Integrated bioprocesses
Karl Schügerl1 and Jürgen Hubbuch2
Integrated bioprocesses have been developed to optimise
yield and cost-effectiveness of production of low and high
molecular weight molecules. Low molecular weight products
are removed from the cultivation medium with in situ extraction,
in situ adsorption or crystallisation to avoid product inhibition.
One-pot processes are being developed to replace two-stage
reactions. Recent developments in the integrated purification
of high molecular weight products focus mainly on the
integration of solid/liquid separation and initial product
recovery such as expanded bed adsorption or extraction in
aqueous two-phase systems. Additionally, new approaches for
a more efficient processing of inclusion bodies have been
developed.
Addresses
1
Institute for Technical Chemistry, University Hannover, Callinstr. 3,
D-30167 Hannover, Germany
2
Institute of Biotechnology 2, Research Center Juelich GmbH,
D-52425 Jülich, Germany
Corresponding author: Schügerl, Karl (Schuegerl@iftc.uni-hannover.de)
Current Opinion in Microbiology 2005, 8:294–300
This review comes from a themed issue on
Ecology and industrial microbiology
Edited by Sergio Sánchez and Betty Olson
Available online 10th May 2005
1369-5274/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.mib.2005.01.002
Introduction
Recent years have seen a rise in the economic pressure on
the production of biotechnological products. Thus, bio-
processes in general, but especially downstream proces-
sing (i.e. the recovery and purification of the product), are
faced with a strong demand for intensification and inte-
gration of process steps to increase yield, to reduce of
process time and to cut down in running costs and capital
expenditure.
Several new and integrated approaches have been devel-
oped to optimise productivity and cost-effectiveness of
different bioprocesses. For example, recombinant DNA
technology efforts are being undertaken to ease product
recovery by addition of specific tags onto the respective
product molecule and thus facilitating a direct capture
based on high affinity interactions. Several biotechnolo-
gical processes are impaired by product inhibition or by
formation of toxic by-products during cultivation, and
Current Opinion in Microbiology 2005, 8:294–300
sometimes product decomposition during its formation
lessens the productivity and product yield. To avoid these
drawbacks, the product is removed from the reactor
during its formation by a procedure known as in situ
product removal (ISPR). This approach includes opera-
tional steps such as separation of cells from process liquor,
product extraction or conversion followed by re-integra-
tion of cells and supernatant into the cultivation. For
larger molecules, such as proteins, antibodies or DNA
products, a common approach aims to integrate the solid/
liquid separation process with an initial capture or pur-
ification step. The methods used to achieve this task can
be divided into adsorptive techniques, such as expanded
bed adsorption (EBA) and high gradient magnetic fishing
(HGMF), membrane based applications and extraction
using aqueous two-phase systems. Besides the removal of
cells and cell debris from a cultivation broth, the ability to
process solids containing feedstocks has led to a variety of
new unit operations for inclusion body processing. Solids
containing feedstock refers to the fact that processing of
liquids in not hampered by solids present in the feedstock
— of course, within certain ranges.
In recent years several reviews have dealt with recovery of
biotechnological products; some of these reports also
discussed integrated processes [1–4,5,6]. In this review
we discuss reports of new integrated processes, and
additionally we present new approaches for a more effi-
cient processing of inclusion bodies.
Primary and secondary metabolite production
Most of the recent research describes the integration of
processes, especially the ISPR technique, during the
production of low molecular weight compounds. To
achieve a continuous product recovery (i.e. ISPR), tech-
niques based on extraction (i.e. the most commonly used
method), adsorption, electrodialysis and crystallisation
are used. Other integrated processes include integrated
product formation in which the breakdown product of a
polymer is converted to a low molecular compound in a
one-pot process, and integrated extraction and enzymatic
conversion.
In situ product removal (ISPR)
In situ extraction of phenylalanine and conversion products
There have been several recent reports describing the
production of phenylalanine from model-based analysis
through to laboratory to pilot scale processes. The aro-
matic amino acid L-phenylalanine is of pharmaceutical
importance for parental nutrition and represents a neces-
sary building block for the production of the low-calorie
sweetener aspartame.
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Figure 1
Tyrosine QI
O2
Exhaust gas
Glucose
QIC
pH
NH3
QIC
DO
Aeration
Overall process set-up of fully integrated reactive extraction in a technical scale fed-batch process for L-phenylalanine production using
recombinant E. coli. DO, dissolved oxygen; QI, concentration measurement; QIC, concentration measurement and flow control.
Takors and colleagues used model-based analysis and
optimisation of an ISPR approach for L-phenylalanine
production [7,8,9,10]. L-Phenylalanine was produced
by L-tyrosine auxotropic Escherichia coli W3110-4(pF20)
mutant in a fed-batch process. The product was in situ
extracted by di-2-ethylhexil-phosphoric acid (DEHPA)
in 10% kerosene and 1 M sulphuric acid in a hollow fibre
module (Figure 1). The L-phenylalanine titre was
increased from 30 g/l to 110 g/l by means of in situ
extraction. This process was performed in a 300 l reactor
with E. coli F4/pF81 auxotropic strain with the same
extractant, but the phase separation was performed by
centrifugal extractions. The reactive extraction process
was mathematically modelled [7,8,9,10].
