Pilot-scale process sensitivity studies for the scaleup of a fungal fermentation for the production of pneumocandins

The filamentous fungus Glarea lozoyensis produces a novel, pharmaceutically important pneumocandin (B(0)) that is used to synthesize a lipopeptide which demonstrates cidal activity against clinically relevant pathogens. A range of unwanted pneumocandin analogs are also produced by the organism. To maintain the unwanted impurities to acceptable levels upon scaleup, a good understanding of the impact of chemical and physical environment on the cell physiology is required, which benefits downstream processing. Pilot-scale studies were performed to determine the impact of dissolved oxygen, temperature, pH, and carbon dioxide on the process. Experiments included multiple fermenters (up to seven) at 0.07 and 0.8 m(3) scale using single source medium sterilization and inoculum. Gas blending was used to separate effects of dissolved oxygen from agitation. The process was significantly influenced by dissolved oxygen level. The critical dissolved oxygen tension (C(crit)) for growth was below 2% air saturation. The C(crit) for production of pneumocandin B(0) was 20% air saturation, with a significant reduction of the specific production rate below this value. In contrast, low dissolved oxygen levels produced a substantial increase of pneumocandins B(1), B(5), and E(0), while high dissolved oxygen levels produced a disproportionate increase of D(5). This sensivity to dissolved oxygen was independent of agitation within a power range of 2-15 kW/m(3). Broth viscosity was impacted below 10% dissolved oxygen, suggesting an effect on morphology. The process was shown to be sensitive to temperature but relatively insensitive to pH and carbon dioxide (in the exhaust gas) within the ranges studied. This scaledown analysis explained phenomena seen at pilot scale and helped define operating boundary conditions for successful scale up to 19 m(3).     

Enclosed outdoor photobioreactors: light regime,photosynthetic efficiency,scale-up,and future prospects

Enclosed outdoor photobioreactors need to be developed and designed for large-scale production of phototrophic microorganisms. Both light regime and photosynthetic efficiency were analyzed in characteristic examples of state-of-the-art pilot-scale photobioreactors. In this study it is shown that productivity of photobioreactors is determined by the light regime inside the bioreactors. In addition to light regime, oxygen accumulation and shear stress limit productivity in certain designs. In short light-path systems, high efficiencies, 10% to 20% based on photosynthetic active radiation (PAR 400 to 700 nm), can be reached at high biomass concentrations (>5 kg [dry weight] m(-3)). It is demonstrated, however, that these and other photobioreactor designs are poorly scalable (maximal unit size 0.1 to 10 m(3)), and/or not applicable for cultivation of monocultures. This is why a new photobioreactor design is proposed in which light capture is physically separated from photoautotrophic cultivation. This system can possibly be scaled to larger unit sizes, 10 to >100 m(3), and the reactor liquid as a whole is mixed and aerated. It is deduced that high photosynthetic efficiencies, 15% on a PAR-basis, can be achieved. Future designs from optical engineers should be used to collect, concentrate, and transport sunlight, followed by redistribution in a large-scale photobioreactor.     

Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k(L)a

Fermentation optimization experiments are ideally performed at small scale to reduce time, cost and resource requirements. Currently microwell plates (MWPs) are under investigation for this purpose as the format is ideally suited to automated high-throughput experimentation. In order to translate an optimized small-scale fermentation process to laboratory and pilot scale stirred-tank reactors (STRs) it is necessary to characterize key engineering parameters at both scales given the differences in geometry and the mechanisms of aeration and agitation. In this study oxygen mass transfer coefficients are determined in three MWP formats and in 7.5 L and 75 L STRs. k(L)a values were determined in cell-free media using the dynamic gassing-out technique over a range of agitation conditions. Previously optimized culture conditions at the MWP scale were then scaled up to the larger STR scales on the basis of matched k(L)a values. The accurate reproduction of MWP (3 mL) E. coli BL21 (DE3) culture kinetics at the two larger scales was shown in terms of cell growth, protein expression, and substrate utilization for k(L)a values that provided effective mixing and gas-liquid distribution at each scale. This work suggests that k(L)a provides a useful initial scale-up criterion for MWP culture conditions which enabled a 15,000-fold scale translation in this particular case. This work complements our earlier studies on the application of DoE techniques to MWP fermentation optimization and in so doing provides a generic framework for the generation of large quantities of soluble protein in a rapid and cost-effective manner.     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale-up

