Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.     

Inoculum size influences bacterial cross contamination between surfaces

Many factors have been shown to influence bacterial transfer between surfaces, including surface type, bacterial species, moisture level, pressure, and friction, but the effect of inoculum size on bacterial transfer has not yet been established. Bacterial cross contamination rates during performance of common food service tasks were previously determined in our laboratory using nalidixic acid-resistant Enterobacter aerogenes. Eight different transfer rates were determined, each involving a minimum of 30 volunteers. The influence of source inoculum level on the percentage of bacteria transferred (percent transfer rates) and log10 CFU per recipient surface was determined using statistical analysis. The effect of inoculum size on transfer rate was highly statistically significant (P < 0.0001) for all transfer rate data combined (352 observations) and for each individual cross contamination rate, except for data on contamination via transfer from chicken to hand through a glove barrier (P = 0.1643). Where inoculum size on the source was greater, transfer rates were lower, and where inoculum size on the source was less, transfer rates were higher. The negative linear trend was more obvious for activities that had a larger range of inoculum sizes on the source surface. This phenomenon has serious implications for research seeking to determine bacterial cross contamination rates, since the different transfer efficiencies that were previously shown to be associated with certain activities may actually be the result of differing initial inoculum levels. The initial inoculum size on the source and the amount of bacteria transferred must both be considered to accurately determine bacterial transfer rates.     

Inoculum Size Influences Bacterial Cross Contamination between Surfaces

Many factors have been shown to influence bacterial transfer between surfaces, including surface type, bacterial species, moisture level, pressure, and friction, but the effect of inoculum size on bacterial transfer has not yet been established. Bacterial cross contamination rates during performance of common food service tasks were previously determined in our laboratory using nalidixic acid-resistant Enterobacter aerogenes. Eight different transfer rates were determined, each involving a minimum of 30 volunteers. The influence of source inoculum level on the percentage of bacteria transferred (percent transfer rates) and log₁₀ CFU per recipient surface was determined using statistical analysis. The effect of inoculum size on transfer rate was highly statistically significant (P < 0.0001) for all transfer rate data combined (352 observations) and for each individual cross contamination rate, except for data on contamination via transfer from chicken to hand through a glove barrier (P = 0.1643). Where inoculum size on the source was greater, transfer rates were lower, and where inoculum size on the source was less, transfer rates were higher. The negative linear trend was more obvious for activities that had a larger range of inoculum sizes on the source surface. This phenomenon has serious implications for research seeking to determine bacterial cross contamination rates, since the different transfer efficiencies that were previously shown to be associated with certain activities may actually be the result of differing initial inoculum levels. The initial inoculum size on the source and the amount of bacteria transferred must both be considered to accurately determine bacterial transfer rates.     

Evaluation of criteria for bioreactor comparison and operation standardization for mammalian cell culture

Development of bioprocesses with mammalian cell culture deals with different bioreactor types and scales. The bioreactors might be intended for generation of cell inoculum and production, research, process development, validation, or transfer purposes. During these activities, not only the difficulty of up and downscaling might lead to failure of consistency in cell growth, but also the use of different bioreactor geometries and operation conditions. In such cases, criteria for bioreactor design and process transfer should be carefully evaluated in order to select appropriate cultivation parameters. In this work, power input, mixing time, impeller tip speed, and Reynolds number have been compared systematically for the cultivation of the human cell line AGE1.HN within three partner laboratories using five different bioreactor systems. Proper operation ranges for the bioreactors were identified using the maximal cell‐specific growth rate (μ_max) as indicator. Common optimum values for process transfer criteria were found in these geometrically different bioreactors, in which deviations of μ_max between cultivation systems can be importantly reduced. The data obtained in this work are used for process standardization and comparability of results obtained in different bioreactor systems, i.e. to guarantee lab‐to‐lab consistency for systems biology approaches using mammalian cells.     

Application of scale-down experiments in the study of citric acid biosynthesis

Problems associated with the development of submersed citric acid biosynthesis on sugar beet molasses for process improvement were solved by scale-down experiments: the determination of optimal temperature profile, optimal pH value, influence of OTR and inoculum quality. The optimal pH value at the start of the process was 6.4 with 3% of inoculum volume and temperature profile between 28–30°C. In the case of OTR which is scale-dependent, we prefer to use DOC pilot plant data for the transfer of technology.     

