Bioreaction engineering principles 3rd edition pdf

Did you find this document useful? Is this content inappropriate? Report this Document. Flag for inappropriate content. Download now. Bioreaction Engineering Principles. Related titles. Carousel Previous Carousel Next. Biochemical Engineering Harvey W. Blanch, Douglas S. Jump to Page. Search inside document. The answer 0. Should read p. Ruben Marquez. Nathan Galicia. Vipin Gupta. Abhinay Batchu. Ashish Jha. Vitra Wahyu Pradana. Tushar Patil. Ren Jamal Bo. TSTC Publishing. Biochemistry Laboratory.

Modern Theory and Techniques. Bubble Column Bioreactors. Modeling of the Beer Fermentation Process. Lactic Acid Production. Page 1 Navigate to page number of 2. In Part A, General principles and techniques with regard to reactor and process models, process control, and metabolic flux analysis are presented.

A virtual bioreactor system is presented as well, which can be used for the training of students and operators of industrial plants and for the development of advanced automation tools. In Part B, the General principles are applied for particular bioreactor models.

In the near future, lignin is likely to be combusted to provide process heat. In the following only glycerol and the sugars derived from biopolymers will be considered as building blocks. In , a group of researchers at Pacific Northwest National Laboratory PNNL and National Renewable Energy Laboratory NREL in the USA made a detailed screening of sugars as potential candidates for building blocks, secondary chemicals, and intermediates to produce final consumer goods in the industry sectors which have traditionally been served by the petroleum industry.

As argued by Werpy and Petersen , all 12 building blocks have a high potential for substituting building blocks derived from oil and gas. The first three building blocks are made from sugars by simple chemical processes. Thus, glucaric acid is obtained from D-glucose or D-gulose by oxidation with dilute Table 2. Newer reviews are Haveren et al. The list of 12 building blocks in Table 2. Thus, glutamic acid is the starting point of the biosynthesis of a family of amino acids, including aspartic acid, and the fermentation can be conducted such that aspartic acid or lysine is the final amino acid product.

In Sect. Glucose is shown as the starting point for the synthesis, but other sugars resulting from enzymatic hydrolysis and saccharification of biomass could also have been used. Directly aimed toward polymers is the Cargill-Dow lactic acid to polylactide facility , annual ton production inaugurated in in Blair Nebraska. The production of polymers from biobased raw materials will be further discussed, in this chapter, and in Chaps. It is interesting that together the building blocks in Table 2.

With at least two of these present in a particular building block this is an attractive platform for building other compounds with a C3, C4, C5, or C6 carbon backbone. This property is of course also found in the raw materials, the sugars, whereas ethanol with only one reactive group has much less value as a platform chemical. The reactivity of the building blocks makes it possible, mostly by chemical routes, to synthesize, e. Environmentally friendly 2. Whole families of pharmaceuticals, exemplified by penicillins and cephalosporins, are produced by fermentation.

No chemical route is competitive for these bulk chemicals. Finally, specialty drugs, produced in small quantities but at high unit price, are synthesized by microorganisms.

Therapeutic proteins and polyketides are examples of products that can only be produced by bioroutes. In the following, we shall examine the metabolic pathways that lead from the raw material, sugar, to the desired end products, and a few examples of complete synthesis paths will be discussed.

The goal is to understand the general rules of carbon flow in the main arteries of the immensely complicated metabolic network of any living organism. Nothing in the network structure was developed by nature without serving a purpose. Biochemistry and biology has given us an understanding of the purpose of the major pathways, but we are still a long way from understanding the whole, tightly regulated network.

Learning from the pathways and guessing their interconnections are the way forward to design new organisms that serve our needs better. It is through systematic research where new sets of experiments are devised to obtain a better understanding of the rationale for the observed phenomena that we shall eventually reap the full benefit of what living cells offer to us. Physiological Engineering, Metabolic Engineering, Systems Biology — new names are constantly coined for this scientific endeavor — is a happy marriage between the biosciences and the engineering sciences.