Pursell et al. [11] extracted phenylalanine by Aliquate 336
(tri-octyl-methylammoniumchloride) and the extent of
extraction at equilibrium was modelled [11].
Stark et al. [12] described the extractive bioconversion of
2-phenylethanol from L-phenylalanine. L-phenylalanine
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Integrated bioprocesses Schügerl and Hubbuch 295
QI
CO2
1 M H2SO4
QIC
Glucose
Organic NaOH
cycle
Permeate 10% D2EHPA
Kerosene
L-phe slurry
Current Opinion in Microbiology
was converted to 2-phenylethanol by Saccharomyces cere-
visiae in fed-batch culture. Because of the toxicity of the
product, phenylethanol was extracted in situ with oleic
acid, which formed a separate phase in the reactor. The
product enriched in the oleic acid phase to 24 g/l and the
average production rate was increased to 0.26 g/l per hour
[12]. The final concentration of 2-phenylethanol was
increased from 3.8 g/l (with direct contact of the cells with
the oil phase) to 5.6 g/l with encapsulated solvent [13].
In situ extraction of other systems
The optimisation of in situ extraction processes has been
reported for several other systems. Lactic acid was pro-
duced by Lactobacillus rhamnosus subsp. rhamnosus in
plastic-composite-support biofilm reactors and the pro-
duct was extracted across the iso-octane, tributylpho-
sphate containing solid supported disc-shaped liquid
membrane [14]. Butyric acid was produced by Clostridium
tyrobutyricum in a hollow fibre module and the product was
removed by in situ extraction with Alamine 336 in oleyl
alcohol. The extractant (Alamine 336) was simulta-
Current Opinion in Microbiology 2005, 8:294–300
296 Ecology and industrial microbiology
neously regenerated by stripping with NaOH in a second
hollow fibre reactor [15].
Immobilised lipase was used for the enantio-selective
hydrolysis of S-Naproxen from racemic naproxen thioe-
sters, and S-naproxen was in situ extracted by isooctane
trioctylamine solvent [16]. Citric acid was produced by
Aspergillus niger and recovered by extraction with Hos-
tarex in oleyl alcohol simultaneously, but not with an
ISPR technique [17].
In situ adsorption and ion exchange
In ion exchange chromatography, the product is adsorbed
on an ion exchange resin and subsequently desorbed by a
pH or temperature change. Barboza et al. [18] studied the
kinetics of clavulanic acid recovery by ion exchange
chromatography. Clavulanic acid was adsorbed on Amber-
lite IRA400 at 10 8C and desorbed at 30 8C in situ.
In a one-pot reaction, 7-aminodeacetoxy cephalosporanic
acid (7-ADCA) was produced by enzymatic hydrolysis
from adipyl-7-ADCA and converted simultaneously with
phenylglycine methylester to cephalexin. The product
was removed by in situ adsorption after cephalexin formed
a complex with b-naphtol [19].
L-phenylalanine was converted to 2-phenylethanol by S.
cerevisiae in fed-batch experiments and the product was
absorbed in situ in an organic solvent (dibutysebacate)
entrapped into a polymeric matrix of polyethylene [20].
The 2-phenylethanol was back extracted from the com-
posite resin yielding a concentrated product.
Red pigment was removed by in situ adsorption from the
solid state and submerged cultivation of the fungus
Monascus [21].
In situ electrodialysis
Two recent papers described the use of in situ electro-
dialysis to achieve continuous product recovery. Pyruvate
was produced by recombinant E. coli YYC202 IdhA:Kan-
strain and the product was removed after cell retention
with in situ electrodialysis [22]. In situ electrodialysis was
also used to recover lactic acid produced by Lactobacillus
rhamonosus IFO 3863 [23].
In situ crystallisation
Buque-Taboada et al. [24] have shown that in situ product
removal using a crystallization loop has improved produc-
tion of 6R-dihydro-oxoisophorone (DOIP). During asym-
metric reduction of 4-oxoisosphoroe (OIP) to DOIP using
bakers yeast, actinol, an unwanted by-product, is formed
by further degeneration of the target molecule. By estab-
lishing an integrated fermentation-crystallisation process,
this degradation process could be significantly reduced to
4% whereas DOIP productivity was improved by 50% and
selectivity rose from 87% to 96% [24].
Current Opinion in Microbiology 2005, 8:294–300
Integrated product formation
The decomposition of a polymer (starch, cellulose, hemi-
cellulose, protein) and the conversion of the monomer are
performed sometimes in a one-pot process. Such a process
is the simultaneous saccharification of corncobs by the
enzyme of Trichoderma reesei and the conversion of the
product by L. rhamnosus to lactic acid [25,26].
