Showing papers in "Bioprocess Engineering in 2013"

Bioreactor design and operation

Abstract: For a well mixed reactor, there are three common operational modes: batch, steady CSTR and fed-batch. The selection of the operational modes depends on the catalyst stability, substrate supply, product demand and regulatory requirements. Batch operation has the best flexibility, while CSTR has the highest productivity. There are three basic reactor types for aerobic cultivation of suspended cells: internal mixing; bubble columns and loop reactors, each with different properties. Scale-up is difficult because conditions in a large vessel are more heterogeneous than in a small vessel. Scale-up problems are all related to transport processes. Scale-down techniques are useful in identifying the controlling regime at the smaller scale, where many parameters can be tested more quickly and less expensively than at the production scale. Bioreactor instrumentation and control are important for bioreactor operations. Improvements in sensor technology and the dynamical models of bioreactors are critical to improvements in control technology. Sterilization is the removal of biocontaminants or foreign organisms for a fermentation system and the surrounding equipment. Industrial fermenters and associated pipings are designed for in situ steam sterilization under pressure, or steam in place (SIP). The use of saturated steam is common and advantageous over other hot fluids. Sterilization of process fluids is dependent on the expectation of probability of successful fermentations. Stringent requirement leads to longer reduced or dimensionless sterilization time t S . Bacterial spores are known to be more thermal resistant than vegetative forms of yeast, bacteria and bacterophages. One particularly resistant undesired biocontaminant is prions, or infectious proteins. Sterilizing prions requires special attention. To maintain aceptic operations, special considerations are needed for sealing the opening where stirrer shaft entering the vessel, control valves and sampling ports. Microbes, especially shear sensitive bacteria and mammalian cells, tend to attach to surfaces. The attachment of suspended microbes to surfaces can be modeled as adsorption. Surface adhesion delays washout in a chemostat. The cells adhered on the vessel surfaces are less likely to be taken out of the reactor by the outflowing stream. However, the adhesion - desorption balance eventually leads to the washout if the flowrate exceeds the critical limit. The washout is more gradual if cells adhere to the vessel surfaces, and a log tail could exist if the adhesion is strong.

An Overview of Biological Basics

Abstract: Cell chemistry and cell feed are important aspects of biological basics that interest bioprocess engineers. Cells are the basic unit of living organisms. Living organisms can be single celled (unicellular) or multicellular. Therefore, living organisms are commonly cellular. Stem cell is the mother cell of the multicellular organisms. All cells contain at least one of the three macromolecules: protein, RNA , and DNA . The two major groups of cells are prokaryotic and eukaryotic , with eukaryotic cells being more complex. Both prokaryotes and eukaryotes contain both DNA and RNA. Viruses are replicating particles that are obligate parasites. Some viruses use DNA to store genetic information, while others use RNA. Viruses specific to bacteria are called bacteriophages or phages. A prion is an infectious agent composed of protein in a misfolded form. This is in contrast to all other known infectious agents which must contain nucleic acids (either DNA, RNA, or both). Other essential components of these cells are constructed from lipids and carbohydrates. The exception to the cellular living organism is a prion, which consists of protein only. Cells are usually very small, but big ones exist and can weigh over a kilogram. There are rich varieties of chemicals and materials associated with cells. These chemicals and materials present opportunities for bioprocess engineers to make sustainable products that meet the needs of humanity. This chapter introduces the various chemical compositions in cell, structures of protein, RNA and DNA, as well as the nutritional needs of cells.

Fed-Batch Cultivation

Abstract: Fed-batch reactor is based on feeding of a growth limiting nutrient substrate to a culture. Cell growth and fermentation can be controlled by the feeding strategy. The fed-batch strategy is typically used to reach a high cell density in the bioreactor or mimic a continuous culture. Mostly the feed solution is highly concentrated. In essence, fed-batch reactor is applied in such a fashion that a chemostat is simulated with a seemingly batch operation. The controlled addition of the nutrient directly affects the growth rate of the culture and allows to avoid overflow metabolism (formation of side metabolites, such as acetate for Escherichia coli , lactic acid in cell cultures, ethanol in Saccharomyces cerevisiae ), oxygen limitation (anaerobiosis). Substrate limitation offers the possibility to control the reaction rates to avoid technological limitations connected to the cooling of the reactor and oxygen transfer. Substrate limitation also allows the metabolic control, to avoid osmotic effects and catabolite repression. Therefore there are different operation strategies for fed-batch: 1) constant feed rate; 2) exponential feed rate or constant specific growth rate. The exponential growth with fed-batch operation can be at any rate, up to the maximum rate in the exponential growth phase of a batch growth. Usually, maximum growth rate is not wanted for undesired by-product production can be high at maximum growth. Analysis of fed-batch reactors is more complex than either a chemostat or a batch reactor. Solutions of differential equations are usually required. When long operation time is employed, a pseudo-steady state may be assumed, simplifying the analysis. The pseudosteady state is found to correspond to biomass steady state or time derivative of biomass concentration is zero. Some solutions to the biomass concentration and product formation are listed for constant feed and exponential feed at pseudo-steady state conditions.