In the perspective of this textbook all the tools discussed in the following chapters serve the purpose of understanding how to exploit as fully as possible the flux of carbon through the pathways and to engineer the fluxes by quantitative means. Ultimately, we shall have the perfect organism, optimized with respect to its performance in the reactor as well as in the subsequent down-stream processes toward the final product.

All the carbon in the substrate can end up in the final product or it may partly be lost through a number of diverging pathways which branch off from the main pathway. Carbon can also be fed into the pathway by converging pathways. Any of the intermediates of the pathway may be excreted from the cell and can be recovered from the medium sometimes called the fermentation broth from which the cell receives its substrates. Both processes occur by transport through the cell membrane.

The final product of the pathway can serve as a substrate for one or more pathways, and the notion of a separate pathway is more or less fictitious in the overall picture of a highly connected metabolic network.

We shall largely use the latter names. In the first pathway to be discussed Sect. No carbon is lost in the form of CO23 When furthermore no metabolic intermediates are drained away from the pathway as substrates in other reactions, one obtains two molecules of pyruvic acid4 from one glucose molecule.

In the following Sect. The change in free energy DGR for each reaction must be negative. As will be discussed in Chap. When we write stoichiometries e. The need to add acid or base to keep the desired pH is implicitly assumed.

The tri-ester with H3PO4 is formed at the 50 carbon of the sugar. The ribophosphate structure of ATP is also found in NADH, but one of the phosphate groups is substituted with another nucleotide where nicotine amide 2 is attached to the 10 carbon of the C5 sugar. D that is accompanied by a sufficient loss of free energy to make the sum of the two reactions occur with a negative DGR.

In cell reactions, the reaction C! D is in most cases the hydrolysis of an energyrich compound adenosine triphosphate ATP , Fig. B to proceed against its thermodynamically preferred direction as long as DGA! B Ysx 0. The yield coefficient of the missing carbon is shown in the column Ysp1 of the table. Thus, for both cases the degree of reduction for P1 is close to 6. There is good reason to believe that P1 is ethanol.

A loss of carbon of this magnitude is of course unacceptable. Taking the averages Example 3. It is an enormously important product from the bioindustry 1. As carbon source sucrose, cane juice, sugar beet waste, corn steep liquor, and many other cheap substrates are used. The most common producing organism is Aspergillus niger, but other organisms such as yeast have been applied. Production is usually by fed-batch fermentation Sect.

The optimal pH is between 1. Now the cheap bulk chemical is produced in the Far East and in India. A well-designed citric acid fermentation process can give a carbon yield of 0. A reasonable guess is that P1 is another TCA cycle metabolite. Also 2 CO2 would have been lost in the process, which would not give a combined degree of reduction of 3 for P1.

An obvious possibility is that some of the precursors to citric acid have been used to make other products in a process that could formally involve decomposition of 0. The database also refers to recent literature in which the enzyme is studied. It is quite likely that this enzyme which is almost completely inactive at the low pH of 2. The presence of even small amounts of the toxic oxalic acid in citric acid used as a food preservative is unacceptable, and industry carefully monitors and controls the pH at 2.

An inherently safe method of avoiding oxalic acid contamination is to knock out the single gene for oxaloacetate hydrolase in Aspergillus niger. A gene deletion in the production organism does not prevent the use of the product citric acid in food. Among several gene modification studies of A. Bioremediation has a completely different 3. Although the design of bioreactors for this purpose follows the same principles as those applied in Chaps.

Hence, subjects relevant to bioremediation will only receive cursory attention, mostly in the form of problems and examples in the different chapters. Still, the application of the basic material of this chapter can be valuable for Environmental Engineers who might recognize that some of their design concepts are based on the general framework of mass and redox balances, although the nomenclature and approach in Environmental Biotechnology often looks different.

