BIOCHEMICAL ENGINEERING A Concise Introduction

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BIOCHEMICAL ENGINEERINGA Concise IntroductionbyMohamad Hekarl UzirMashitah Mat DonSchool of Chemical EngineeringUniversiti Sains MalaysiaEngineering CampusSeri Ampangan14300 Nibong TebalPenangMALAYSIA

Preface to the First ManuscriptBiochemical Engineering has been offered as one of the elective courses to the Universiti Sains Malaysia’s Chemical Engineering undergraduates since 1998 under the topic ofBioprocess Engineering. The change of name from Bioprocess to Biochemical Engineering shows that the School of Chemical Engineering is very much aware of the currentdevelopment of the area that combines biology and biochemistry with engineering andtechnology. The course might have changed its name, however, the core ingredients ofBiochemical Engineering remain intact. New findings that evolve through research anddevelopment have been included so that the students are up-to-date with the most recenttechnology within the field.This Lecture Notes Series has been used to cover Biochemical Engineering coursethat was usually offered to the Fourth Year, Chemical Engineering undergraduates. Thelectures combine the topics which were handled by both authors as well as previous fewacademics before us, to name a few; Associate Professor Ghasem Najapour (currently alecturer in Iran), Dr. Long Wei Sing (currently a Humbolt Fellow in Germany) and Dr.Jyoti Prasad Chaudhury (currently a lecturer in India). We greatly appreciate their helpand guidance towards compiling this manuscript. Not to forget, the second author whomentirely involved during the first arrangement of the course outline as well as its syllabusnearly a decade ago, and without her support and encouragement, this manuscript wouldnot have come into existence.It is hoped that this manuscript would be of use to the undergraduate students whoare taking the course as an elective or other similar courses that have some elements ofBiochemical Engineering. It could also be an additional reference to the postgraduatestudents undertaking research work that relate either entirely or only a small fraction ofBiochemical Engineering field. This manuscript summarises and simplifies into a conciseform most of the details the topics that discussed in lengthy paragraphs within the mainBiochemical Engineering textbooks.The manuscript could be easily downloaded form the website of the School of ChemicalEngineering, Universiti Sains Malaysia under the tes/ekc471 notes.pdfBoth authors would like to welcome any comments from the readers both students andacademics alike so that the contents of this manuscript could be greatly improved. Yourhelp and cooperation are very much appreciated.M.H. UzirM. Mat Don(December 2007)School of Chemical EngineeringUniversiti Sains MalaysiaPenangi

About the AuthorsMASHITAH MAT DON, PhD. obtained her Bachelor Degree, B.Sc.(Hons.) inBotany in 1988 from the University of Malaya. Right after her first degree, she remainedwith the University of Malaya for her Master Degree (MPhil.) in the area of Biotechnology. In 1992, she joined the Forest Research Institute of Malaysia, (FRIM) for the periodof 3 years where she was actively involved with the research and development focusingon the exploration of Malaysia’s tropical forest for the production of pharmaceutical andagrochemical products. Most of her research while at FRIM, were based upon applyingthe core engineering and biological disciplines to the real life problems. Two main areaswhich include; microbial fermentation technology and process modelling have been hermajor work within the field of Biochemical Engineering. She left FRIM in 1995 and joinedthe School of Chemical Engineering, Universiti Sains Malaysia where she was appointedas the Programme Chairman of Bioprocess and Environmental Group for the period of 2years. Being a Programme Chairman at the time, she was assigned to compile a syllabusof Bioprocess Engineering Course to be introduced as an elective within the Bachelor Degree at the School of Chemical Engineering. She was one of the pioneers in establishingBioprocess Engineering to the undergraduate students which through years of revisionshas changed its name to Biochemical Engineering until this present days. She has writtenmany research articles for journals and proceedings both locally and internationally whileworking with FRIM as well as with Universiti Sains Malaysia and has also graduated anumber of postgraduate students since then. She received her Doctorate Degree, (PhD.)in 2005 from the University of Malaya in the area of Biochemical Engineering and hasrecently been elected as an Associate Professor at the School of Chemical Engineering,Universiti Sains Malaysia.MOHAMAD HEKARL UZIR, PhD. obtained his Bachelor Degree, B.Eng.(Hons.)in Chemical Engineering in 1999 from the University of Leeds, United Kingdom. Aftergraduation, he joined Universiti Teknologi PETRONAS, (UTP) as a Trainee Lectureruntil July 2000. He then left UTP and joined Universiti Sains Malaysia where he receivedthe Fellowship Scheme to pursue for the higher degree courses. He received his MasterDegree, (MSc.) in Advanced Chemical Engineering in 2001 from the University of Londonand a Diploma of Imperial College, (DIC.) from Imperial College of Science, Technologyand Medicine, London. He completed his Doctorate Degree, (PhD.) in 2005 in the areaof Biochemical Engineering from the University of London where he was attached as aResearch Associate at the Advanced Centre for Biochemical Engineering, (ACBE) University College London, United Kingdom. He is now a Senior Lecturer at the School ofChemical Engineering, Universiti Sains Malaysia.ii

