Flow Pattern and Mixing in the Multi‐Turbine Agitated Bioreactor Filled with Poly(γ‐Glutamic Acid) Solution

We investigated the flow pattern and mixing behavior of a poly(γ‐glutamic acid) (γ‐PGA) solution in a bioreactor equipped with two Rushton turbines by simulation and experiment. Computational fluid dynamics (CFD) is used to solve the three‐dimensional hydrodynamics in the bioreactor and to obtain the flow patterns and tracer concentration at every point. The flow circulation patterns by inter‐impeller clearance and viscosity and their effects on overall mixing time were studied. Based on the results we can conclude that the impeller clearance should not be larger than 0.2 D for the efficient mixing under non‐aerated condition when the liquid viscosity is above 20 cp, which corresponds to concentrations of 20 g/L or above for γ‐PGA.     

新型生物反应器结构研究进展

生物反应器是生物工程的核心设备,其结构的合理性直接决定反应器生物加工的效率。生物反应器的研究一直是生物工程的核心问题之一。随着青霉素的工业化生产,机械搅拌式生物反应器应运而生,此后,随着动植物细胞培养,高等真菌培养,藻类培养等生物过程的发展,人们相应开发了大量的生物反应器,其中以机械搅拌式生物反应器和气升式样生物反应器尤为突出,本文总结了近年来文献报道的新型生物反应器,主要阐述了机械搅拌式和气升式两类生物反应器结构的研究进展,对目前国内外报道的11种新型反应器典型结构进行了总结与分析。     

A Robust Test for Two‐Stage Design in Genome‐Wide Association Studies

Summary A two‐stage design is cost‐effective for genome‐wide association studies (GWAS) testing hundreds of thousands of single nucleotide polymorphisms (SNPs). In this design, each SNP is genotyped in stage 1 using a fraction of case–control samples. Top‐ranked SNPs are selected and genotyped in stage 2 using additional samples. A joint analysis, combining statistics from both stages, is applied in the second stage. Follow‐up studies can be regarded as a two‐stage design. Once some potential SNPs are identified, independent samples are further genotyped and analyzed separately or jointly with previous data to confirm the findings. When the underlying genetic model is known, an asymptotically optimal trend test (TT) can be used at each analysis. In practice, however, genetic models for SNPs with true associations are usually unknown. In this case, the existing methods for analysis of the two‐stage design and follow‐up studies are not robust across different genetic models. We propose a simple robust procedure with genetic model selection to the two‐stage GWAS. Our results show that, if the optimal TT has about 80% power when the genetic model is known, then the existing methods for analysis of the two‐stage design have minimum powers about 20% across the four common genetic models (when the true model is unknown), while our robust procedure has minimum powers about 70% across the same genetic models. The results can be also applied to follow‐up and replication studies with a joint analysis.     

Statistical Inference for a Two‐Stage Outcome‐Dependent Sampling Design with a Continuous Outcome

Summary The two‐stage case–control design has been widely used in epidemiology studies for its cost‐effectiveness and improvement of the study efficiency ( White, 1982 , American Journal of Epidemiology 115, 119–128; Breslow and Cain, 1988 , Biometrika 75, 11–20). The evolution of modern biomedical studies has called for cost‐effective designs with a continuous outcome and exposure variables. In this article, we propose a new two‐stage outcome‐dependent sampling (ODS) scheme with a continuous outcome variable, where both the first‐stage data and the second‐stage data are from ODS schemes. We develop a semiparametric empirical likelihood estimation for inference about the regression parameters in the proposed design. Simulation studies were conducted to investigate the small‐sample behavior of the proposed estimator. We demonstrate that, for a given statistical power, the proposed design will require a substantially smaller sample size than the alternative designs. The proposed method is illustrated with an environmental health study conducted at National Institutes of Health.     

