Defining process design space for biotech products: case study of Pichia pastoris fermentation

The concept of "design space" has been proposed in the ICH Q8 guideline and is gaining momentum in its application in the biotech industry. It has been defined as "the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality." This paper presents a stepwise approach for defining process design space for a biologic product. A case study, involving P. pastoris fermentation, is presented to facilitate this. First, risk analysis via Failure Modes and Effects Analysis (FMEA) is performed to identify parameters for process characterization. Second, small-scale models are created and qualified prior to their use in these experimental studies. Third, studies are designed using Design of Experiments (DOE) in order for the data to be amenable for use in defining the process design space. Fourth, the studies are executed and the results analyzed for decisions on the criticality of the parameters as well as on establishing process design space. For the application under consideration, it is shown that the fermentation unit operation is very robust with a wide design space and no critical operating parameters. The approach presented here is not specific to the illustrated case study. It can be extended to other biotech unit operations and processes that can be scaled down and characterized at small scale.     

Application of balancing methods in modeling the penicillin fermentation

This paper shows the application of elementary balancing methods in combination with simple kinetic equations in the formulation of an unstructured model for the fed-batch process for the production of penicillin. The rate of substrate uptake is modeled with a Monod-type relationship. The specific penicillin production rate is assumed to be a function of growth rate. Hydrolysis of penicillin to penicilloic acid is assumed to be first order in penicillin. In simulations with the present model it is shown that the model, although assuming a strict relationship between specific growth rate and penicillin productivity, allows for the commonly observed lag phase in the penicillin concentration curve and the apparent separation between growth and production phase (idiophase-trophophase concept). Furthermore it is shown that the feed rate profile during fermentation is of vital importance in the realization of a high production rate throughout the duration of the fermentation. It is emphasized that the method of modeling presented may also prove rewarding for an analysis of fermentation processes other than the penicillin fermentation.     

Online monitoring of Mezcal fermentation based on redox potential measurements

We describe an algorithm for the continuous monitoring of the biomass and ethanol concentrations as well as the growth rate in the Mezcal fermentation process. The algorithm performs its task having available only the online measurements of the redox potential. The procedure combines an artificial neural network (ANN) that relates the redox potential to the ethanol and biomass concentrations with a nonlinear observer-based algorithm that uses the ANN biomass estimations to infer the growth rate of this fermentation process. The results show that the redox potential is a valuable indicator of the metabolic activity of the microorganisms during Mezcal fermentation. In addition, the estimated growth rate can be considered as a direct evidence of the presence of mixed culture growth in the process. Usually, mixtures of microorganisms could be intuitively clear in this kind of processes; however, the total biomass data do not provide definite evidence by themselves. In this paper, the detailed design of the software sensor as well as its experimental application is presented at the laboratory level.     

噬菌体在食品工业中的应用与危害防控

近年来,噬菌体由于其特异性侵染细菌的特性,在食品加工及保藏过程中有害微生物的控制和检测方面展现出良好的应用前景。例如在食品表面喷洒噬菌体或将噬菌体与食品包装材料结合,对食源性致病菌及腐败菌加以控制,以及利用基因工程手段构建报告噬菌体对食源性致病菌进行快速检测等。然而,噬菌体也是危害食品发酵的重要因素之一,轻则减产,重则引起整个发酵过程失败,造成巨大的经济损失。目前主要通过噬菌体消毒及灭活、发酵菌种变换等方式防止噬菌体污染。本文综述了食品工业中噬菌体应用及危害的研究现状,以期为拓宽噬菌体在食品工业中的应用途径及开发噬菌体污染防治的新技术提供理论依据。     

Application of the macroscopic electric charge balance in fermentation modeling

This article treats the application of the macroscopic electric charge balance in fermentation modeling. From the presented calculations it follows that the definition formula of the so-called degree of reduction is changed due to the ionic character of the fermentation reactants. It is also shown that the macroscopic electric charge balance, together with the ionic equilibria between fermentation reactants, leads to an expression for the calculation of the pH during fermentation. Finally it is concluded that one should be very careful in the estimation of biomass production from the acid or base feed rates which are necessary for pH control.     