ContentsPreface to the First ManuscriptiAbout the AuthorsiiContentsiii1 Batch and Continuous Cultures1.1 Batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2 Continuous culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.1 Material balance for a continuous cultivation . . . . . . . . . . . .1.3 Advantages and disadvantages of different modes of operation of the stirredtank reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2245.52 Growth Rate: The Kinetics of Cell Growth2.1 Monod growth kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . .773 Measurement of Cell Growth124 Effects of Environment on Cell Growth4.1 Effect of Temperature . . . . . . . . . . . . .4.2 Effect of pH . . . . . . . . . . . . . . . . . . .4.3 Effect of Oxygen . . . . . . . . . . . . . . . .4.3.1 Oxygen Uptake Rate (OUR) . . . . . .4.3.2 Heat Generation by Microbial Growth1515171921215 Viable Cell Growth: The Stoichiometry of5.1 Medium Formulation and Yield Factors . .5.2 Material Balance of Cell Growth . . . . . .5.3 Degree of Reduction . . . . . . . . . . . .Microbial Reactions27. . . . . . . . . . . . . . . . . . 27. . . . . . . . . . . . . . . . . . 28. . . . . . . . . . . . . . . . . . 316 Fed-Batch Culture6.1 Fed-Batch Model Formulation . . . . . . . . . .6.2 Comparison Between Fed-Batch and Continuous6.3 Advantages of Fed-Batch System . . . . . . . .6.4 Application of Fed-Batch System . . . . . . . . . . . . . .Bioreactors. . . . . . . . . . . . .32323434347 Mixing and Mass Transfer367.1 Macro-mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367.2 Micro-mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387.3 Methods for Characterising Mixing . . . . . . . . . . . . . . . . . . . . . . 39iii

8 Oxygen Transfer8.1 Gas-Liquid Mass Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2 Oxygen Transfer Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.1 Oxygen Consumption in Cell Growth . . . . . . . . . . . . . . . . .8.2.2 Factors Affecting Cellular Oxygen Demand . . . . . . . . . . . . . .8.3 Measurement of kl a in Continuous-Stirred-Tank Bioreactor and Airlift Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.1 Continuous-Stirred-Tank Bioreactor . . . . . . . . . . . . . . . . . .8.3.2 Airlift Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . .40404244449 Liquid Mixing9.1 Types of Mixing and Stirrers . . . . . .9.2 Types of Flows in Agitated Tanks . . .9.3 The Mechanism of Mixing . . . . . . .9.4 Power Requirement for Mixing . . . . .9.4.1 Un-gased Newtonian Fluids . .9.4.2 Un-gased non-Newtonian Fluids9.4.3 Gased Fluids . . . . . . . . . .5454565758586060.10 Kinetics of Substrate Utilisation, Product Formation and Biomass Production in Cell Cultures10.1 The kinetics of substrate consumption in cellular growth and enzymecatalysed reaction and their relationship with bioreactor modelling . . . . .10.2 Unstructured Batch Growth Models . . . . . . . . . . . . . . . . . . . . . .10.3 Structured Kinetic Models . . . . . . . . . . . . . . . . . . . . . . . . . . .10.3.1 Compartmental models . . . . . . . . . . . . . . . . . . . . . . . . .10.3.2 Metabolic models . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.4 Product Formation Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . .10.4.1 Unstructured model . . . . . . . . . . . . . . . . . . . . . . . . . .10.4.2 Structured product formation kinetic modelling . . . . . . . . . . .10.5 Microbial and enzyme kinetic models and their applications in bioreactordesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10.5.1 Plug-flow-tubular bioreactor with immobilised enzyme . . . . . . .474751626265676769696971727211 Sterilisation8611.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8611.1.1 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8611.1.2 Heat Sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8712 Bioreactor Design8813 Downstream Processing in Biochemical Engineering13.1 Introduction to downstream processing . . . . . . . . .13.2 Solid-Liquid Separation . . . . . . . . . . . . . . . . . .13.2.1 Filtration/Ultrafiltration . . . . . . . . . . . . .13.2.2 Centrifugation . . . . . . . . . . . . . . . . . . .13.3 Liquid-liquid Extraction . . . . . . . . . . . . . . . . .13.3.1 Introduction . . . . . . . . . . . . . . . . . . . .13.3.2 Fundamental of Liquid-Liquid Extraction . . . .13.4 Cell Rupture/Disruption . . . . . . . . . . . . . . . . .iv.8989909097999999101

