Table Of ContentKarl Schiigerl
Solvent Extraction
in Biotechnology
Recovery of Primary
and Secondary Metabolites
With 130 Figures and 48 Tables
Springer-Verlag Berlin Heidelberg GmbH
Prof. Dr. Dr. h. c. Karl Schiigerl
University of Hannover
Institut fiir Technische Chemie
CallinstraBe 3
30167 Hannover I FRG
ISBN 978-3-642-08190-3
Library ofCongress Cataioging-in-Publication Data
SchUgerl, K. (Karl)
Solvent extraction in biotechnology : recovery of primary and sencondary metabolites IKarl
SchUgerl.
Includes index.
ISBN 978-3-642-08190-3 ISBN 978-3-662-03064-6 (eBook)
DOI 10.1007/978-3-662-03064-6
1. Extraction (Chemistry) 2. Biotechnology--Methodology. 3. MetaboIites--Separation. 1. Title.
TP248.25.E88S38 1994 660'.284248--dc20
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Preface
Two excellent books on extraction are available:
Handbook of solvent extraction T.e. Lo, M.H.!. Baird and e. Hanson, eds.,
John wiley & Sons, New York, 1983, and
"Science and Practice of Liquid-Liquid Extraction" by J.D. Thornton, ed.,
Clarendon Press Oxford, 1992, which also consider extraction in biotechnology,
but in only one chapter. On the whole, however, these chapters cover only a very
small part of relevant biotechnological aspects.
On the other hand, several books deal with separation in biotechnology:
E.g.,
"Bioseparations. Downstream Processing for Biotechnology" by P.A. Belter,
E.L. Cussler and W.S. Hu, John Wiley & Sons, New York, 1988,
"Separation and Purification Techniques in Biotechnology" by F.J. Dechow,
Noyes Publications, New Yersey, 1989, and
"Separations for Biotechnology" M.S. Verral and M.J. Hudson, eds., Ellis
Horwood Ltd., Chichester, 1987.
However, none of them treat extraction in detail.
Also excellent handbooks:
"Biochemical Engineering and Biotechnology Handbook" by B. Atkinson and
F. Mavitune, The Nature Press, New York, 1983, and
"Biotechnology of Industrial Antibiotics" E.1. Vandamme, ed., Marcel Dekker,
Inc., New York, 1984,
consider only special cases of extraction and do not go into detail.
On account of our laboratory and pilot plant experience with the recovery of
primary and secondary metabolites by extraction and the invitation of the
Springer Verlag to participate in publishing with an actual topic, I decided to
write this book. Particular attention was paid to modern extractants with high
selectivity and modeling of extraction processes.
This volume should provide the reader with a comprehensive overview of
this subject and reference material for students of biotechnology and biochemi
caljbioprocess engineering.
Hannover, March 1994 Karl Schiigerl
Contents
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 Reaction Engineering Principles................................ 2
2.1 Definitions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.1 Mass Action Law Equilibria.............. .. .. .. .. ... . .... . . .... 2
2.1.2 Phase Diagrams............................................... 6
2.1.3 Diffusion and Mass Transfer... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10
2.1.4 Stagewise Counter-Current Multiple Contact
with a Single Solvent .......................................... 12
2.1.5 System Models................................................ 14
2.1.6 Symbols for Sect. 2.1 . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 16
2.2 Mechanisms of Mass Transfer Between Two Phases. . . . . . . . . . . . .. 19
2.2.1 Symbols for Sect. 2.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21
2.3 Mass Transfer During Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22
2.3.1 Mass Transfer Within the Droplet. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22
2.3.2 Mass Transfer in the Continuous Phase. . . . . . . . . . . . . . . . . . . . . . . .. 30
2.3.3 Resistances of Both Phases under Consideration. . . . . . . . . . . . . . . .. 34
2.3.4 Comparison of the Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36
2.3.5 Symbols for Sect. 2.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 42
2.4 Experimental Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 44
2.4.1 Holdup and Drop Size Distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 44
2.4.2 Mass Transfer Coefficient of Single Drops. . . . . . . . . . . . . . . . . . . . . .. 45
2.4.3 Residence Time Distributions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 49
2.4.4 Symbols for Sect. 2.4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51
3 Extraction Equipment......................................... 52
3.1 Centrifugal Extraction Equipment.............................. 52
3.2 Extraction Columns........................................... 58
4 Extraction of Metabolites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 66
4.1 Recovery of Alcohols from Fermentation Broths. . . . . . . . . . . . . . . .. 66
4.2 Extraction of Aliphatic Carboxylic Acids. . . . . . . . . . . . . . . . . . . . . . .. 72
4.3 Extraction of Aromatic Carboxylic Acids. . . . . . . . . . . . . . . . . . . . . . .. 91
4.3.1 Symbols for Sect. 4.3 ........................................... 111
4.4 Extraction of Amino Acids ..................................... 113
VIII Contents
4.4.1 Symbols for Sect. 4.4 ........................................... 125
4.5 Recovery of Antibiotics from Fermentation Broths ............... 125
4.6 Recovery of Penicillin from Fermentation Broths ................. 129
4.6.1 Symbols for Sect. 4.6 ........................................... 183
4.7 Integrated Processes ........................................... 184
4.8 Separation of Complex Mixtures ................................ 190
References .......................................................... 198
Subject Index ....................................................... 209
1 Introduction
Cultivation media for microorganisms consist of carbon-, nitrogen-, phosphate
sources and nutrient salts with trace elements and vitamins. In laboratories,
synthetic media with well-defined composition can be used. In industrial pro
duction, complex media consisting of agricultural (by)products and wastes
(molasses, pharma medium, cornsteep liquor, yeast extract, soybean meal,
peanut flour, cottonseed meal, fish meal, etc.) with poorly defined composition
are employed. During the cultivation and production process, the medium
components are consumed and converted into other compounds. The aim of the
downstream processing is the recovery and purification of the valuable products
from this complex fermentation broth.