Scalable Manufacturing of Human Mesenchymal Stromal Cells in the Vertical‐Wheel Bioreactor System: An Experimental and Economic Approach

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering applications and cell‐based therapies. Large cell doses (>1 × 10⁶ cells kg^?1) and Good Manufacturing Practices (GMP)‐compliant processes are however required for clinical purposes. Here, a serum‐ and xenogeneic‐free (S/XF) microcarrier‐based culture system is established for the expansion of human umbilical cord matrix (UCM)‐ and adipose tissue (AT)‐derived MSC using the Vertical‐Wheel system (PBS‐0.1 MAG; PBS Biotech). UCM and AT MSC are expanded to maximum cell densities of 5.3 ± 0.4 × 10⁵ cell mL^?1 (n = 3) and 3.6 ± 0.7 × 10⁵ cell mL^?1 (n = 3), respectively, after 7 days of culture, while maintaining their identity, according to standard criteria. An economic evaluation of the process transfer from T‐flasks to PBS‐0.1 MAG shows a reduction in the costs associated with the production of a dose for an average 70 kg adult patient (i.e., 70 million cells). Costs decrease from $17.0 K to $11.1 K for UCM MSC and from $21.5 K to $11.1 K for AT MSC, proving that the transition to Vertical‐Wheel reactors provides a cost‐effective alternative for MSC expansion. The present work reports the establishment of a scalable and cost‐effective culture platform for the manufacturing of UCM and AT MSC in a S/XF microcarrier‐based system.     

Combined Two‐Stage Xanthate Processes for the Treatment of Copper‐Containing Wastewater

Heavy metal removal is mainly conducted by adjusting the wastewater pH to form metal hydroxide precipitates. However, in recent years, the xanthate process with a high metal removal efficiency, attracted attention due to its use of sorption/desorption of heavy metals from aqueous solutions. In this study, two kinds of agricultural xanthates, insoluble peanut‐shell xanthate (IPX) and insoluble starch xanthate (ISX), were used as sorbents to treat the copper‐containing wastewater (Cu concentration from 50 to 1,000 mg/L). The experimental results showed that the maximum Cu removal efficiency by IPX was 93.5 % in the case of high Cu concentrations, whereby 81.1 % of copper could rapidly be removed within one minute. Moreover, copper‐containing wastewater could also be treated by ISX over a wide range (50 to 1,000 mg/L) to a level that meets the Taiwan EPA's effluent regulations (3 mg/L) within 20 minutes. Whereas IPX had a maximum binding capacity for copper of 185 mg/g IPX, the capacity for ISX was 120 mg/g ISX. IPX is cheaper than ISX, and has the benefits of a rapid reaction and a high copper binding capacity, however, it exhibits a lower copper removal efficiency. A sequential IPX and ISX treatment (i.e., two‐stage xanthate processes) could therefore be an excellent alternative. The results obtained using the two‐stage xanthate process revealed an effective copper treatment. The effluent (C_e) was below 0.6 mg/L, compared to the influent (C₀) of 1,001 mg/L at pH = 4 and a dilution rate of 0.6 h^–1. Furthermore, the Cu‐ISX complex formed could meet the Taiwan TCLP regulations, and be classified as non‐hazardous waste. The xanthatilization of agricultural wastes offers a comprehensive strategy for solving both agricultural waste disposal and metal‐containing wastewater treatment problems.     

Structure and Dynamics of a Two‐Helix SNARE Complex in Live Cells

SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q_a‐, Q_b‐, Q_c‐ and R‐SNARE‐motif form a complex. The core complex represents a Q_aQ_bQ_cR‐SNARE‐motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)‐studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q_a), SNAP25 (Q_b/Q_c) and synaptobrevin 2 (R)]. We find that the Q_b‐SNARE‐motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q_b‐SNARE‐motif and depends on its position within the molecule. We conclude that a zippered Q_aQ_b‐SNARE complex represents a short‐lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.     