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale-up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164-176]. In order to enable the predictive scale-up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale-up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, P(g)/P(ug), was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631-2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215-224]. A correlation relating the measured oxygen mass transfer coefficient, k(L)a, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale-up studies at matched k(L)a (0.06-0.11 s(-1)) and P(g)/V (657-2,960 W m(-3)) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant k(L)a was shown to be the most reliable basis for predictive scale-up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of k(L)a values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development.     

生态学中的尺度问题：内涵与分析方法

尺度问题已成为现代生态学的核心问题之一.尺度问题主要涉及3个方面：尺度概念、尺度分析和尺度推绎.主要评述前两个方面.生态学尺度有三重概念：维数、种类和组分,其中每重概念又包含了多个定义,有必要进行澄清、分类和统一.尺度分析涉及尺度效应分析和多尺度空间格局分析.格局、过程及它们之间的关系,以及某些景观特性均表现出尺度效应,因此多尺度研究非常必要和重要.多尺度空间格局分析（尤其是特征尺度的识别）是进行尺度效应分析和跨尺度推绎的基础.多尺度分析需要特定的方法,景观指数法是最常用和最简单的方法,但也常产生误导;空间统计学方法（如半方差分析法、尺度方差分析法、空隙度指数法和小波分析法等）和分维分析法在最近十几年发展起来,并逐渐应用于生态学,在尺度分析上具有很大的应用潜力.各种方法在尺度分析上各有优势和不足,有必要同时使用两种或两种以上方法进行比较和评估.总之,有关尺度分析的研究需要进一步加强,从而为下一步的尺度推绎提供可靠的依据.     

Pattern analysis techniques to process fermentation curves: application to discrimination of enological alcoholic fermentations

In fermentation processes, kinetic curves are generally aimed at control purposes. However, these curves could also contain information about inherent features of the product (such as origin, quality, etc.). This article presents several pattern analysis techniques used to classify fermentation curves. An application to alcoholic fermentation is presented as an illustration: it aims at retrieving the origin of a must from its fermentation curve. The fermentation kinetics of five vineyard musts, harvested over 9 years on the same parcels, were recorded. From these curves two sets of variables were generated: The first (p(1)) gathers all the kinetic curve points. The second (p(2)) contains a restrained number of variables, generated by the expert knowledge of the enologist. The set p(2) was processed by two very different techniques: a linear one (factorial discriminant analysis) and a nonlinear one (artificial neural networks). The set p(1) was processed by a new chemometric technique, the discriminant partial least-squares regression. For all the sets and the techniques used the selection of variables was studied. The interest in the latter is largely demonstrated both by theoretical and practical discussions. The discrimination results (up to 94% of good predictions) enhance the interest of the on-line measurements and their use in such pattern analysis tools.     

Comparative technoeconomic analysis of a softwood ethanol process featuring posthydrolysis sugars concentration operations and continuous fermentation with cell recycle