Ideal Flow Reactors

Abstract: There is one important parameter used for flow reactors, τ, the reactor space time or its inverse, D , the dilution rate. The space time is the time needed to feed a reactor full of reaction mixture through the reactor. There are two basic types of ideal flow reactors: PFR (plug flow reactor) and CSTR (continuous stirred tank reactor). PFR represents a one dimensional flow reactor, like a piston. At steady state, the concentration of reactants as well as products changes along the direction of flow in a PFR. The concentrations of the reactants are the highest in a PFR presented to reaction. The analysis to a PFR is similar to that for a batch reactor, the only difference being the independent variable: the residence time or distance along the reactor flow direction, rather than the time for a batch reactor. CSTR represents a reactor that is well mixed such that no concentration gradient exits. CSTR is also termed chemostat. At steady state, only algebraic equations are involved in CSTR analysis. The concentrations of reactants in a CSTR are at their lowest level in a CSTR and thus least available to reaction. Graphical and analytical solution methods are demonstrated with examples for CSTR and PFR. In flow reactor design, our interest is not only on the reactor volume, but type of reactors. Usually, a whole bioprocess system optimization (economic analysis) is applied to determine whether CSTR or PFR is employed. In some cases, one can save time by knowingly selecting the right type of reactors. Selection of PFR or CSTR depends on the nature of the reaction mixture and type of reaction (kinetics). The reactor feed strategy greatly affects the product mixture. A reactor with optimum feed distribution and / or product separation can yield significantly more desired product. Methods of how these “complicated” flow reactors as well as simple flow reactors are demonstrated in the chapter.

Chemical Reactions on Solid Surfaces

Abstract: Catalysts are substances or materials that increase the reaction rates while emerged unaltered after reaction. Solid catalysts are particularly important because of their ease of separation from reaction mixture and stability. In other occasions, solid substrates are employed to produce a desired product. Reactions involving solid surface are thus common. Reactions occurring on solid surface start with reactants or catalyst from the fluid phase collide and associate with active centers (or sites) on the surface, i.e. adsorption. Adsorption can be described as Langmuir isotherm if uniform surface activity (or ideal surface) is assumed. Nonideal surfaces can be modeled by a distribution of interaction energy or multiple adsorption layers. Exland and UniLan are two models based on exponential and uniform distributions of active sites with adsorption energy. The adsorption isotherms exhibit different dependence on the bulk adsorbate concentration: Types I through V. LHHW kinetics is derived from Langmuir adsorption isotherm. PSSH is more general and a better approximation than FES as PSSH can be traced back to the overall effect of all the fluxes on the reaction network or pathway. However, the final rate expressions are quite similar when the rate constants are lumped together. Both ExLan and UniLan isotherms can also be employed to derive at reaction rate expressions. The rate expression can also appear to be of power-law form if Freundlich or Temkin approximation were applied to UniLan isotherms. The multilayer adsorption can also be applied to describe reactions on nonideal surfaces as steric interactions and interaction potential differences can be effectively modeled by multilayers. Rate expressions for non catalytic reactions involving solids can be derived in the same manner as LHHW. The participation of the surface active centers in actual reactions can affect the site balance, either active center renewal or depletion occurs. For catalytic surfaces, the activity can decrease with increase duration of service. The surface activity decline can be treated the same way as the concentration of a reacting component in the mixture.