ANAMMOX process start up and stabilization with an anaerobic seed in Anaerobic Membrane Bioreactor (AnMBR)

ANaerobic AMMonium OXidation (ANAMMOX) process, an advanced biological nitrogen removal alternative to traditional nitrification--denitrification removes ammonia using nitrite as the electron acceptor without oxygen. The feasibility of enriching anammox bacteria from anaerobic seed culture to start up an Anaerobic Membrane Bioreactor (AnMBR) for N-removal is reported in this paper. The Anammox activity was established in the AnMBR with anaerobic digester seed culture from a Sewage Treatment Plant in batch mode with recirculation followed by semi continuous process and continuous modes of operation. The AnMBR performance under varying Nitrogen Loading Rates (NLR) and HRTs is reported for a year, in terms of nitrogen transformations to ammoniacal nitrogen, nitrite and nitrate along with hydrazine and hydroxylamine. Interestingly ANAMMOX process was evident from simultaneous Amm-N and nitrite reduction, consistent nitrate production, hydrazine and hydroxylamine presence, notable organic load reduction and bicarbonate consumption.     

On Estimating the Relationship between Longitudinal Measurements and Time‐to‐Event Data Using a Simple Two‐Stage Procedure

Summary Ye, Lin, and Taylor (2008, Biometrics 64 , 1238–1246) proposed a joint model for longitudinal measurements and time‐to‐event data in which the longitudinal measurements are modeled with a semiparametric mixed model to allow for the complex patterns in longitudinal biomarker data. They proposed a two‐stage regression calibration approach that is simpler to implement than a joint modeling approach. In the first stage of their approach, the mixed model is fit without regard to the time‐to‐event data. In the second stage, the posterior expectation of an individual's random effects from the mixed‐model are included as covariates in a Cox model. Although Ye et al. (2008) acknowledged that their regression calibration approach may cause a bias due to the problem of informative dropout and measurement error, they argued that the bias is small relative to alternative methods. In this article, we show that this bias may be substantial. We show how to alleviate much of this bias with an alternative regression calibration approach that can be applied for both discrete and continuous time‐to‐event data. Through simulations, the proposed approach is shown to have substantially less bias than the regression calibration approach proposed by Ye et al. (2008) . In agreement with the methodology proposed by Ye et al. (2008) , an advantage of our proposed approach over joint modeling is that it can be implemented with standard statistical software and does not require complex estimation techniques.     

A Comparison of Procedures for Adaptive Choice of Location Tests in Flexible Two‐Stage Designs

Although linear rank statistics for the two‐sample problem are distribution free tests, their power depends on the distribution of the data. In the planning phase of an experiment, researchers are often uncertain about the shape of this distribution and so the choice of test statistic for the analysis and the determination of the required sample size are based on vague information. Adaptive designs with interim analysis can potentially overcome both problems. And in particular, adaptive tests based on a selector statistic are a solution to the first. We investigate whether adaptive tests can be usefully implemented in flexible two‐stage designs to gain power. In a simulation study, we compare several methods for choosing a test statistic for the second stage of an adaptive design based on interim data with the procedure that applies adaptive tests in both stages. We find that the latter is a sensible approach that leads to the best results in most situations considered here. The different methods are illustrated using a clinical trial example.     

Multiple Comparisons of Treatments with Stable Multivariate Tests in a Two‐Stage Adaptive Design,Including a Test for Non‐Inferiority

The application of stabilized multivariate tests is demonstrated in the analysis of a two‐stage adaptive clinical trial with three treatment arms. Due to the clinical problem, the multiple comparisons include tests of superiority as well as a test for non‐inferiority, where non‐inferiority is (because of missing absolute tolerance limits) expressed as linear contrast of the three treatments. Special emphasis is paid to the combination of the three sources of multiplicity – multiple endpoints, multiple treatments, and two stages of the adaptive design. Particularly, the adaptation after the first stage comprises a change of the a‐priori order of hypotheses.     