Primary and secondary metabolites are excreted into the cultivation broths
by the microorganism. Their recovery can be performed by different techniques:
crystallization (e.g., tetracycline), evaporation (e.g., ethanol) or after removal of
the cells and solid particles (by means of filtration or centrifugation) by precipi
tation (e.g., Ca-salt of citric acid and lactic acid), fractional distillation (e.g.,
acetone/butanol), adsorption (e.g., cephalosporin C), ion exchange (e.g., amino
acids), ultrafiltration (e.g., acetic acid), electrodialysis (e.g., lactic acid) or solvent
extraction.
Solvent extraction is the most called-for method for the recovery of hy
drophilic substances, and, therefore, a method for separating well-soluble
metabolites from the cultivation medium. The classical extraction processes use
organic solvents, which are often rarely suitable for effective recovery of the
solute. Recently, new extractants were developed which form specific adducts
(complexes or compounds) with a metabolite in question and allow its recovery
with high efficiency and selectivity.
After considering the reaction engineering principles of extraction in
Chapter 2, and extraction equipment in Chapter 3, the extraction of primary
and secondary metabolites with different extractants are treated in Chapter 4.
Besides solvent extraction, novel separation techniques with liquid membrane,
microemulsion, and reversed micelles are also presented.
2 Reaction Engineering Principles
2.1 Definitions
2.1.1 Mass Action Law Equilibria
Equilibrium partitioning of nonelectrolytes between two immiscible phases is
achieved, when the chemical potential of the solute is equal in the two phases
J.la - J.lo, (2.1)
where the potential in the aqueous phase is given by
J.la = J.l~ + RTlnxaYa (2.2)
and that of the organic phase by
J.lo = J.l~ + RTlnxaYo (2.3)
In Eqs. (2.2) and (2.3), J.l~ and J.l~ are the standard potentials, Xa, Xo are the mol
fractions and Ya, Yo are the activity coefficients in the aqueous and organic
phases. At low mol fractions (usually below 0.1 moll-1) the activity coefficients
are unity (ideal solutions),
(2.4)
The standard energy of the transfer of the solute is given by
AG~ = J.lo - J.la = - RTlnPx, (2.5)
where
(2.6)
is the mol fraction based partition coefficient. The molar concentration based
partition coefficient
P =~ (2.7)
c ,
Ca
where Co and Ca are the molar concentrations of the solute (moll-1) in the
organic and aqueous phases, and the solute mols and phase masses based
2.1 Definitions 3
partition coefficient are
(2.8)
where Wo and Wa are the molar concentrations of the solute with regard to the
mass of the phases (mol kg~ 1) and are more frequently used than the mole
fraction based partition coefficient. The relationship between these partition
coefficients are
(2.9)
(2.10)
where Va, Vo are the molar volumes and Pa, Po are the densities of the aqueous
and organic phases.
Since the mutual solubilities of the aqueous and organic phases are usually
not negligible, the two phases have to be mutually saturated for the determina
tion of the partition coefficients. When using the molar concentration based
partition coefficient Pc, the standard free energy of the solute transfer is given by
LlG~ = - RTlnPc = LlG~ + RTln(vo/va), (2.11)
where vo' Va are the molar volumes of the organic and aqueous phases.
The distribution ratio Dc is often used in literature
(2.12)
where Cot, Cat (moll ~ 1) are the total measured solute concentrations in the
organic and aqueous phases without any correction.
Because of the necessary corrections, Pc and Dc are generally not identical.
Some solutes interact in this way with the solvent (extractant Ex). The
stoichiometry and the equilibrium constant are
Ken
SOa + n Exo ~ (SoExn)o (2.13)
K = [SoExn]o
(2.14)
en [So]a [Ex]o
The square brackets denote equilibrium concentrations in moll ~ 1.
The distribution ratio Dc is given by
D = [SoExn]o + [So]o = -=--[s_o=]o'---+-----::::K-:c:en~[S-O-=-].::....::o['-E---=x]:..;c~
c [So]a [So]a
= Pc(1 + Ken[Ex]~), (2.15)
where
(2.16)
4 2 Reaction Engineering Principles
A plot of log [(Dc/Pc) - 1] vs log [Ex]o yields a straight line with the slope of
n and an intercept of log Ken.
Some solutes (e.g. organic acids HS) dissociate in the aqueous phase
(2.17)
where H+ is the proton and S - the acid anion.
[H+] [S-]
K = [HS] . (2.18)
The partition coefficient is based on the undissociated solute
(2.19)
When the solute forms dimers in the organic phase, the following relationships
hold true:
2HSo ~ (HS)z.o (2.20)
K _ [(HS)z]o
(2.21)
[HS];
di -
The distribution ratio Dc is again based on the total concentrations of the solute
in all its possible forms in the aqueous phase CHAat and in the organic phase
CHAot:
D = CHSot = [HS]o + 2[(HS)z]o = [HS]aPc + 2K [HS];
di
c CHSat [HS]a + [S-]a [HS]a + K[HS]./[H+]a
[HS]aPc + 2K P.?[HS]; Pc + 2P;K [HS]a
di di (2.22)
[HS]a(l + K/[H+ ]a) 1 + K/[H+]a
The Mass Action Law Equilibria for the extraction of monocarboxylic acid
by strong solvating extract ants, such as organo-phosphorous compounds, are
strongly influenced by the dissociation of the acid in the aqueous solution
(2.23)
(2.24)
and by the formation of the acid solvating extractant Ex in the organic phase
Ken
HSa + nExo ~ HSExno, (2.25)
(2.26)