The removal of cellulosic material from the environment by microbial action is easy to model: Cellulose is degraded to sugars or to fatty acids. In an aerobic waste water plant these compounds are degraded to CO2. In anaerobic reactors the fatty acids are converted to the final products CH4 and CO2 by redox-neutral processes.

Thus, by Metabolite Balancing one obtains, e. Sometimes CO2 can act as a redox sink, and it is reduced to CH4, but a potent energy source such as H2 must be present the H2 ends up as H2O to overcome the large positive DG of the reaction see Example 4. The presence of microorganisms that allow sulfate or nitrate reduction will be treated below. At first the true Metabolite Balancing situation will be discussed.

It will be seen that quite complicated calculations on anaerobic bioremediation reactors can be done in analogy to the examples in 3. Note 3. The major goal of domestic waste treatment is to reduce BODn. This is defined as the amount of O2 required to oxidize all the reduced matter solids or solutes in the sample of waste water to redox neutral compounds with the help of a consortium of microorganisms, either naturally present or added as standards.

BOD is measured in a sealed reactor at 20 C, and the index n refers to the duration of the experiment. The dissolved oxygen level in the reactor is measured before and after the experiment to calculate the amount of O2 used. A text on waste water treatment or environmental microbiology, e. For our purpose, we only need to 94 3 Elemental and Redox Balances consider BOD as the amount of O2 measured in units of kg used by the consortium of microorganisms in the anaerobic waste water plant to convert the organic compounds in the waste water to CO2, H2O, Volatile Fatty Acids usually HAc , and gaseous metabolic products such as CH4.

An example will show how mass balances can be set up and solved for the plant, and BOD is used to express the redox level of the substrate and the products. The feed to an anaerobic waste water plant operating as a continuous, stirred tank reactor is a mixture of animal fats represented by tri-palmitin, C51H98O6.

The microorganisms represented as CH1. The output streams from the digester are 1. A gas composed of CH4 and CO2. A liquid effluent which contains no residual fats. For simplicity the liquid effluent is assumed to contain only HAc. Calculate the rates of production of CO2 and CH4, and determine the stoichiometry of the total bioreaction. Consequently, Effluent stream 3: kg biomass as CH1,. The NH3 consumed is consequently 0. Under strictly anaerobic conditions certain obligate anaerobic bacteria can use inorganic ions as electron acceptors.

One such group of microorganisms is the sulfur-reducing bacteria that are widespread in Nature. The sulfur compounds are 3. Typical sulfur-reducing bacteria such as Desulphobacter and Desulphovibrio do not incorporate the sulfur and convert it to sulfur-containing amino acids such as cysteine , but run the sulfur reduction process in parallel with cell growth on a carbon source.

Much lower biomass yields are of course desired, and 0. Here, the waste water treatment unit removes nitrate at the expense of a cheap carbon source, e. Some denitrifiers e. Needless to say, there are also naturally occurring microorganisms that oxidize N and S containing compounds, e. Here oxygen is a substrate, and both biomass formation and oxidation of the inorganic compound are energetically favored.

In removal of nitrogen from waste water an aerobic bioreactor is coupled in series with an anaerobic reactor. Problem 3. Sulfide-oxidizing bacteria are used in the mining industry to access, e. The yield coefficients could be calculated manually using a carbon — and a degree of reduction balance. In the general case with N substrates and M metabolic products, some of which might contain nitrogen, a more systematic procedure to calculate the stoichiometry is, however, needed.

For a standard biomass y is 1 for carbon, 1. If an element does not appear in the compound y is zero, e. Equation 3. In each column the elemental composition of one of the reaction species is written, i. Now 3. If other elements, such as S, appear in some of the reactant compositions cystein would be a case in point the extension of 3. Typical cases where the procedure fails due to a singularity in Ec is when substrates or products with the same composition per C-mol are included in Ec, e.