13.5 Product Recovery . . . . . . .13.5.1 Extraction . . . . . . .13.5.2 Adsorption . . . . . .13.6 Purification . . . . . . . . . .13.6.1 Chromatography . . .13.6.2 Precipitation . . . . .13.6.3 Electrophoresis . . . .13.6.4 Membrane Separation13.7 Solvent Recovery . . . . . . .13.8 Drying . . . . . . . . . . . . .13.9 Crystallisation . . . . . . . . .1.102102102102102104104104104104104

Chapter 1Batch and Continuous Cultures1.1Batch cultureThere are a number of biochemical processes that involve batch culture/growth of cell.This type of culture requires enough nutrient to maintain the growth. A typical growthprofile is given in the figure below.ExponentialLag phasegrowth phaseStationary phaseDeath phase(b)Number of cells (g/l)(c)(a)Time (min)Figure 1.1: Growth curve of a batch culture. (a) Acceleration phase, (b) Retardationphase and (c) Declining phase.The figure shows an increase of cell at the start of the cultivation (fermentation)process. This is due to the presence of enough nutrient for the cell to grow. At thesame time the amount of nutrient decreases as it being consumed by the cell. Other sideproducts such as carbon dioxide or ethanol is also formed simultaneously.In batch cultures, the cell properties such as; size of cells internal nutrient metabolic functionvary considerably during the above growth phases. No apparent increase of the amountof cell at the start of cultivation, this is termed as the lag phase. After this period (can2

Growth phase Rate of growthCommentsLagZeroInnoculum adapting withthe changing condition (temperature, tPopulation growth changesthe environment of the cellsRetardationDecreasingThe effect of changing conditions appearStationaryZeroOne or more nutrients are exhaustedto the threshold level of the cellDeclineNegativeThe duration of stationary phase and therate of decline are stronglydependent on the kind of organismDeath phaseNegativeCells lyse due to lack of nutrientTable 1.1: Summary of the growth phases shown in Figure 1.1.be between 10 to 15 mins) the number of cells increases exponentially thus, this stage iscalled the exponential growth phase; the cell properties tend to be constant last for a short period of timeThe next stage is the stationary phase where the population of cell achieves it maximumnumber. This is because: all nutrient in the closed system has been used up by the cell. lack of nutrient will eventually stop the cell from multiplying.The final stage of cell cultivation is the death phase. The decrease of the number of celloccurs exponentially which happens when the cell breaks open (lysed). The rate of deathnormally follows the first-order kinetics given by;dN0 kd Ndtwhich upon integration leads to0N Ns e kd twhere Ns is the concentration of cells at the end of the stationary phase and at the0beginning of the death phase and kd is the first order death rate constant. In bothstationary and death phase, it is important to recognise that there is a distribution ofproperties among the cells in a population. A summary of the different phases of cellgrowth is given in Table 1.1.Material balance for a batch cultivationThe balance of a batch reactor is given by the rate of accumulation of product equals tothe rate of formation of the product due to chemical reaction or can be simply written as;d(VR · c) VR · rdtdc rdt3(1.1)

Sterile air oroxygenVFigure 1.2: A batch reactor configuration.where c is the amount of the component and r is the reaction rate. VR in the first line ofthe equation is the total volume of the culture in the reactor. The configuration of simplebatch reactor is given in Figure 1.21.2Continuous cultureBatch and continuous culture systems differ in that, in a continuous culture system,nutrients are supplied to the cell at a constant rate and in order to maintain a constantvolume of biomass in the reactor, an equal volume of cell culture is removed. This willallow the cell population to reach a steady-state condition. The reactor configuration ofa continuous process is given in Figure 1.3.Sterile air oroxygenNutrient InletVBiomass OutletFigure 1.3: A continuous stirred tank reactor (CSTR) configuration.Similar to the batch cultivation, the air is pumped into the culture vessel through asterile filter. Bubbling of air provides: supplying air for the growth of aerobic culture4

