单细胞转录组数据批次效应评测的研究进展

单细胞转录组测序(Single cell RNA sequencing,ScRNA seq)是一种变革性的生物技术,以前所未有的高分辨率来解析组织复杂性,解决了普通转录组测序(Bulk RNA sequencing)无法回答的问题。但单细胞数据的高通量及复杂性给分析带来极大难度,批次效应(Batch effects,BEs)的处理便是主要挑战之一。批次效应是高通量生物数据分析中的技术性偏倚,其来源及处理具有高复杂性和研究依赖性。根据组织类型、测序技术及实验设计的不同,测序数据需采用不同的评估、分析、测量及处置流程来实现有效的批次效应处理。评测批次效应在单细胞数据分析中极易被忽略,但却有助于判断批次效应的来源、对数据变异的解释度、对数据分析结果的影响度及处理方法,是有效处理批次效应的基础。因此,本篇综述聚焦单细胞转录组数据的批次效应,分别论述批次效应的概念、与普通转录组批次效应的区别、评测方法及面临的挑战,并对未来发展做出展望。     

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

Introducing fed‐batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed‐batch fermentation processes. Therefore, cost efficient and easy to use small‐scale fed‐batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer‐based controlled‐release fed‐batch microtiter plate is described. In this work, the polymer‐based controlled‐release fed‐batch microtiter plate is used to investigate fed‐batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online‐monitored within each well of the polymer‐based controlled‐release fed‐batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer‐based controlled‐release fed‐batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed‐batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed‐batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale‐up criterion, microtiter plate‐ and shake flask‐based fed‐batch cultivations are highly comparable. On basis of the small‐scale fed‐batch cultivations, a mechanistic model is established and validated. Model‐based simulations coincide well with the experimentally acquired data.     

基于遗传算法的麦角固醇分批发酵动力学参数估算

把遗传算法应用于求解麦角固醇分批发酵动力学模型参数,能进一步提高麦角固醇分批发酵过程状态变量的计算值与实验值的吻合程序,在计算机上对动力学模型进行了拟合,模拟值与实验值对比显示,该动力学模型能很好地反映麦角固醇分批发酵过程。     

The practical effect of batch on genomic prediction

Measurements from microarrays and other high-throughput technologies are susceptible to non-biological artifacts like batch effects. It is known that batch effects can alter or obscure the set of significant results and biological conclusions in high-throughput studies. Here we examine the impact of batch effects on predictors built from genomic technologies. To investigate batch effects, we collected publicly available gene expression measurements with known outcomes, and estimated batches using date. Using these data we show (1) the impact of batch effects on prediction depends on the correlation between outcome and batch in the training data, and (2) removing expression measurements most affected by batch before building predictors may improve the accuracy of those predictors. These results suggest that (1) training sets should be designed to minimize correlation between batches and outcome, and (2) methods for identifying batch-affected probes should be developed to improve prediction results for studies with high correlation between batches and outcome.     

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

One essential task in bioprocess development is strain selection. A common screening procedure consists of three steps: first, the picking of colonies; second, the execution of a batch preculture and main culture, e.g., in microtiter plates (MTPs); and third, the evaluation of product formation. Especially during the picking step, unintended variations occur due to undefined amounts and varying viability of transferred cells. The aim of this study is to demonstrate that the application of polymer‐based controlled‐release fed‐batch MTPs during preculture eliminates these variations. The concept of equalizing growth through fed‐batch conditions during preculture is theoretically discussed and then tested in a model system, namely, a cellulase‐producing Escherichia coli clone bank containing 32 strains. Preculture is conducted once in the batch mode and once in the fed‐batch mode. By applying the fed‐batch mode, equalized growth is observed in the subsequent main culture. Furthermore, the standard deviation of cellulase activity is reduced compared to that observed in the conventional approach. Compared with the strains in the batch preculture process, the first‐ranked strain in the fed‐batch preculture process is the superior cellulase producer. These findings recommend the application of the fed‐batch MTPs during preculture in high‐throughput screening processes to achieve accurate and reliable results.     