The concept of the systematic procedure is illustrated in Example 3. Consider anaerobic fermentation of S. Let the carbon source be glucose and the nitrogen source NH3. The biomass composition is CH1. With 7 reacting species and four constraints one needs to measure three rates. The remaining rates can be calculated.

The yield coefficient for NH3 is immediately given to be 0. Basically this is the result obtained in 2. If desired Ysw can be found from an H or an O balance. With NH3 as nitrogen source and a standard biomass composition the stoichiometry of the total reaction is given in 1. Some carbon is lost to the undesired byproduct acetic acid. Four rates can be calculated, and the systematic method 3. Calculations such as these are made as a preliminary to a process design.

It is of first importance to see whether a sufficiently high yield of the desired product can be obtained to make the process economically viable. In this respect, the Black Box model is too crude. In actual lysine production the biomass yield is far from zero — it is in fact higher than for most aerobic processes. Consequently, the theoretical limit for Ysp1 is not at all approached in practice — but carbon yields of 0. Furthermore, several essential amino acids must be supplied, but the metabolic product lysine obtains its nitrogen directly by uptake of the nitrogen source NH3 as discussed in Sect.

It would, however, be a very poor policy to use only the minimum number of rates when fermentation results at a given set of environmental conditions are to be interpreted in terms of a stoichiometric equation. First of all compounds may be missing from the stoichiometric equation as was the case in Example 3.

But even small — and quite unavoidable — experimental inaccuracies will lead to substantial errors in the calculated rates.

The data of Duboc , which were analyzed in Example 3. The sum of yield coefficients of products is 0. Still, a calculation of two yield coefficients Ysx and Ysg based on Ysc, Yse, and using the carbon — and the redox balances leads to significant errors due to the ill-conditioned nature of the linear algebraic problem.

Inserting the round-off values for Ysc and Yse in 3. This is done by least-squares fitting of the coefficients, see also Section 5. But the product of ETc and Ec is a quadratic matrix of order less than four but larger than or equal to zero: 3. Inserting 3. This case will be treated shortly for the general case of L measurements with L bounded as shown above. Usually some of the rates can, however, not be found with satisfactory precision.

Consequently, 3. We now assume that all the measured rates contain random errors. The estimate in 3. In Note 3. Application of 3. When d is normally distributed, the function to be minimized is the same for the least-square minimization problem and for the maximum-likelihood minimization problem.

If the error vector is not normally distributed the estimate in 3. We return to the experimental data of von Meyenburg , which were presented in Example 3. We want to find better estimates for the measured variables. First consider the data for low dilution rates where no ethanol is formed.

In Example 3. At low dilution rates the measured rates are qs ; qo ; qc and qx. There are two nonmeasured rates — ammonia utilization qn and formation of water qw. Thus, with the biomass composition specified in 1 of Example 3. From 3. We will, however, try to obtain even better estimates for the measured rates.

In order to find the variance—covariance matrix, we need to know something about the size of the measurement errors. It is, however, justified to make the small corrections, and we can next calculate the variance covariance matrix for the estimated rates by using 6 of Note 3.

Whereas the errors of the raw measurements are uncorrelated, it is observed from 10 that the errors of the estimated rates are correlated through the constraints in 3. In the analysis we have used the volumetric rates, whereas yield coefficients were used in Example 3.

The present error analysis could, however, just as well be carried out using the yield coefficients simply replace the volumetric rate vector with a vector containing the yields. Normally the variance—covariance matrix is assumed to be diagonal, i. However, the volumetric rates are seldom measured directly, but they are based on measurements of so-called primary variables, which may influence more than one of the measured volumetric rates.

An example is measurement of the oxygen uptake rate and the carbon dioxide production rate, which are based on measurement of the gas flow rate through the bioreactor together with measurement of the partial pressure of the two gases in the exhaust. If there is an error in the measured gas flow rate, this will influence both of the above-mentioned rates, and errors in the measured rates are therefore indirectly correlated.