A correlative evaluation of morphology and rheology of<Emphasis Type="Italic">Aspergillus terreus</Emphasis> during lovastatin fermentation

Lovastatin, a secondary metabolite, was produced by fermentation process usingAspergillus terreus in an internal loop airlift reactor. It is a highly aerobic fermentation process. Biomass concentration and cell morphology were evaluated and observed to contribute significantly to the high viscosity and pseudoplastic non-Newtonian behavior of the broth. Typical morphological changes over 10 days in the fermentation broth were studied. The viscosity increased from the start of the fermentation with an increasing cell mass content, reached to a maximum of 60 N/m²·s at 160 h and then declined after the branching of the hyphae with the formation of arthrospores. Rheological parameters like consistency index and fluidity index were evaluated. The consistency index was observed to increase from 9.8 to 66.85 N/m², while fluidity index decreased from 0.69 to 0.48 s⁻¹ during 10 days of lovastatin production. A correlation between growth and consistency index of the broth has been evaluated.     

Effect of reproductive modes and environmental heterogeneity in the population dynamics of a geographically widespread clonal desert cactus

The dynamics of plant populations in arid environments are largely affected by the unpredictable environmental conditions and are fine-tuned by biotic factors, such as modes of recruitment. A single species must cope with both spatial and temporal heterogeneity that trigger pulses of sexual and clonal establishment throughout its distributional range. We studied two populations of the clonal, purple prickly pear cactus, Opuntia macrocentra, in order to contrast the factors responsible for the population dynamics of a common, widely distributed species. The study sites were located in protected areas that correspond to extreme latitudinal locations for this species within the Chihuahuan Desert. We studied both populations for four consecutive years and determined the demographic consequences of environmental variability and the mode of reproduction using matrix population models, life table response experiments (LTREs), and loop and perturbation analyses. Although both populations seemed fairly stable (population growth rate, λ∼1), different demographic parameters and different life cycle routes were responsible for this stability in each population. In the southernmost population (MBR) LTRE and loop and elasticity analyses showed that stasis is the demographic process with the highest contributions to λ, followed by sexual reproduction, and clonal propagation contributed the least. The northern population (CR) had both higher elasticities and larger contributions of stasis, followed by clonal propagation and sexual recruitment. Loop analysis also showed that individuals in CR have more paths to complete a life cycle than those in MBR. As a consequence, each population differed in life history traits (e.g., size class structure, size at sexual maturity, and reproductive value). Numerical perturbation analyses showed a small effect of the seed bank on the λ of both populations, while the transition from seeds to seedlings had an important effect mainly in the northern population. Clonal propagation (higher survival and higher contributions to vital rates) seems to be more important for maintaining populations over long time periods than sexual reproduction.     

Influence of some environmental factors on Rhizopus nigricans submerged growth in the form of pellets

Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH below 4 and above 7, the presence of Ca²⁺ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was mainly influenced by the inoculum size and the presence of baffles and Ca²⁺ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation, resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

The DNA restriction endonuclease of Escherichia coli B. I. Studies of the DNA translocation and the ATPase activities

Electron microscopic examination of DNA intermediates formed by the restriction endonuclease of Escherichia coli B revealed supercoiled loops that are presumably formed during an ATP-dependent DNA translocation process in which the enzyme remains bound to the recognition site while tracking along the DNA helix to a cleavage site. The rate of DNA translocation during this process is at least 5000 base pairs/min at 37 degrees C. Even after all cleavages have been completed, complexes are seen that contain terminal loops or loop plus tail structures. During this later phase of the reaction, ATP is hydrolyzed at a rate which is dependent upon the size of the largest possible loop (or loop plus tail); this ATP hydrolysis can be terminated by one double-strand cleavage within the loop region between the recognition site and the terminus. To explain these results, it is hypothesized that after cleavage the enzyme cycles between a tracking (and possibly back-tracking) mode which is fueled by ATP hydrolysis and a relatively long static period in which ATP hydrolysis does not occur. While tracking, the enzyme would be bound both to the recognition site and to a distal site but, while static, the enzyme would be bound only at the recognition site of nonlooped molecules. This post-nuclease phase of the reaction is hypothesized to reflect a reaction whereby the enzyme initially scans DNA molecules before making a strand cleavage.     

Rabbit Ileal Loop Response to Strains of Clostridium perfringens

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.     

Selection for resistance to Cochliobolus sativus in tall fescue (Festuca arundinacea Schreb.)

Summary Cochliobolus sativus (Ito and Kurib.) Drechsl. ex Dastur is a major foliar pathogen of tall fescue (Festuca arundinacea Schreb.) which can greatly reduce the quantity and quality of forages available for animal consumption. A greenhouse screening program was initiated to determine the inheritance of resistance to C. sativus in tall fescue over several cycles of mass selection. Resistance to C. sativus in four tall fescue cultivars was increased with 2–3 cycles of mass selection. Realized heritabilities were low to moderate (0.04 to 0.58) indicating that environmental influences on the expression of resistance are quite high. Variances were unchanged by selection, indicating that further improvement should be possible. However, progress with mass selection can be expected to be slow. Lesion size was decreased in each cultivar by selecting for lesion coverage. Lesion size, being independent of inoculum load and therefore less subject to environmental variation, should be considered as an additional selection criteria to improve the rate of progress.Journal article No. 6370 of the Mississippi Agricultural and Forestry Experiment Station     

Crystal structure of SecB from Escherichia coli

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 A resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.     