Enhanced process monitoring of fed-batch penicillin cultivation using time-varying and multivariate statistical analysis

On-line monitoring of penicillin cultivation processes is crucial to the safe production of high-quality products. In the past, multiway principal component analysis (MPCA), a multivariate projection method, has been widely used to monitor batch and fed-batch processes. However, when MPCA is used for on-line batch monitoring, the future behavior of each new batch must be inferred up to the end of the batch operation at each time and the batch lengths must be equalized. This represents a major shortcoming because predicting the future observations without considering the dynamic relationships may distort the data information, leading to false alarms. In this paper, a new statistical batch monitoring approach based on variable-wise unfolding and time-varying score covariance structures is proposed in order to overcome the drawbacks of conventional MPCA and obtain better monitoring performance. The proposed method does not require prediction of the future values while the dynamic relations of data are preserved by using time-varying score covariance structures, and can be used to monitor batch processes in which the batch length varies. The proposed method was used to detect and identify faults in the fed-batch penicillin cultivation process, for four different fault scenarios. The simulation results clearly demonstrate the power and advantages of the proposed method in comparison to MPCA.     

Phospholipase D production using immobilized cells of Streptoverticillium cinnamoneum

Summary Production of phospholipase D (PLD) by Streptoverticillium cinnamoneum immobilized within porous particles was investigated in repeated batch fermentation. The enzyme productivity in repeated batch fermentation was 2.2-fold that obtained in batch fermentation without immobilization, since many of the immobilized cells could be utilized as seed cells for each subsequent batch cycle.     

2-酮-L-古洛酸的补料分批发酵的研究

通过对２－酮－Ｌ－古洛酸的一般发酵与补料分批发酵过程比较研究,发现了补糖分批发酵的一些表观特征,为其发酵控制提供了依据。     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Unstructured model for L-lysine fermentation under controlled dissolved oxygen

An unstructured model was developed for batch cultivation of Corynebacterium lactofermentum (ATCC 21799) under controlled dissolved oxygen. The model is capable of predicting batch experiments performed at various initial substrate concentrations. By extending the batch culture model to a fed-batch model and using a heuristic approach to optimize the fed-batch cultivation, it is shown that fed-batch cultivation is superior to batch operation due to increased productivity at high substrate concentrations.     

Bioactivity of immobilized microalgal cells: application potential of vegetable sponge in microbial biotechnology

Bioactivity of immobilized cells, as evidenced by the production of intracellular phycoerythrin and extracellular polysaccharides by Porphyridium cruentum , in batch and repeated batch culture, respectively, is reported. Cells of P. cruentum immobilized within the vegetable sponge of Luffa cylindrica were grown in batch culture for 24 d and maintained at the stationary phase of growth in repeated batch cultures in a growth-limiting medium for 50 d. The study indicates the application potential of this recently reported biostructure as an immobilization matrix in microbial biotechnology.     

Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported K(s) vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NH(x)/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures. (c) 1994 John Wiley & Sons, Inc.     

Investigation of vinegar production using a novel shaken repeated batch culture system

Nowadays, bioprocesses are developed or optimized on small scale. Also, vinegar industry is motivated to reinvestigate the established repeated batch fermentation process. As yet, there is no small‐scale culture system for optimizing fermentation conditions for repeated batch bioprocesses. Thus, the aim of this study is to propose a new shaken culture system for parallel repeated batch vinegar fermentation. A new operation mode — the flushing repeated batch — was developed. Parallel repeated batch vinegar production could be established in shaken overflow vessels in a completely automated operation with only one pump per vessel. This flushing repeated batch was first theoretically investigated and then empirically tested. The ethanol concentration was online monitored during repeated batch fermentation by semiconductor gas sensors. It was shown that the switch from one ethanol substrate quality to different ethanol substrate qualities resulted in prolonged lag phases and durations of the first batches. In the subsequent batches the length of the fermentations decreased considerably. This decrease in the respective lag phases indicates an adaptation of the acetic acid bacteria mixed culture to the specific ethanol substrate quality. Consequently, flushing repeated batch fermentations on small scale are valuable for screening fermentation conditions and, thereby, improving industrial‐scale bioprocesses such as vinegar production in terms of process robustness, stability, and productivity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1158–1168, 2013     