it also circulate and agitate the culture pressurise the head space of the culture vessel such that to provide a force duringthe removal of the media (and cells) from the vessel for analysis (OD, cell viabilityetc.).However it is highly difficult to control the delivery of the nutrient and the removal of thecell so that equal amounts of medium is maintain in the vessel. This can be tackled bychanging the configuration of the reactor into a semi-continuous or fed-batch type reactor.The rate of flow of medium into a system of continuous culture is known as thedilution rate. When the number of cells in the culture vessel remains constant overtime, the dilution rate is said to equal the rate of cell division in the culture, since thecells are being removed by the outflow of medium are being replaces by an equal numberthrough cell division in the culture.1.2.1Material balance for a continuous cultivationSimilar to that of the batch cultivation, the material balance for a continuous culture canbe written as;d(VR · c) Fo co Fi ci VR · r(1.2)dtin order to maintain the volume within the vessel;Fi Fo Fthus,d(VR · c) F (co ci ) VR · rdtdcFdVR (co ci ) r cdtVRdt(1.3)for a reactor without a recycle system,dVR 0dttherefore,let the termdcF (co ci ) rdtVRFVR(1.4)denote as D, the final equation leads to,dc D(co ci ) rdt(1.5)where D is the dilution rate of a CSTR cultivation system.1.3Advantages and disadvantages of different modesof operation of the stirred tank reactorBy far, the stirred tank reactor is the most common type of bioreactor used in industry.A summary of the advantages and disadvantages of different kinds of stirred tank reactoris given in Table 1.2.5

6Combines the advantagesof batch and continuousoperation. Excellent forcontrol and optimisationof a given agesHigh labour cost:skilled labour is requiredMuch idle time: Sterilisation, growth ofinoculum, cleaning after fermentationSafety problem: whenfilling, emptying, cleaningOften disappointing: promisedcontinuous production for months fails due to(a) infection, e.g. a short interruptionof the continuous feed sterilisation.(b) spontaneous mutation of microorganismto non producing strain.Very inflexible: can rarely beused for other productionswithout substantial retrofitting.Downstream: all the downstreamprocess equipment must be designed forlow volumetric rate,continuous operation.Some of the advantages of bothbatch and continuous operationbut the advantagesfar outweigh the disadvantages,and fed-batch is used toproduce both biomass(baker’s yeast) and importantsecondary metabolites (e.g. penicillin).Table 1.2: A summary of advantages and disadvantages of different modes of operation of the stirred tank reactor. [Adapted from Neilsenand Villadsen, esVersatile: can be used fordifferent reaction everydaySafe: can be properly sterilised.Little risk of infectionor strain mutation. Completeconversion of substrate is possibleWorks all the time: low labourcost, good utilisation of reactorOften efficient: due tothe autocatalytic nature ofmicrobial reactions, theproductivity can be high.Automation maybe very appealing.Constant product quality.Type of operationBatch

Chapter 2Growth Rate: The Kinetics of CellGrowthIn a usual way, the kinetics of any cellular growth can be simply described by unstructuredmodels. The net rate of the biomass growth is given by µx, where x represents the biomassper unit culture volume and µ is the specific growth rate of the cells with the units ofreciprocal time. This can be written as;rx µx(2.1)and using a similar equation for the continuous stirred tank reactor in equation (1.5) atsteady-state for the cell balance;dx rx D(xo xi )dtrx D(xo xi ) 0(2.2)Dxi Dx µx (D µ)x(2.3)rearranging this gives;since the inlet stream of the continuous culture should be sterile, therefore, xi 0 andD µfrom the above equation. This shows that the cell population in a vessel can be maintainedat a certain level higher than zero given that the above criteria is achieved.2.1Monod growth kineticsThe growth of most of the bacterial cells is in the form of hyperbolic curve. A simplegrowth model describing such a curve was first proposed by Monod in 1942 by linking thespecific growth rate and the concentration of the nutrient used by the cells. The modelis similar to that of the Langmuir isotherm and the famous Michaelis-Menten model ofenzyme-catalysed reactions. It is given

Bioprocess Engineering. The change of name from Bioprocess to Biochemical Engineer-ing shows that the School of Chemical Engineering is very much aware of the current development of the area that combines biology and biochemistry with engineering and technology. The course might have changed its name, however, the core ingredients of