Pilot-scale culture ofCoffea arabica in a novel loop fluidised bed reactor

A novel bubble free loop fluidized bed reactor for plant cell cultures was developed and tested usingCoffea arabica as a model cell line. The effects of main operational parameters like morphology and size of inoculum, oxygen supply as well as recirculation of sparingly soluble gases on cell growth and alkaloid production rates in this reactor were studied and the results were compared with standard shake flask experiments. By on-line monitoring of biomass and oxygen uptake rates the main kinetic parameters for cell growth and alkaloid production were evaluated. It was demonstrated that the novel reactor is easy to run and is particularly adequate for measuring kinetic parameters necessary for scale up.     

De-risking Pharmaceutical Tablet Manufacture Through Process Understanding,Latent Variable Modeling,and Optimization Technologies

In pharmaceutical tablet manufacturing processes, a major source of disturbance affecting drug product quality is the (lot-to-lot) variability of the incoming raw materials. A novel modeling and process optimization strategy that compensates for raw material variability is presented. The approach involves building partial least squares models that combine raw material attributes and tablet process parameters and relate these to final tablet attributes. The resulting models are used in an optimization framework to then find optimal process parameters which can satisfy all the desired requirements for the final tablet attributes, subject to the incoming raw material lots. In order to de-risk the potential (lot-to-lot) variability of raw materials on the drug product quality, the effect of raw material lot variability on the final tablet attributes was investigated using a raw material database containing a large number of lots. In this way, the raw material variability, optimal process parameter space and tablet attributes are correlated with each other and offer the opportunity of simulating a variety of changes in silico without actually performing experiments. The connectivity obtained between the three sources of variability (materials, parameters, attributes) can be considered a design space consistent with Quality by Design principles, which is defined by the ICH-Q8 guidance (USDA 2006). The effectiveness of the methodologies is illustrated through a common industrial tablet manufacturing case study.     

Dependence of phenotypic variance in body size on environmental quality

The recent "overhead threshold" model for optimal age and body size at maturity (Day and Rowe 2002 ) predicts that phenotypic variability in adult body size will be low under inferior environmental quality and will increase with improving conditions. The model is, however, based on a potentially restrictive assumption of a monotone increase of fecundity with increasing body size. On the basis of a numerical model, we show that introducing the concept of maximum adult body size changes the predictions of the model. The dependence of variability in adult body size on environmental quality becomes a concave function with a maximum at intermediate values. Depending on the range of environmental conditions considered, one may therefore expect to observe both increasing and decreasing functions. We test the predictions of our model on a literature-based database of 131 insect species covering all major orders. We demonstrate that, in most species, relative phenotypic variation in body size decreases when environment-specific average of adult body size increases. In the majority of cases at least, such a relationship can be interpreted as a decreased relative variation in better growing conditions. With some potentially meaningful exceptions (e.g., females of capital-breeding insects), the general pattern was largely invariable across different taxa, ecological subdivisions, and sexes.     

Elimination of thermodynamically infeasible loops in steady-state metabolic models

The constraint-based reconstruction and analysis (COBRA) framework has been widely used to study steady-state flux solutions in genome-scale metabolic networks. One shortcoming of current COBRA methods is the possible violation of the loop law in the computed steady-state flux solutions. The loop law is analogous to Kirchhoff's second law for electric circuits, and states that at steady state there can be no net flux around a closed network cycle. Although the consequences of the loop law have been known for years, it has been computationally difficult to work with. Therefore, the resulting loop-law constraints have been overlooked. Here, we present a general mixed integer programming approach called loopless COBRA (ll-COBRA), which can be used to eliminate all steady-state flux solutions that are incompatible with the loop law. We apply this approach to improve flux predictions on three common COBRA methods: flux balance analysis, flux variability analysis, and Monte Carlo sampling of the flux space. Moreover, we demonstrate that the imposition of loop-law constraints with ll-COBRA improves the consistency of simulation results with experimental data. This method provides an additional constraint for many COBRA methods, enabling the acquisition of more realistic simulation results.     

