture and Home Economics Expriment Station (Journal Paper No. J-16109; Project 2985), and Agreculture and Agri-Food Canada. Salaries and research support for S. K. St. Martin Provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University     

Hierarchy in the assembly of HLA-B27 and HLA-Cw3 molecules in transgenic mice

Cell surface expression of human class I molecules in transgenic mice is dependent upon the available pool of ₂-microglobulin (₂m) and the affinity between mouse ₂m and human class I molecules. HLA-B27 and HLA-Cw3 transgenes can be expressed in mouse strains of the H-2 haplotypes b,f,k, and s which encode two endogenous class I genes mapping to H-2K and H-2D. The human class I genes cannot be expressed on H-2 ^dand H-2 ^qhaplotypes which encode three endogenous class I molecules (K,D,L). This suggests that there may be only enough mouse ₂m molecules to support three class I molecules. When both the HLA-B27 and HLA-Cw3 genes are introduced into H-2 ^bmice, only HLA-Cw3 reaches the cell surface. This suggests that HLA-Cw3 has a higher affinity than HLA-B27 for mouse ₂m. The possible implications of our findings regarding the assembly, transport, and expression of class I MHC molecules in vivo are discussed.     

Nitrogenase — hydrogenase relationships in Rhizobium japonicum

The conditions necessary for coordinate derepression of nitrogenase and O₂-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate -ketoglutarate was able to support the highest levels of both nitrogenase and hydrogenase activities. When cells were incubated in -ketoglutarate-containing medium, without added H₂ but in the presence of acetylene (to block H₂ evolution from nitrogenase) significant hydrogenase activity was still observed. Complete inhibition of nitrogenase-dependent H₂ evolution by acetylene was verified by the use of a Hup^- mutant. Hydrogen is therefore not required to induce hydrogenase. The presence of 10% acetylene inhibited derepression of hydrogenase. Constitutive (Hup^c) mutants were isolated which contained up to 9 times the level of hydrogenase acitivity than the wild type in nitrogenase induction medium. These mutants did not have greater nitrogenase activities than the wild type.This is contribution number 1254 from the Department of Biology and the McCollum-Pratt Institute Abbreviations: -Ketoglutarate-containing medium (LOKG) and pre-adaptation medium (SRM) as described in Materials and methods     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Estimation of oxygen penetration depth in immobilized cells

Summary A simple method is proposed for calculating oxygen pentration depth in immobilized cells by assuming zero order kinetics in the presence of several external oxygen transport resistances. Calculations indicate that typical penetration depths of oxygen for immobilized microbial cells are in the range of 50–200 and those for immobilized or encapsulated animal and plant tissue culture are about 500–1000 . Based on calculations, oxygen transport in microencapsulation and microcarriers for tissue cultures are not transport-limited, but a slight limitation is expected for those in a hollow fiber reactor.Nomenclature a_s specific area of a support (cm) - Bi Biot number - dimensionless - C_b oxygen concentration in the bulk liquid (mM) - C _b C _b ^* -C_cr (mM) - C _b ^* bulk oxygen concentration in equilibrium with air (mM) - C_cr critical oxygen concentration (mM) - C_s oxygen concentration in the solid phase (mM) - d_p diameter or thickness of a support (cm) - D_eff effective diffusivity of oxygen in the solid phase (cm²/s) - k_m membrane permeability of oxygen (cm/s) - k _m ^* D_eff/_m - k_La_L liquid phase mass transfer rate coefficient (1/s) - k_sa_s solid phase mass transfer rate coefficient (1/s) - (OUR)_v volumetric oxygen uptake rate (mmol O₂/l) - p geometry parameter, p=0 for slab, p=1 for cylinder, p=2 for sphere - P_d oxygen penetration depth (cm) - P _d oxygen penetration depth in the absence of external diffusion limitation (cm) - Q volumetric oxygen uptake rate, (mmol O₂/l·h) - specific oxygen uptake rate (mmol O₂gm biomass (dry)·h) - r length coordinate (cm) - r_c oxygen penetration depth for sphere (cm) - r _c r_c in the absence of external diffusion limitation (cm) - r _c ^* oxygen penetration depth for cylinder (cm) - r _c ^* r _c ^* in the absence of external diffusion limitation (cm) - r_com combined mass transfer rate resistance (s) - r_d location where C_s becomes zero or C_cr (cm) - r_i radius of cylinder or sphere, half thickness of slab (cm) - U_sg superficial gas velocity (cm/s) - X cell concentration (g/l) Greek letters Thiele modulus, dimensionless - _L, _s liquid and solid phase volume fraction, respectively, dimensionless - effectiveness factor On sabbatical leave from KAIST, Seoul, Korea     