Economical production of second generation ethanol from Ponderosa pine is of interest due to widespread mountain pine beetle infestation in the western United States and Canada. The conversion process is limited by low glucose and high inhibitor concentrations resulting from conventional low‐solids dilute acid pretreatment and enzymatic hydrolysis. Inhibited fermentations require larger fermentors (due to reduced volumetric productivity) and low sugars lead to low ethanol titers, increasing distillation costs. In this work, multiple effect evaporation (MEE) and nanofiltration (NF) were evaluated to concentrate the hydrolysate from 30 g/l to 100, 150, or 200 g/l glucose. To ferment this high gravity, inhibitor containing stream, traditional batch fermentation was compared with continuous stirred tank fermentation (CSTF) and continuous fermentation with cell recycle (CSTF‐CR). Equivalent annual operating cost (EAOC = amortized capital + yearly operating expenses) was used to compare these potential improvements for a local‐scale 5 MGY ethanol production facility. Hydrolysate concentration via evaporation increased EAOC over the base process due to the capital and energy intensive nature of evaporating a very dilute sugar stream; however, concentration via NF decreased EAOC for several of the cases (by 2 to 15%). NF concentration to 100 g/l glucose with a CSTF‐CR was the most economical option, reducing EAOC by $0.15 per gallon ethanol produced. Sensitivity analyses on NF options showed that EAOC improvement over the base case could still be realized for even higher solids removal requirements (up to two times higher centrifuge requirement for the best case) or decreased NF performance. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:946–956, 2015     

Numerical simulation and PEPT measurements of a 3D conical helical-blade mixer: a high potential solids mixer for solid-state fermentation

Helical-blade solids mixers have a large potential as bioreactors for solid-state fermentation (SSF). Fundamental knowledge of the flow and mixing behavior is required for robust operation of these mixers. In this study predictions of a discrete particle model were compared to experiments with colored wheat grain particles and positron emission particle tracking (PEPT) measurements. In the discrete particle model individual movements of particles were calculated from interaction forces. It was concluded that the predicted overall flow behavior matched well with the PEPT measurements. Differences between the model predictions and the experiments with wheat grains were found to be due to the assumption that substrate particles were spherical, which was in the model. Model simulations and experiments with spherical green peas confirmed this. The mixing in the helical-blade mixer could be attributed to (1) the transport of particles up and down in the interior of the mixer, and (2) dispersion or micro-mixing of particles in the top region of the mixer. It appeared that the mixing rate scaled linearly with the rotation rate of the blade, although the average particle velocity did not scale proportionally. It may be that the flow behavior changes as a function of the rotation rate (e.g., changing thickness of the top region); further study is required to confirm this. To increase the mixing performance of the mixer, a larger blade or a change in the shape of the mixer (larger top surface/volume ratio) is recommended.     

一株兼具秸秆腐解能力玉米促生菌的筛选、鉴定及发酵优化

【背景】砂姜黑土地区存在秸秆腐解缓慢、秸秆还田后作物幼苗生长不良等问题。【目的】从砂姜黑土区农田筛选一株兼具秸秆腐解能力的玉米促生菌MC29,以促进秸秆腐解和玉米作物生长。【方法】通过16S rRNA基因序列分析对该菌株进行鉴定;采用液态摇瓶及盆栽试验验证菌株实际促腐、促生能力及土壤养分的提升效果,并且探究菌株的最佳生长及产吲哚乙酸(indole-3-acetic acid, IAA)条件;采用电击转化法将绿色荧光蛋白(green fluorescent protein, GFP)基因导入菌株细胞,并通过PCR琼脂糖凝胶电泳进行验证。【结果】分离筛选的玉米促生菌鉴定为纤维化纤维微细菌(Cellulosimicrobium cellulans)。该菌株MC29羧甲基纤维素(carboxymethyl cellulose, CMC)酶活可达13.32 U/mL,产IAA量为8.63 mg/L。与对照相比,施用菌株MC29后秸秆腐解率显著提高24.8%;玉米soil and plant analyzer development (SPAD)值、植株总重、根表面积和根长分别提高7.6%、21.3%、30.9%和18.3%;土壤碱解氮含量显著提高68.1%,土壤速效磷及土壤速效钾含量分别提高5.8%及6.0%。菌株MC29最佳生长条件为pH 7.0、装液量为25/250 mL、碳源为麦芽糖、氮源为酵母粉;最佳产IAA条件为pH 7.0、装液量为50/250 mL、碳源为果糖、氮源为硝酸钾;成功构建荧光标记菌株MC29-GFP,并据此追踪到其接入砂姜黑土15 d后定殖量为2.8×10⁵-9.5×10⁵ copies/g。【结论】所筛选的纤维化纤维微细菌MC29对于指导砂姜黑土区多功能秸秆促腐菌剂、微生物菌肥的研制及提升作物产量有一定的积极意义,并为探究其在砂姜黑土中的实际应用奠定基础。     