Treatment of Process Water Containing Heavy Metals with a Two‐Stage Electrolysis Procedure in a Membrane Electrolysis Cell

The capability of a two‐stage electrochemical treatment for the regeneration of acidic heavy‐metal containing process water was examined. The process water came from sediment bioleaching and was characterized by a wide spectrum of dissolved metals, a high sulfate content, and a pH of about 3. In the modular laboratory model cell used, the anode chamber and the cathode chamber were separated by a central chamber fitted with an ion exchanger membrane on either side. The experiments were carried out applying a platinum anode and a graphite cathode at a current density of 0.1 A/cm². The circulation flow of the process water in the batch process amounted to 35 L/h, the electrolysis duration was 5.5 h at maximum and the total electrolysis current was about 1 A. In the first stage, the acidic process water containing metals passed through the cathode chamber. In the second stage, the cathodically pretreated process water was electrolyzed anodically. In the cathode chamber the main load of dissolved Cu, Zn, Cr and Pb was eliminated. The sulfuric acid surplus of 3–4 g/L decreased to about 1 g/L, the pH rose from initially 3.0 to 4–5, but the desired pH of 9–10 was not achieved. Precipitation in the proximity to the cathode evidently takes place at a higher pH than farther away. The dominant process in the anode chamber was the precipitation of amorphous MnO₂ owing to the oxidation of dissolved Mn(II). The further depletion of the remaining heavy metals in the cathodically pretreated process water by subsequent anodic treatment was nearly exhaustive, more than 99 % of Cd, Cr, Cu, Mn, Ni, Pb, and Zn were removed from the leachate. The high depletion of heavy metals might be due to both the sorption on MnO₂ precipitates and/or basic ferrous sulfate formed anodically, and the migration of metal ions through the cation exchanger membrane via the middle chamber into the cathode chamber. In the anode chamber, the sulfuric acid content increased to 6–7 g/L and the pH sank to 1.7. All heavy metals contained, with the exception of Zn, were removed to levels below the German limits for discharging industrial wastewaters into the receiving water. Moreover, the metal‐depleted and acid‐enriched process waters could be returned to the leaching process, hence reducing the output of wastewater. The results indicated that heavy metals could be removed from acidic process waters by two‐stage electrochemical treatment to a large extent. However, to improve the efficiency of metal removal and to establish the electrochemical treatment in practice, further work is necessary to optimize the operation of the process with respect to current density, energy consumption, discharging of metal precipitates deposited in the electrode chambers and preventing membrane clogging.     

A Bootstrap Procedure for Adaptive Selection of the Test Statistic in Flexible Two‐Stage Designs

Adaptive two‐stage designs allow a data‐driven change of design characteristics during the ongoing trial. One of the available options is an adaptive choice of the test statistic for the second stage of the trial based on the results of the interim analysis. Since there is often only a vague knowledge of the distribution shape of the primary endpoint in the planning phase of a study, a change of the test statistic may then be considered if the data indicate that the assumptions underlying the initial choice of the test are not correct. Collings and Hamilton proposed a bootstrap method for the estimation of the power of the two‐sample Wilcoxon test for shift alternatives. We use this approach for the selection of the test statistic. By means of a simulation study, we show that the gain in terms of power may be considerable when the initial assumption about the underlying distribution was wrong, whereas the loss is relatively small when in the first instance the optimal test statistic was chosen. The results also hold true for comparison with a one‐stage design. Application of the method is illustrated by a clinical trial example.     

Production of Laccase by Trametes hirsuta Grown in an Immersion Bioreactor and its Application in the Docolorization of Dyes from a Leather Factory

An alternative system for producing laccase on a bioreactor scale by the white‐rot fungus Trametes hirsuta is proposed. The experiments were performed in an immersion bioreactor (employing cuttings of stainless steel sponges as a support) and the culture medium was supplemented with copper sulfate (1 mM). Operating under these conditions, it was possible to obtain a maximum laccase activity of nearly 5,000 U/L within 9 days. In addition, the ability of the crude laccase produced to decolorize two synthetic acid dyes utilized in the leather industry (Luganil Green and Sella Solid Red) was investigated. The effect of the pH and the enzyme activity on decolorization was analyzed. It was found that a pH of 4.0 and a laccase activity of 300 U/L were optimal for Luganil dye decolorization (16.2 % in 2 hours). Sella Solid Red showed its highest decolorization (around 40 % in 2 hours) when used at pH 5.0 and at a laccase activity of 1,000 U/L.     