Reactor design for the enzymatic isomerization of glucose to fructose

A comprehensive methodology is presented for the design of reactors using immobilized enzymes as catalysts. The design is based on material balances and rate equations for enzyme action and decay and considers the effect of mass transfer limitations on the expression of enzyme activity. The enzymatic isomerization of glucose into fructose with a commercial immobilized glucose isomerase was selected as a case study. Results obtained are consistent with data obtained from existing high-fructose syrup plants. The methodology may be extended to other cases, provided sound expressions for enzyme action and decay are available and a simple flow pattern within the reactor might be assumed.List of Symbols C kat/kg specific activity of the catalyst - D m²/s substrate diffusivity within the catalyst particle - Dr m reactor diameter - d d operating time of each reactor - E kat initial enzyme activity - E _i kat initial enzyme activity in each reactor - F m³/s process flowrate - F _i m³/s reactor feed flowrate at a given time - F ₀ m³/s initial feed flowrate to each reactor - H number of enzyme half-lives used in the reactors - K mole/m³ equilibrium constant - K _S mole/m³ Michaelis constant for substrate - K _P mole/m³ Michaelis constant for product - K _m mole/m³ apparent Michaelis constant f(K, K _s, K_p, s₀) - k mole/s · kat reaction rate constant - k _d d^–1 first-order thermal inactivation rate constant - L m reactor height - L _r m height of catalyst bed - N _R number of reactors - P _i kg catalyst weight in each reactor - p mole/m³ product concentration - R m particle radius - R _P ratio of minimum to maximum process flowrate - r m distance to the center of the spherical particle - s mole/m³ substrate concentration - s _0i mole/m³ substrate concentration at reactor inlet - s ₀ mole/m³ bulk substrate concentration - s mole/m³ apparent substrate concentration - T K temperature - t d time - t _i d operating time for reactor i - t _s d time elapsed between two successive charges of each reactor - V m³ reactor volumen - V _m mole/m³ s maximum apparent reaction rate - V _p mole/m³ s maximum reaction rate for product - V _R m³ actual volume of catalyst bed - V _r m³ calculated volume of catalyst bed - V _S mol/m³ s maximum reaction rate for substrate - v mol/m³ s initial reaction rate - v _i m/s linear velocity - v _m mol/m³ s apparent initial reaction rate f(K_m, s,V_m) - X substrate conversion - X _eq substrate conversion at equilibrium - =s/K dimensionless substrate concentration - ₀=s₀/K bulk dimensionless substrate concentration - _eq=s_eq/K dimensionless substrate concentration at equilibrium - local effectiveness factor - mean integrated effectiveness factor - Thiéle modulus - =r/R dimensionless radius - _s kg/m³ hydrated support density - substrate protection factor - s residence time     

Leaf water potential,component potentials and relative water content in a xeric grass,Agropyron dasystachyum (Hook.) Scribn.

Summary Leaf water potential ( _l ), osmotic potential ( _s ), pressure potential ( _p , turgor pressure), relative water content (R) and their interrelationships were determined for a xeric grass (Agropyron dasystachyum) found in the grasslands of Canada. Thermocouple psychrometers were used to measure _l and _s ; _p was obtained by subtraction. _l dropped from near 0 bars to about-28 bars as R went from 90% to 75%. R greater than 90% was not observed, perhaps because of a systematic error in determination of turgid water content. R remained relatively high in A. dasystachyum, even at low _l . The slope of the _l -R relationship was similar to other species which are generally considered to be drought tolerant. _p as high as 14 bars was observed. Most of the decrease in _l was accounted for by a decline in _p . The ability of A. dasystachyum to adjust to fluctuating water stress over the growing season is probably as much related to changes in tissue structure and turgor relationships as to simple changes in osmotic potential.