高产纤溶酶菌株CNY16发酵条件优化及其酶学特性初步研究

【目的】通过响应面试验对产纤溶酶菌株CNY16发酵条件进行优化,并对其酶学特性进行初步研究。【方法】采用Plackett-Burman设计得出酵母膏、氯化钠、转速3个最重要影响因素,通过最陡爬坡实验逼近酶活的最高区域,然后根据Box-Behnken中心组合设计实验对显著因素进行优化分析,最后对该酶学性质进行初步分析。【结果】最终得到3个因素的最优组合:酵母膏3.28%,氯化钠1.14%,转速166 r/min,在此培养条件下,纤溶酶活达到875.932 U/mL,比优化前提高了46%;该菌株产纤溶酶最适温度为30°C,最适pH为6.5。【结论】确定了高产纤溶酶菌株CNY16的最优发酵条件及其部分酶学性质,为该酶的进一步深入研究及中试实验奠定基础。     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

CFD analysis of the human airways under impedance-based boundary conditions: application to healthy,diseased and stented trachea

A computational fluid dynamics model of a healthy, a stenotic and a post-operatory stented human trachea was developed to study the respiration under physiological boundary conditions. For this, outflow pressure waveforms were computed from patient-specific spirometries by means of a method that allows to compute the peripheral impedance of the truncated bronchial generation, modelling the lungs as fractal networks. Intratracheal flow pattern was analysed under different scenarios. First, results obtained using different outflow conditions were compared for the healthy trachea in order to assess the importance of using impedance-based conditions. The resulted intratracheal pressures were affected by the different boundary conditions, while the resulted velocity field was unaffected. Impedance conditions were finally applied to the diseased and the stented trachea. The proposed impedance method represents an attractive tool to compute physiological pressure conditions that are not possible to extract in vivo. This method can be applied to healthy, pre- and post-operatory tracheas showing the possibility of predicting, through numerical simulation, the flow and the pressure field before and after surgery.     

Dynamic modeling of syngas fermentation in a continuous stirred-tank reactor: Multi-response parameter estimation and process optimization

Syngas fermentation is one of the bets for the future sustainable biobased economies due to its potential as an intermediate step in the conversion of waste carbon to ethanol fuel and other chemicals. Integrated with gasification and suitable downstream processing, it may constitute an efficient and competitive route for the valorization of various waste materials, especially if systems engineering principles are employed targeting process optimization. In this study, a dynamic multi-response model is presented for syngas fermentation with acetogenic bacteria in a continuous stirred-tank reactor, accounting for gas–liquid mass transfer, substrate (CO, H₂) uptake, biomass growth and death, acetic acid reassimilation, and product selectivity. The unknown parameters were estimated from literature data using the maximum likelihood principle with a multi-response nonlinear modeling framework and metaheuristic optimization, and model adequacy was verified with statistical analysis via generation of confidence intervals as well as parameter significance tests. The model was then used to study the effects of process conditions (gas composition, dilution rate, gas flow rates, and cell recycle) as well as the sensitivity of kinetic parameters, and multiobjective genetic algorithm was used to maximize ethanol productivity and CO conversion. It was observed that these two objectives were clearly conflicting when CO-rich gas was used, but increasing the content of H₂ favored higher productivities while maintaining 100% CO conversion. The maximum productivity predicted with full conversion was 2 g·L⁻¹·hr⁻¹ with a feed gas composition of 54% CO and 46% H₂ and a dilution rate of 0.06 hr⁻¹ with roughly 90% of cell recycle.     