Sustainability: Humanity Perspective

Abstract: Sustainability is the capacity to endure in ecological terms. A sustainable system is one that has a "stable" nontrivial "end" state. Monotonous change is an indication that the system is not sustainable. The Sun's energy powers the entire ecosystem on earth: water cycle and biomass cycle. The water cycle provides opportunity for hydroelectric power development. While both hydroelectricity and wind electricity are renewable and sustainable, hydroelectricity power plants have a more predictable load factor. The water storage reservoirs can have a stabilizing effect on the "local climate", improving biomass cycles. Biomass utilization brings a sustainable state change. Switching from fossil energy use to biomass use will see an increase in carbon dioxide emission into the atmosphere initially. However, there is a new sustainable state foreseeable with biomass use, in direct contrast to fossil energy use. Apart from the sustainability impact, the depletion of fossil sources has made it imperative to move away from its utilization. Wood or forest biomass has the highest saturation standing biomass, while algae have the highest production rate. High saturation biomass species are more advantageous than low saturation biomass species due to the collection and transportation restrictions. The lower standing biomass also leads to more CO 2 staying in the atmosphere. Depending on the life-span of the final product, the effect of biomass management on the carbon storage can be either positive or negative. Solar energy is vastly available and its use is less disturbing to the ecosystems. It is clear that in the direct solar energy utilization cycle; there is no water, carbon dioxide, or any other than substance emission involvement. Efficient capture of solar energy is the key for its sustainable exploitation. However, solar energy capture cannot interfere with plant biomass. Geothermal energy is most reliable as it is stored deep underneath the earth. It can be exploited from any point on the earth surface by deep drilling into the rocks. However, geothermal energy is neither sustainable nor renewable. Just because there is vast amount of geothermal energy is not the reason for us to exploit. It is a more desperate resource than anything else on earth.

An Overview of Chemical Reaction Analysis

Abstract: This chapter is a survey of chemical reaction engineering basics: chemical species, reaction rates, representations of chemical reactions, chemical stoichiometry, thermodynamics, energy regularity for biotransformations, multiple reactions, conversion, yield, selectivity and yield factor, mass and energy balances, ideal chemical reactors, and economics of reaction engineering. Basic concepts are introduced, for example, elementary reaction, approximate reaction, coupled reaction, concentrations, specific reaction rate, Roels formula, transitional state, activation energy, chemical equilibrium, Gibbs free energy, enthalpy, and entropy. Also, included are how reaction rates are related between different substrates, different products; why there is only one independent variable in every reaction; what yield factors are, and how all are linked through stoichiometry. You should understand why A → B or S → P is used commonly in a reaction engineering text, instead of more realistic or real-life examples after reading this chapter. The understanding of how chemical reactions are approximated and represented, for example, is the key in understanding the representation of metabolic pathways in microbiology.

Kinetic Theory and Reaction Kinetics

Abstract: While reaction rates are empirical in nature, collision theory does provide a means to estimate the reaction rate. Collision theory gives rise to the correct reaction rate dependence on concentrations and temperature. Based on collision theory, most viable reactions are bimolecular. Reactions with more than three molecules are unfavorable. Therefore, an elementary reaction usually involves three or less molecules on either side of the reaction. Nonelementary reactions can be decoupled into elementary reaction steps or reaction pathways. The reaction system that forms a nonelementary reaction can be analyzed as one were dealing with a multiple reaction system. In many occasions, we find that simplification of the nonelementary reaction is necessary. Some reaction steps are fast than others. Some or all the reaction intermediates are present in the reaction system in only trace amount. These observations lead to two methods of simplifying reaction rate expressions: FES and PSSH. FES stands for fast equilibrium step approximation, while PSSH stands for pseudosteady state hypothesis. Active reaction intermediates are common as they are means to overcome the activation energies. Active intermediates can be active complex, transitional state (or activated molecules), and free radicals. Nonelementary reactions usually have complicated reaction rate expressions than the simple power law form. Fractional order (1/2, 3/2, ..) can be observed for free-radical reactions. Simple kinetic expressions can be obtained for otherwise a complicated problem when proper assumptions are made. For acid hydrolysis of polymers, the reaction kinetics may be approximated by three “first order reaction rate” relationships of lumped components despite a complicated composition of the reaction mixture.