The same objection holds for measurements of many other 3 Elemental and Redox Balances volumetric rates, which are in reality obtained by combination of a concentration and a flow-rate measurement. In all these cases of indirect error correction it is difficult to specify the true variance—covariance matrix F. Madron et al. The diagonal F matrix is preferable, partly because the calculations are simplified but also because we usually know little beyond the order of magnitude of the measurement errors.

Normally the measured rates are determined from several measurements of socalled primary variables, e. Specification of the variance-covariance matrix is therefore not straightforward, but Madron et al. The accuracy of the computed variances is limited by the accuracy of the linear approximation in 2 involved in the computation of the sensitivities.

Using 3. If any components in the residual vector are significantly different from zero, either there must be a significant error in at least one of the measurements or the applied model is not correct. To quantify what is meant by residuals significantly different from zero, we introduce the test function h given by the sum of weighted squares of the residuals, i. Heijden et al. Table 3. With the data in Example 3.

However, in many cases one may suspect that one of the measurements has a systematic error, and if by leaving this measurement out of the analysis the errors become nonsignificant it is reasonable to assume that this particular measurement contains a systematic error. Thus the approach illustrated above can be used for error diagnosis, but only when the system is over-determined by at least two measurements, i.

Otherwise, when one measurement is left out the system is no longer over-determined, and the analysis cannot be carried out. The concept of eliminating one measurement at a time is very simple, as illustrated in Example 3. A more systematic approach for error diagnosis is found in Heijden et al.

We now consider the data of von Meyenburg for high dilution rates. As in Example 3. We can therefore carry out the error diagnosis described above, i. The results of this analysis give the test function for each case, i. It is evident that when the ethanol measurement is left out, all signs of trouble disappear from the data set. It seems beyond doubt that there is a systematic error in the ethanol measurement, and we can obtain a nice statistical confirmation of the somewhat more qualitative argument of Example 3.

This is identical to the sum of Ysp and the calculated Ysp1 found in Example 3. The method for error diagnosis illustrated here is very simple and quite powerful. It is, however, advisable not to embark on a mechanical error analysis without first using the intuitively simple engineering approach of Example 3. Problems Problem 3. Consider a well-stirred laboratory reactor of volume 2 L used for aerobic continuous cultivation of S. The oxygen volume fraction in vgf is Determine the rate of oxygen transfer qto when the volume fraction of oxygen in the exhaust gas is 0.

Determine the mass transfer coefficient kla. Different models for the degree of mixing of the gas between bubbles should be considered: No mixing completely segregated flow of the gas bubbles , partly mixed, and fully mixed gas phase completely mixed as we would assume is the case for the liquid phase.

The inlet concentration is zero. Also determine the mole fraction of ethanol in the liquid. Problems e From physical chemistry the following relation is obtained between the liquid mole fraction x and the gas-phase mole fraction y.

The activity coefficient for ethanol in water is quite high. Rarey and Gmehling give the value 6. The off-gas should be dried before pO2 is measured. Christensen et al. If a reaction component here CO2 can be easily measured, then measure it. The microorganism Mycobacterium vaccae is able to grow with ethane as the sole source of carbon and energy and with NH3 as the nitrogen source. Except for small amounts of S and P an elemental analysis of dry cell mass is C, The ash content is 4.

Determine the elemental composition of the ash-free biomass and its formula weight per C-atom. Write the stoichiometry for conversion of ethane to SCP. The yield Ysx is that calculated above, but the value of RQ is 0. Show that an extra metabolic product must be formed besides CO2 and H2O. Determine the degree of reduction of this extra metabolic product. Using your general chemical knowledge you should be able to identify the extra metabolic product. In the last step of the pathway phenoxyacetic acid forms an amide with the amino group of 6-APA.