Dilute-and-shoot analysis of therapeutic monoclonal antibody variants in fermentation broth: a method capability study

Monoclonal antibodies (mAbs) are widely applied as highly specific and efficient therapeutic agents for various medical conditions, including cancer, inflammatory and autoimmune diseases. As protein production in cellular systems inherently generates a multitude of molecular variants, manufacturing of mAbs requires stringent control in order to ensure safety and efficacy of the drugs. Moreover, monitoring of mAb variants in the course of the fermentation process may allow instant tuning of process parameters to maintain optimal cell culture conditions. Here, we describe a fast and robust workflow for the characterization of mAb variants in fermentation broth. Sample preparation is minimal in that the fermentation broth is shortly centrifuged before dilution and HPLC-MS analysis in a short 15-min gradient run. In a single analysis, N-glycosylation and truncation variants of the expressed mAb are identified at the intact protein level. Simultaneously, absolute quantification of mAb content in fermentation broth is achieved. The whole workflow features excellent robustness as well as retention time and peak area stability. Additional enzymatic removal of N-glycans enables determination of mAb glycation levels, which are subsequently considered in relative N-glycoform quantification to correct for isobaric galactosylation. Several molecular attributes of the expressed therapeutic protein may thus be continuously monitored to ensure the desired product profile. Application of the described workflow in an industrial environment may therefore substantially enhance in-process control in mAb production, as well as targeted biosimilar development.     

Long‐term changes in hypoxia and soluble reactive phosphorus in the hypolimnion of a large temperate lake: consequences of a climate regime shift

The (Lower) Lake of Zurich provides an ideal system for studying the long‐term impact of environmental change on deep‐water hypoxia because of its sensitivity to climatic forcing, its history of eutrophication and subsequent oligotrophication, and the quality and length of its data set. Based on 39 years (1972–2010) of measured profiles of temperature, oxygen concentration and phosphorus (P) concentration, the potentially confounding effects of oligotrophication and climatic forcing on the occurrence and extent of deep‐water hypoxia in the lake were investigated. The time‐series of Nürnberg's hypoxic factor (HF) for the lake can be divided into three distinct segments: (i) a segment of consistently low HF from 1972 to the late‐1980s climate regime shift (CRS); (ii) a transitional segment between the late‐1980s CRS and approximately 2000 within which the HF was highly variable; and (iii) a segment of consistently high HF thereafter. The increase in hypoxia during the study period was not a consequence of a change in trophic status, as the lake underwent oligotrophication as a result of reduced external P loading during this time. Instead, wavelet analysis suggests that changes in the lake's mixing regime, initiated by the late‐1980s CRS, ultimately led to a delayed but abrupt decrease in the deep‐water oxygen concentration, resulting in a general expansion of the hypoxic zone in autumn. Even after detrending to remove long‐term effects, the concentration of soluble reactive P in the bottom water of the lake was highly correlated with various measures of hypoxia, providing quantitative evidence supporting the probable effect of hypoxia on internal P loading. Such climate‐induced, ecosystem‐scale changes, which may result in undesirable effects such as a decline in water quality and a reduction in coldwater fish habitats, provide further evidence for the vulnerability of large temperate lakes to predicted increases in global air temperature.     

Expression of a collagen‐binding domain fusion protein: Effect of amino acid supplementation,inducer type,and culture conditions

Collagen binding domain fusion proteins are of significant importance because of their potential as therapeutic biomaterials. In this paper, we investigate the production of such therapeutic proteins via fermentation of Escherichia coli on both an undefined medium and a defined medium. Defined media with amino acid supplementation provided higher amounts of therapeutic protein than undefined media with no supplementation. Additionally, utilizing lactose instead of isopropyl‐β‐d ‐thio‐galactoside (IPTG) for induction and extending batch time yielded higher amounts of the model therapeutic. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:503–509, 2015     