Kinetics of the conversion of glucose to gluconic acid by Pseudomonas ovalis

The concept of a “critical oxygen concentration” is conventionally considered to hold for the submerged aerobic fermentation of glucose to gluconic acid. Above the critical level the fermentation rate is supposedly independent of oxygen concentration. In this work it is shown that, at a given agitation rate, the fermentation is independent of dissolved oxygen when above the critical. However, an increase in the agitation rate results in an increase in the fermentation rate. This increase was shown to be accompanied by an increase in the gluconolactone concentration in the broth. Gluconolactone, an intermediate in the reaction pathway, is hydrolyzed nonenzymatically to gluconic acid. Evidence is presented to suggest that the increased gas-liquid interfacial area brought about by increased agitation causes an increased net rate of lactone formation. This in turn results in an increased rate of hydrolysis of the lactone to gluconic acid. A model is presented hypothesizing that negatively charged cells adsorb at the gas-liquid interface. These cells attract hydrogen ions, causing a lowering of the pH in the film around the bubbles. It is this lowered pH which is considered to bring about increased fermentation rates when the interfacial area is increased. Supporting evidence is presented.     

Temperature effects on anaerobic fermentation of domestic refuse

Anaerobic fermentation of organic solid waste can provide a significant source of fuel gas (methane). Application of this process requires a better understanding of the kinetics of the biological system. The literature is replete with kinetic studies of this process as applied to waste solids from water pollution control systems. Much of this work has been conducted in the mesophilic temperature range. Increased temperatures yield higher reaction rates that will improve the economics of the process. The rate limiting step in the fermentation of refuse is the hydrolysis of the complex organic solids, in particular cellulose. Cellulose is a major component of the refuse. A laboratory study employing domestic refuse has shown the effect of temperature on the rate of methane fermentation. The optimum mesophilic temperature was found to be 42°C, while the optimum thermophilic temperature was at least 60°C. No data was obtained beyond the 60°C temperature. Reaction rate constants are presented for anaerobic fermentation of domestic refuse. Because of the characteristics of the substrate it^?was not possible to obtain the necessary measurements for evaluation of constants in the Monod model. An overall system constant was developed.     

Ionic liquid-based in situ product removal design exemplified for an acetone–butanol–ethanol fermentation

Selecting an appropriate separation technique is essential for the application of in situ product removal (ISPR) technology in biological processes. In this work, a three-stage systematic design method is proposed as a guide to integrate ionic liquid (IL)-based separation techniques into ISPR. This design method combines the selection of a suitable ISPR processing scheme, the optimal design of an IL-based liquid–liquid extraction (LLE) system followed by process simulation and evaluation. As a proof of concept, results for a conventional acetone–butanol–ethanol fermentation are presented (40,000 ton/year butanol production). In this application, ILs tetradecyl(trihexyl)phosphonium tetracyanoborate ([TDPh][TCB]) and tetraoctylammonium 2-methyl-1-naphthoate ([TOA] [MNaph]) are identified as the optimal solvents from computer-aided IL design (CAILD) method and reported experimental data, respectively. The dynamic simulation results for the fermentation process show that, the productivity of IL-based in situ (fed-batch) process and in situ (batch) process is around 2.7 and 1.8fold that of base case. Additionally, the IL-based in situ (fed-batch) process and in situ (batch) process also have significant energy savings (79.6% and 77.6%) when compared to the base case.     

Rheometry of fermentation liquids

This article is concerned with the measurements of rheological properties of nonhomogeneous fermentation liquids. In order to determine the flow curves of such liquids, an impeller measuring system of own design was used, and the method of calibration of the system is presented. The experimental verification was carried out using samples of fermentation broth of Aureobasidium pullulans and it has been shown that the applied measuring technique gives internally consistent results. The sensitivity of the technique proves its applicability for monitoring and control purposes in fermentation technology.     

Model‐based identifiable parameter determination applied to a simultaneous saccharification and fermentation process model for bio‐ethanol production

In this work, a methodology for the model‐based identifiable parameter determination (MBIPD) is presented. This systematic approach is proposed to be used for structure and parameter identification of nonlinear models of biological reaction networks. Usually, this kind of problems are over‐parameterized with large correlations between parameters. Hence, the related inverse problems for parameter determination and analysis are mathematically ill‐posed and numerically difficult to solve. The proposed MBIPD methodology comprises several tasks: (i) model selection, (ii) tracking of an adequate initial guess, and (iii) an iterative parameter estimation step which includes an identifiable parameter subset selection (SsS) algorithm and accuracy analysis of the estimated parameters. The SsS algorithm is based on the analysis of the sensitivity matrix by rank revealing factorization methods. Using this, a reduction of the parameter search space to a reasonable subset, which can be reliably and efficiently estimated from available measurements, is achieved. The simultaneous saccharification and fermentation (SSF) process for bio‐ethanol production from cellulosic material is used as case study for testing the methodology. The successful application of MBIPD to the SSF process demonstrates a relatively large reduction in the identified parameter space. It is shown by a cross‐validation that using the identified parameters (even though the reduction of the search space), the model is still able to predict the experimental data properly. Moreover, it is shown that the model is easily and efficiently adapted to new process conditions by solving reduced and well conditioned problems. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1064–1082, 2013     