There are two carbon substrates, glucose S1 and phenoxyacetic acid S2 a On a C-mole basis what is the yield coefficient Yps2? Also find RQ. Discuss whether this assumption appears to be reasonable. If the assumption holds, then what would be the effect on the specific penicillin production rate if during the cultivation Yso starts to increase. After h when growth of the Problems fungus has virtually ceased RQ increases rapidly above 1.

Discuss probable reasons for this observation. Are other products formed? Could there be some straightforward experimental error? The CO2 production is measured by a photoacoustic method. The CO2 concentration in the effluent gas in the head space is found to be 2.

On a flow-meter the total gas flow 30 C, 1 atm is determined to 0. Show that the data are not consistent, and calculate the yield coefficient of a missing metabolite. In your opinion what is the source of the error? When growing under Nlimitation but with an excess of the carbon source the organism can build up a large carbon storage that can be used to support active growth when, or if the N-limitation is lifted.

This is part of the clean-up cycle in waste water treatment plants, but PHB has an independent and increasing interest as a biodegradeable polymer — in competition with particularly polylactides. Although PHB is a part of the biomass it can just as well be regarded as a metabolic product. It grows aerobically, and some CO2 is produced together with X and P.

Also calculate the inlet concentrations of acetic acid and ammonia. Nissen et al. TN21 is a transformant of TN1 which expresses the transhydrogenase gene tdh from Azotobacter vinlandii to a high level, 4.

TN26 is also a transformant of TN1 with the same multicopy expression vector as in TN1, but without ligation of the tdh gene. TN26 is consequently genetically identical to TN21, except that it cannot express transhydrogenase. The table below shows product yields from steady state, glucose limited continuous cultivations of the three strains. Note the high accuracy of the data, and also that TN26 behaves exactly as TN1. There are, however, small but significant differences between TN21 and TN Repeat the calculation of a with this assumption.

What could be the reason for the higher yield of 2-oxaloacetate? The dominant cofactor for conversion of 2-oxoglutarate to glutamic acid is NAPDH in all three strains. With a less than perfect aeration there will be anoxic regions within the otherwise aerated reactor, and nitrification aerobic of NH3 to nitrate occurs simultaneously with anaerobic denitrification of nitrate to N2.

Three types of organisms live together: 1. Aerobic organisms that respire the carbon source e. Aerobic organisms that convert NH3 to nitrate using CO2 as carbon source and the oxidation of inorganic N NH3 to nitrate as the energy source.

These nitrifying organisms belong to the so-called chemolithotrophs i. Denitrifying organisms that use, e. The liquid effluent contains 0. The exhaust gas from the plant contains N2 and CO2 if air was used instead of O2 it would be N2 in excess of that in the incoming air. Write the complete stoichiometry for the process. How would the stoichiometry change if the liquid effluent contained no HNO3 while the values of Ysx and Yso remained the same as in the previous case? Consequently, it is interesting to see if Ys;NH3 is higher in the variation of the problem.

A 1 L working volume fermentor is sparged with air composition Determine the respiratory quotient RQ. Calculate the composition of CO2 and O2 in the exit gas. Determine the value of kla in the two experiments. Is it now correct to assume that the true rate of the bioreaction can be calculated based on the composition of O2 in the exit gas?

What happens when vgf is reduced too much? Determine the stoichiometry for the bioreaction. How many redox equivalents are contained in this molecule? What is the redox content of the two molecules?

What is the redox level per carbon atom in benzene? What is the redox content in this molecule? References d Now construct 4-hydroxy phenyl pyruvic acid by addition of pyruvic acid to p-hydroxy benzene. Again find the redox content of the molecule.

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Duboc, P. Systematic errors in data evaluation due to ethanol stripping and water vaporization. Erickson, L. Application of mass and energy balance regularities in fermentation. Goldberg, I. Biology of Methylotrophs, Butterworth-Heinemann, Boston. Heijden, R. Linear constraint relations in biochemical reaction systems: I, Classification of the calculability and the balanceability of conversion rates.

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