Residual ridge atrophy in complete denture wearers and relationship with densitometric values of a cervical spine: a hierarchical regression analysis

doi: 10.1111/j.1741‐2358.2011.00589.x Residual ridge atrophy in complete denture wearers and relationship with densitometric values of a cervical spine: a hierarchical regression analysis Background: The rate of residual ridge atrophy (RRR) and its association with mineral density of other bones have not yet been fully explained. Objective: To measure RRR over a 5‐year period in complete denture wearers and relate it to the density of a cervical spine (CSBD). Materials and methods: Sixty‐two patients (different gender, age, body mass index, duration of edentulousness (DE) and different denture‐wearing habits) participated. A copper stepwedge was attached to the cassette, and 50 lateral radiograms met the criteria to be included. Results: A significant decrease in vertical height was observed in all measured sites. The amount of RRR was highest in frontal areas of both jaws and decreased gradually towards lateral regions. Hierarchical regression analysis revealed that the amount of RRR in the maxillary frontal area could be explained up to 48.4% by the variable DE and only up to 6.1% by the CSBD, while gender had almost no influence (1%). Similar results were obtained for the lateral maxillary RRR (33.9%; 7%; 2%), frontal mandibular RRR (40; 8.4; 0.4%) and lateral mandibular RRR (31.5%; 3.4%; 7.7%). Conclusion: Skeletal bone density, reflecting systemic and hereditary factors, is weakly related to RRR (3.4–8.4%).     

Structural stability of Sclerotium rolfsii ATCC 201126 β-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.
Methods and Results:  Fermenter-scale production led to productivity ( P _r) and yield ( Y _P/C) values higher at 48 h ( P _r = 0·542 g l⁻¹ h⁻¹; Y _P/C = 0·74) than at 72 h ( P _r = 0·336 g l⁻¹ h⁻¹; Y _P/C = 0·50). Both EPSs were neutral glucose-homopolysaccharides with a β-(1,3)-glycosidic backbone and single β-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm⁻¹ confirmed β-glycosidic linkages, while gentiobiose released by β-(1,3)-glucanases confirmed single β-1,6-glycosidic branching for both EPSs.
Conclusions:  The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour.
Significance and Impact of the Study:  Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.     

长期不同磷肥施用量对砂姜黑土真菌多样性、群落组成和种间关系的影响

以安徽蒙城砂姜黑土氮磷钾肥肥效长期定位试验为平台,选取P0(不施磷肥)、P1(P_2O_5 45 kg/hm~2)和P2(P_2O_5 90 kg/hm~2)3个磷肥施用梯度,采用现代高通量测序技术,探索长期施用磷肥对真菌多样性、群落组成和种间关系的影响。结果发现真菌多样性随磷肥施入量增加而降低,且与全磷和有效磷均成显著负相关。磷肥施用导致真菌群落组成发生明显改变,冗余分析结果表明这与全磷和可溶性有机碳密切相关。P1和P2中子囊菌门和子囊菌纲显著升高,而被孢霉菌门和被孢霉菌纲则显著降低。在属水平上,施磷处理中腐生营养型真菌显著升高,而病原型和共生型真菌显著降低:其中P0中Sagenomella、Simplicillium、Magnaporthiopsis、Schizothecium和Nigrospora等5个属相对丰度最高;P1中Plenodomus、Penicillium和Arthrobotrys相对丰度最高;P2中Cyphellophora、Zopfiella、Guehomyces、Mortierella和Mucor等5个属相对丰度最高。网络分析发现施磷后真菌网络复杂度和稳定性升高,物种间正相关关系增加,负相关关系减少。因此在砂姜黑土区长期施用磷肥可降低真菌多样性,增加土壤中腐生营养型真菌、减少共生或病原菌数量,同时提高物种间网络复杂度和稳定性。以上结果表明磷肥施用对促进土壤养分循环,控制植物病害、维持地下生态系统稳定性具有重要作用。