亚麻脱胶菌种的选育及脱胶过程的初步研究

从沤麻主生物期的水中分离产果胶酶的菌株经初筛、复筛获得了三株专性厌氧细菌,初步鉴定为费氏芽孢杆菌,对其亚麻脱胶性能进行了初步研究,确定人工加菌沤麻的最适工艺条件为：加菌量2％,加菌时间：沤麻进入主生物期零时,菌株A优于其它菌株．结果表明：采用上述工艺进行沤麻实验,可缩短沤麻时间30％,并可提高麻纤维质量。     

Batch-to-Batch Quality Consistency Evaluation of Botanical Drug Products Using Multivariate Statistical Analysis of the Chromatographic Fingerprint

Botanical drug products have batch-to-batch quality variability due to botanical raw materials and the current manufacturing process. The rational evaluation and control of product quality consistency are essential to ensure the efficacy and safety. Chromatographic fingerprinting is an important and widely used tool to characterize the chemical composition of botanical drug products. Multivariate statistical analysis has showed its efficacy and applicability in the quality evaluation of many kinds of industrial products. In this paper, the combined use of multivariate statistical analysis and chromatographic fingerprinting is presented here to evaluate batch-to-batch quality consistency of botanical drug products. A typical botanical drug product in China, Shenmai injection, was selected as the example to demonstrate the feasibility of this approach. The high-performance liquid chromatographic fingerprint data of historical batches were collected from a traditional Chinese medicine manufacturing factory. Characteristic peaks were weighted by their variability among production batches. A principal component analysis model was established after outliers were modified or removed. Multivariate (Hotelling T ² and DModX) control charts were finally successfully applied to evaluate the quality consistency. The results suggest useful applications for a combination of multivariate statistical analysis with chromatographic fingerprinting in batch-to-batch quality consistency evaluation for the manufacture of botanical drug products.     

Jet aeration as alternative to overcome mass transfer limitation of stirred bioreactors

In industrial biotechnology increasing reactor volumes have the potential to reduce production costs. Whenever the achievable space time yield is determined by the mass transfer performance of the reactor, energy efficiency plays an important role to meet the requirements regarding low investment and operating costs. Based on theoretical calculations, compared to bubble column, airlift reactor, and aerated stirred tank, the jet loop reactor shows the potential for an enhanced energetic efficiency at high mass transfer rates. Interestingly, its technical application in standard biotechnological production processes has not yet been realized. Compared to a stirred tank reactor powered by Rushton turbines, maximum oxygen transfer rates about 200% higher were achieved in a jet loop reactor at identical power input in a fed batch fermentation process. Moreover, a model‐based analysis of yield coefficients and growth kinetics showed that E. coli can be cultivated in jet loop reactors without significant differences in biomass growth. Based on an aerobic fermentation process, the assessment of energetic oxygen transfer efficiency [kgO₂ kW^?1 h^?1] for a jet loop reactor yielded an improvement of almost 100%. The jet loop reactor could be operated at mass transfer rates 67% higher compared to a stirred tank. Thus, an increase of 40% in maximum space time yield [kg m^?3 h^?1] could be observed.     

Feedbacks of Soil Inoculum of Mycorrhizal Fungi Altered by N Deposition on the Growth of a Native Shrub and an Invasive Annual Grass

Anthropogenic nitrogen (N) deposition causes shifts in vegetation types as well as species composition of arbuscular mycorrhizal (AM) fungi and other soil microorganisms. A greenhouse experiment was done to determine whether there are feedbacks between N-altered soil inoculum and growth of a dominant native shrub and an invasive grass species in southern California. The region is experiencing large-scale loss of Artemisia californica shrublands and replacement by invasive annual grasses under N deposition. Artemisia californica and Bromus madritensis ssp. rubens were grown with soil inoculum from experimental plots in a low N deposition site that had (1) N-fertilized and (2) unfertilized soil used for inoculum, as well as (3) high-N soil inoculum from a site exposed to atmospheric N deposition for four decades. All treatments plus a nonmycorrhizal control were given two levels of N fertilizer solution. A. californica biomass was reduced by each of the three inocula compared to uninoculated controls under at least one of the two N fertilizer solutions. The␣inoculum from the N-deposition site caused the greatest growth depressions. By contrast, B.␣madritensis biomass increased with each of the three inocula under at least one, or both, of the N solutions. The different growth responses of the two plant species may be related to the types of AM fungal colonization. B. madritensis was mainly colonized by a fine mycorrhizal endophyte, while A. californica had primarily coarse endophytes. Furthermore, A. californica had a high level of septate, nonmycorrhizal root endophytes, while B. madritensis overall had low levels of these endophytes. The negative biomass response of A. californica seedlings to high N-deposition inoculum may in part explain its decline; a microbially-mediated negative feedback may occur in this system that causes poor␣seedling growth and establishment of A.␣californica in sites subject to N deposition and B. madritensis invasion.