原位显微镜在细胞生物量在线监测中的发展与应用

随着现代生物技术的快速发展,生物发酵过程在工业生产中的重要性日益增加。为获得质量稳定的发酵产品,通常需要对发酵过程进行监测与调控。生物量可以直接反映生物反应器中细胞代谢的主体——细胞的生长状况,因此实现生物量的在线监测对发酵过程的调控具有重要意义。原位显微镜是一项非侵入式的、基于图像分析的技术,可以实时监测生物过程中的细胞量。文中就原位显微镜的发展及其在细胞生物量实时监测中的应用进行了综述。     

Comparison of techniques for monitoring antibody fragment production in E. coli fermentation cultures

The use of an optical biosensor for monitoring antibody fragment accumulation following induction in a batch fermentation of recombinant E. coli is compared to the more traditional method of ELISA quantification. Using the biosensor, concentration data can be obtained within minutes of sample addition to the device, compared to an average assay time of 3-4 h for the ELISA. We describe two biosensor assays developed as an alternative to ELISA and compare them with ELISA in the ability to provide quantitative product accumulation profiles during fermentation. Discrepancies in titers recorded by the assays are explained by a combination of differences in product variants detected by each assay and interference from sample contaminants. Method of sample preparation is also shown to be important if accurate concentration data is required. Both biosensor assays are shown to be capable of providing product accumulation profiles comparable to those obtained by ELISA. The use of a rapid extraction technique would allow such data to be obtained during process operation, enabling improved fermentation control and more rapid process development.     

Flexible biorefinery for producing fermentation sugars, lignin and pulp from corn stover

A new biorefining process is presented that embodies green processing and sustainable development. In the spirit of a true biorefinery, the objective is to convert agricultural residues and other biomass feedstocks into value-added products such as fuel ethanol, dissolving pulp, and lignin for resin production. The continuous biomass fractionation process yields a liquid stream rich in hemicellulosic sugars, a lignin-rich liquid stream, and a solid cellulose stream. This paper generally discusses potential applications of the three streams and specifically provides results on the evaluation of the cellulose stream from corn stover as a source of fermentation sugars and specialty pulp. Enzymatic hydrolysis of this relatively pure cellulose stream requires significantly lower enzyme loadings because of minimal enzyme deactivation from nonspecific binding to lignin. A correlation was shown to exist between lignin removal efficiency and enzymatic digestibility. The cellulose produced was also demonstrated to be a suitable replacement for hardwood pulp, especially in the top ply of a linerboard. Also, the relatively pure nature of the cellulose renders it suitable as raw material for making dissolving pulp. This pulping approach has significantly smaller environmental footprint compared to the industry-standard kraft process because no sulfur- or chlorine-containing compounds are used. Although this option needs some minimal post-processing, it produces a higher value commodity than ethanol and, unlike ethanol, does not need extensive processing such as hydrolysis or fermentation. Potential use of low-molecular weight lignin as a raw material for wood adhesive production is discussed as well as its use as cement and feed binder. As a baseline application the hemicellulosic sugars captured in the hydrolyzate liquor can be used to produce ethanol, but potential utilization of xylose for xylitol fermentation is also feasible. Markets and values of these applications are juxtaposed with market penetration and saturation.     

Adaptive on-line simulation of bioreactors: Fermentation monitoring and modeling system

Summary In order to study and control fermentation processes, indirect on-line measurements and mathematical models can be used. Here an on-line model for fermentation processes is presented. The model is based on atom and partial mass balances as well as on stability equations for the protolytes. The model is given an adaptive form by including transport equations for mass transfer and expressions for the fermentation kinetics. The state of the process can be estimated on-line using the balance component of the model completed with measurement equations for the input and the output flows of the process. Adaptivity is realized by means of on-line estimation of the parameters in the transport and kinetic expressions using recursive regression analysis. On-line estimation of the kinetic and mass transfer parameters makes model-based predictions possible and enables intelligent process control while facilitating testing of the validity of the measurement variables. A practical MS-Windows 3.1 model implementation called FMMS—Fermentation Monitoring and Modeling System is shown. The system makes it easy to configure the operating conditions for a run. It uses Windows dialogs for all set-ups, model configuration parameters, elemental compositions, on-line measurement devices and signal conditioning. Advanced on-line data analysis makes it possible to plot variables against each other for easy comparison. FMMS keeps track of over 100 variables per run. These variables are either measured or estimated by the model. Assay results can also be entered and plotted during fermentation. Thus the model can be verified almost instantly. Historical fermentation runs can be re-analyzed in simulation mode. This makes it possible to examine different signal conditining filters as well as the sensitivity of the model. Combined, the data analysis and the simulation mode make it easy to test and develop model theories and new ideas.     