Evolution and Genetic Engineering

Abstract: A cell’s genotype represents the cell’s genetic potential, whereas its phenotype represents the expression of a culture’s potential. The genotype of a cell can be altered by mutations. Mutations may be selectable or unselectable . The rate of mutation can be enhanced by the addition of chemicals called mutagens or by radiation. Auxotrophs are of particular use in genetic analysis and as a basis for some bioprocess. Another useful class of mutants is conditional mutants. Gene transfer from one cell to another augments genetic information in ways that are not possible through mutation only. Genetic recombination of different DNA molecules occurs within most cells. Thus, genetic information transferred from another organism may become a permanent part of the recipient cell. The three primary modes of gene transfer in bacteria are transformation, transduction, and conjugation . We can use gene transfer in conjunction with restriction enzymes and ligases to genetically engineer cells. In-vitro procedures to recombine isolated donor DNA gens with vector DNA (for example plasmids, temperate phages, or modified viruses) are called recombinant DNA techniques . The application of recombinant DNA technology at the commercial level requires a judicious choice of the proper host-vector system. E. coli , S. cerevisiae , P. stipitis and Bacillus are commonly selected as hosts because of their unique properties. Animal cell culture is required when posttranslational modifications are essential. The vector must be designed to optimize a desired process. One must be aware of the regulatory constraints on the release of cells with recombinant DNA. These are particularly relevant in plant design, where guidelines for physical containment must be met. Deliberate release of genetically modified cells is possible, but extensive documentation will be required. Two increasingly important applications of genetic engineering are metabolic or pathway engineering for the production or destruction of nonproteins and protein engineering for the production of novel or specifically modified proteins.

Sustainability and Stability

Abstract: One of the important aspects of reactor design is to seek desirable stable steady state reaction conditions as the reactor set point. Stable steady state is a subset of sustainable states that the system operates at a single valued point. While a sustainable system can be weakly stable, exhibiting limit cycle or oscillation between bound and none trivial values, industrial bioprocesses are preferably to be strictly stable. Stability of a reactor operation is usually associated with the existence of multiple state states (MSS). MSS could exist for nonisothermal exothermic reactions (of any kinetics) and isothermal substrate inhibited systems in a CSTR. Feed conditions also affect the existence of MSS. Catalyst instability is another important issue. Catalyst deactivation is affected by the reaction mixture, temperature, and flow conditions. The simplistic root for bio-instability is the difference in specific growth rate (for mixed culture) and mutation. Biostability is of concern for genetically engineered cells. Genetically engineered cells for industrial applications are commonly exploiting the cells to produce protein or products that are not generic to the host cells. Genetic evolution back to the generic cell function looses the desired traits imposed to the cells. The genetic evolution can be modeled quite accurately with kinetic considerations. Populations containing multiple species are important in natural ecosystems, well-defined processes, waste-water treatment, and systems using genetically modified cells. Some examples of interactions among these species are competition, neutralism, mutualism, protocooperation, commensalism, amensalism, predation, and parasitism. Neither pure competition nor pure mutualism gives a stable steady state in a chemostat. Predator and prey model shows that while there are no stable steady states, the bioprocess can be sustainable and the final prey and predator populations oscillate in a confined cycle. Continuous culture can be employed to screen organisms of certain traits based on the growth instability. Spatial heterogeneity, dynamic fluctuations, and the addition of other interactions can lead to the sustained coexistence of species with competitive or mutualistic interactions. One of the major process uses of mixed cultures is waste-water treatment. The activated-sludge system is commonly employed in treating waste waters.

Chapter 4 – Batch Reactor

Abstract: The batch reactor is widely used in industry and is the preferred reactor in laboratories and in pharmaceutical industries. The performance of a batch reactor can be analyzed via material and energy balances. The reactor is initially loaded with substrates; and there is no addition or withdrawal until the end of the reaction. The temperature and concentrations change with time, which are governed by differential equations derived from materials and energy balances. The solutions for these differential equations require a numerical integrator, for which the ODExLIMS routine is introduced for use with Microsoft Excel. For simple kinetics, methods of solution are introduced, with some solutions listed for reactions with simple kinetics. Batch reactor sizing depends on the productivity requirement, that is, processing efficiency as well as the quality requirement, the cost of raw materials, and the value of the product(s). Examples are given on how these problems can be solved in a concise manner. For more complicated reactions based on complex raw materials, a relative time, or H-Factor, concept is introduced for ease of process control and performance characterization. Maximizing productivity in a batch reactor system, as well as reactor sizing based on the time curve of concentration, can be as easy as drawing a pinch line.