Statistical Modelling of Physiological Heat Stress Response by Means of a Nonlinear Two‐Stage Model

In the context of medical or biological studies, very often parameters of interest are measured repeatedly over time under a given set of conditions. This results in a set of (often similarly shaped) time series. Then, the objective is the determination of the functional relationship between the parameter of interest and time on the one hand, and the analysis of the variation of this functional relationship between experiments, on the other hand. This may be done by means of a two‐stage model. The present work describes the theory of the two‐stage model and its application to the increase of human core temperature for a set of 678 experiments where the subjects were exposed to warm and hot environments. The data originating from 6 European research institutes, have been pooled into one database for the Heat Stress Project within the scope of the BIOMED 2 programme of the European Union. A nonlinear two‐stage model was applied, with a logistic function modelling the nonlinear time course of the core temperature, and with its parameters depending on air temperature, mean radiant temperature, air velocity, partial vapour pressure, clothing insulation, metabolic rate, gender, acclimatisation status and body surface area. We conclude that acclimatisation, clothing insulation, body surface area, air temperature, air velocity, partial vapour pressure, metabolic rate, and the difference between mean radiant temperature and air temperature play an important role for work in warm and hot environments. We show how our results can be used for the estimation of allowable exposure times for work in hot environments.     

Growth and biomass production with enhanced β‐glucan and dietary fibre contents of Ganoderma australe ATHUM 4345 in a batch‐stirred tank bioreactor

In this study we maximized biomass production by the basidiomycete Ganoderma australe ATHUM 4345, a species of pharmaceutical interest as it is a valuable source of nutraceuticals, including dietary fibers and glucans. We used the Biolog FF MicroPlate to screen 95 different carbon sources for growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint, which is useful in selecting components for media optimization of maximum biomass production. Response surface methodology, based on the central composite design was applied to explore the optimum concentrations of carbon and nitrogen sources of culture medium in shake flask cultures. When the improved culture medium was tested in a 20‐L stirred tank bioreactor, using 13.7 g/L glucose and 30.0 g/L yeast extract, high biomass yields (10.1±0.4 g/L) and productivity of 0.09 g L^?1 h^?1 were obtained. The yield coefficients for total glucan and dietary fibers on biomass formed were 94.82±6 and 341.15±12.3 mg/g mycelium dry weight, respectively.     

High Density Cultivation of Panax notoginseng Cell Cultures with Methyl Jasmonate Elicitation in a Centrifugal Impeller Bioreactor

True ginseng roots contain “active compounds” called ginsenosides. The enhanced production of useful bioactive ginsenosides by high‐density cell cultures of Panax notoginseng in a self‐developed centrifugal impeller bioreactor (CIB) was achieved by adding methyl jasmonic acid (MJA) during cultivation. The production of the major, individual ginsenosides Rg₁, Re and Rb₁ was significantly enhanced in both 3‐L and 30‐L CIBs. The production titer of Rg₁, Re and Rb₁ ginsenosides in the 30‐L CIB was improved from 42 ± 8, 42 ± 9 and 41 ± 6 mg/L without MJA elicitation, to 104 ± 6, 71 ± 5 and 95 ± 6 mg/L with MJA elicitation, respectively. The ratio of Rb/Rg was slightly improved by MJA treatment in a 3‐L CIB but no apparent difference was observed in a 30‐L CIB. This work is useful for the understanding of the effects of large‐scale production on the individual ginseng saponins produced by plant cell cultures