Dynamic modeling and analyses of simultaneous saccharification and fermentation process to produce bio-ethanol from rice straw

The rice straw, an agricultural waste from Asians’ main provision, was collected as feedstock to convert cellulose into ethanol through the enzymatic hydrolysis and followed by the fermentation process. When the two process steps are performed sequentially, it is referred to as separate hydrolysis and fermentation (SHF). The steps can also be performed simultaneously, i.e., simultaneous saccharification and fermentation (SSF). In this research, the kinetic model parameters of the cellulose saccharification process step using the rice straw as feedstock is obtained from real experimental data of cellulase hydrolysis. Furthermore, this model can be combined with a fermentation model at high glucose and ethanol concentrations to form a SSF model. The fermentation model is based on cybernetic approach from a paper in the literature with an extension of including both the glucose and ethanol inhibition terms to approach more to the actual plants. Dynamic effects of the operating variables in the enzymatic hydrolysis and the fermentation models will be analyzed. The operation of the SSF process will be compared to the SHF process. It is shown that the SSF process is better in reducing the processing time when the product (ethanol) concentration is high. The means to improve the productivity of the overall SSF process, by properly using aeration during the batch operation will also be discussed.     

Optimization of batch fermentation processes by graphical method

A graphical method is proposed for batch fermentation process optimization. Its objective function is the maximum output of the product when the time of process is fixed or free. The technique of optimization is based on Bellman's dynamic programming concepts and the assumption that the process can be described by mathematical models of generalized structure. An example of the proposed technique's application is presented.     

基于神经网络的生化过程预估优化控制

综观历史与现状,生化过程数学模型的建立是很困难的。不多的一些数学模型也往往由于精度低,应用范围窄而无法在实际中应用。这是由于生化过程的机理非常复杂,具有高度非线性和时变特性。并且不同于一般物理过程的是生化过程是个物理上不可逆的过程。近几年来,人工神经网络(ANN)得到了迅速发展,并被广泛应用到各个领域。同样,ANN也为生化过程控制提供了一种新方法。本文以工业生产中发酵过程的补料控制为例,叙述了ANN如何用干生化过程预估和优化控制。相应地,对结果进行了分析讨论。     

Physiologically Motivated Monitoring of Fermentation Processes by Means of an Electronic Nose

An on‐line approach of non‐invasive monitoring of the physiological changes in fermentation processes is presented. In yeast batch and bacterial fed‐batch fermentations it is shown that metabolic state changes can be revealed using an electronic nose. The transient responses of the gas sensors to the changes in the composition of the volatiles emitted from the cell cultures during fermentation are used to retrieve a semi‐quantitative representation of the physiological state of the cultures. With the sensor responses of the electronic nose it is shown that physiological variables such as rates of growth, substrate uptake and product formation can be depicted. The non‐invasive method thus seems as a pertinent alternative to conventional bioreactor monitoring methods.     

以能力培养为导向的基于工厂化生产实践指导的“发酵工程”教学研究与应用

"发酵工程"是生物技术和生物工程等专业的核心基础课程,应用性非常强,其工艺流程包括上游的菌种和培养基,中游发酵动力学和过程控制,以及下游的发酵产物分离制备与精制等。在实际生产过程中,工艺流程的每个阶段都是理论知识在生产实践中的具体应用。授课过程中,如果教师只介绍教材的基础理论知识,而不结合具体生产实践,学生不仅难以掌握知识点,而且对利用发酵工程生产产品的工艺流程没有深刻的认识,更不知道如何去应用理论知识指导实际生产。由于发酵工程应用的重要性,发酵工程教学质量的好坏直接影响学生的专业素质与就业情况。因此,本课程组开展了基于生产实践指导的发酵工程教学方法的研究与应用,在发酵工程课程教学中,结合生产实践中的科研经验,总结教材的理论知识与生产实践的结合点,使教材中较为抽象、复杂的理论知识具体化。这样不仅激发了学生的学习兴趣,加强了学生对理论知识的理解,更为重要的是,结合实际科研、生产过程中曾面临的问题,引导学生如何利用基础理论知识去解决这些问题,这种授人以渔的方法对于指导学生的科研和毕业后的生产实践具有重要的应用价值。