Robust parameter estimation during logistic modeling of batch and fed‐batch culture kinetics

Methods for robust logistic modeling of batch and fed‐batch mammalian cell cultures are presented in this study. Linearized forms of the logistic growth, logistic decline, and generalized logistic equation were derived to obtain initial estimates of the parameters by linear least squares. These initial estimates facilitated subsequent determination of refined values by nonlinear optimization using three different algorithms. Data from BHK, CHO, and hybridoma cells in batch or fed‐batch cultures at volumes ranging from 100 mL–300 L were tested with the above approach and solution convergence was obtained for all three nonlinear optimization approaches for all data sets. This result, despite the sensitivity of logistic equations to parameter variation because of their exponential nature, demonstrated that robust estimation of logistic parameters was possible by this combination of linearization followed by nonlinear optimization. The approach is relatively simple and can be implemented in a spreadsheet to robustly model mammalian cell culture batch or fed‐batch data. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

法夫酵母产虾青素的反复分批及反复分批补料发酵

以生物量和虾青素产量为指标,考察法夫酵母多批次半连续培养产虾青素的稳定性。实验结果显示,在摇瓶上分别以4 d和5 d为周期反复分批培养法夫酵母,虾青素产量呈现先增加再下降的趋势,但第2代至第7代虾青素产量仍高于第1代,并且4 d为周期的虾青素平均产量略高于5 d的。在5 L罐法夫酵母进行反复分批补料发酵中,不管是补加30％的葡萄糖还是补加30％的淀粉水解糖,第2个批次发酵的生物量和虾青素产量均达到第1个批次的水平,表明菌种稳定性较好。     

Monitoring of a sequencing batch reactor using adaptive multiblock principal component analysis

Multiway principal component analysis (MPCA) for the analysis and monitoring of batch processes has recently been proposed. Although MPCA has found wide applications in batch process monitoring, it assumes that future batches behave in the same way as those used for model identification. In this study, a new monitoring algorithm, adaptive multiblock MPCA, is developed. The method overcomes the problem of changing process conditions by updating the covariance structure recursively. A historical set of operational data of a multiphase batch process was divided into local blocks in such a way that the variables from one phase of a batch run could be blocked in the corresponding blocks. This approach has significant benefits because the latent variable structure can change for each phase during the batch operation. The adaptive multiblock model also allows for easier fault detection and isolation by looking at the relationship between blocks and at smaller meaningful block models, and it therefore helps in the diagnosis of the disturbance. The proposed adaptive multiblock monitoring method is successfully applied to a sequencing batch reactor for biological wastewater treatment.     

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.     

Increased lovastatin formation by Aspergillus terreus using repeated fed-batch process

Lovastatin biosynthesis with Aspergillus terreus in batch fermentation reached 160 U/l in 161 h at pH 6.8 and a dissolved O tension maintained at 70%. At the end of repeated fed batch fermentations, the yield of lovastatin was increased by 37% though this took over twice as long as in the batch fermentation.     

Recombinant β-glucanase production and plasmid stability of Bacillus subtilis in cyclic fed batch culture

Recombinant -glucanase production and plasmid stability were higher in cyclic fed batch culture than in batch and chemostat culture. Plasmid stability was maintained indefinitely in cyclic fed batch cultures of Bacillus subtilis when subjected to cycle periods of 2 or 4 hours. Cycle periods greater than 6 hours resulted in loss of the plasmid. In batch and chemostat cultures plasmid loss was rapid. -1,3-glucanase productivity was highest in cyclic fed batch culture.     

δ-endotoxin activity and spore production in batch and fed-batch cultures of Bacillus thuringiensis

Summary The toxicity and the spore count of batch and fed batch cultures of Bacillus thuringiensis var. israelensis were studied. Spore counts reached in both batch and fed batch cultures were as high as those reported in the literature, but the levels of toxicity found in the latter were about one order of magnitude lower than those attained in batch cultures. Avoiding restricted cultures might be necessary to reach high titres of -endotoxin, which are essential if a good product is intended. Furthermore, spore count might not be a good parameter to predict insecticidal activity of Bacillus thuringiensis cultures.