Integrated extraction and enzymatic conversion
A multistage process is the extraction of penicillin G from
the cultivation medium by Amberlite LA-2 at pH 5 in a
hollow fibre module, its re-extraction at pH 8 in another
coupled hollow fibre module and its conversion by peni-
cillin G-amidase (PA) at the same pH 8 to 6-amino
penicillanic acid (6-APA) and phenyl acetic acid (PAA).
Subsequently, PAA and 6-APA are separated in a multiple
electrodialysis cell and 6-APA is converted with phenyl-
glycine ethyl ester (PGE) by immobilised PA at a pH 6 to
ampicillin [27,28,29] in a tubular reactor. All four mod-
ules are connected in-line and operate in a continuous
mode.
High molecular weight products
Adsorptive techniques
Currently, the two most promising adsorptive techniques
for direct product recovery from crude feedstocks are
expanded bed adsorption (EBA) and high gradient mag-
netic fishing (HGMF). EBA is based on the fluidisation of
special designed porous adsorbent resins with a high-
density core. The controlled fluidisation increases the
free space within the bed to allow cells and cell debris to
flow freely through the bed while at the same time
product is recovered similar to a traditional chromato-
graphic process. Biochemical engineering aspects, strate-
gies for process development and new applications of this
technique can be found in a recent review [30].
HGMF is based on batch adsorption of the product
followed by a recovery process of the product-loaded
adsorbent using high gradient magnetic separation [31].
In contrast to chromatographic resins, magnetic adsor-
bents are generally micron-sized and non-porous, and
thus combine a high surface to volume ratio with reduced
fouling tendency. Comparative studies between these
techniques indicate that HGMF expresses a faster
(4–8-fold) and more robust processing (uncoupling of
product capture and separation from the feedstock),
whereas EBA shows the advantage of higher resolution
and probably lower buffer consumption (multistage col-
umn procedure) [32].
Expanded bed adsorption (EBA)
The applicability of molecular engineered tags to facil-
itate downstream processing was shown in a study in
which a highly charged basic domain was used as a tag
for enhanced ion-exchange performance [33]. Using this
methodology, DNA polymerase and viral protease could
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be purified in a single step from E. coli using the cation
exchanger Streamline SP.
The same adsorbent type was used in a study in which
recombinant human calcitonin has been expressed in
Staphylococcus as part of a secreted fusion protein [34].
The integrated operation allowed a single step recovery
and was successfully transferred to pilot scale (100 l). In
further studies, glutathione S-transferase and human epi-
dermal growth factor were directly purified from E. coli
homogenate [35,36]. Finally, it was shown that EBA
performance could be greatly enhanced by optimising
cultivation strategies [37,38] as well as homogenisation
conditions [39,40].
Besides ion-exchange systems, Protein A based affinity
interactions gain more and more attention. Protein A
Streamline adsorbents were thus used for the direct
recovery of antibodies from hybridoma cell cultures
[41], transgenic tobacco plants [42] as well as recombinant
b-secretase coupled to an FC part of human IgG [43].
High gradient magnetic fishing (HGMF)
The applicability of HGMF processes has been recently
shown in systems using affinity adsorbents to recover
trypsin from crude porcine pancreatin [31] and lectins
from legume extracts [44], as well as applications based on
ion-exchange interactions for the recovery of lactoferrin,
lactoperoxidase and superoxide dismutase from whey
[45–47].
Inclusion body processing
Conventional processes for the recovery and conversion
of inclusion bodies into renatured proteins involve several
centrifugation, washing, solubilisation and refolding
steps, and thus lead to an inefficient process because of
the large number of process steps involved. It has, how-
ever, be shown that chemical extraction and simultaneous
solubilisation can be carried out directly in the crude E.
coli process liquor [48]. Despite the resulting highly
complex mixture (i.e. high viscosities because of the
8 M urea, free DNA molecules, cell debris and aggregates
and insoluble inclusion bodies), this approach opened the
door for more sophisticated process routes. Starting from
chemical extraction in the crude cultivation liquor three
possible approaches are discussed. First, direct recovery
of denatured inclusion bodies — all furnished with a poly-
histidine tag — from the crude feedstock using immobi-
lised metal affinity interactions in an EBA [49–51] or
HGMF system [52]. This resulted in recovery yields
between 60–70% and achieved purities of 89% and
95%, while at the same time replacing multiple centri-
fugal steps and reducing buffer consumption signifi-
cantly.
A second approach focused on the renaturing of dena-
tured protein by extensive dilution in a static mixer
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Integrated bioprocesses Schügerl and Hubbuch 297
followed by EBA [53]. This prevents clogging of the
subsequent capture step — traditionally packed bed
adsorption — by inactive or insoluble inclusion bodies.
Finally, recent studies showed the feasibility of refolding
of the denatured protein captured on the EBA column
before its elution [54,55]. By combining chemical extrac-
tion with such an EBA step, numerous conventional steps
described above could be reduced to chemical extraction
followed by a single EBA step. In the latter study [55], a
comparison showed that packed bed mediated and EBA
(expanded bed adsorption) mediated processes led to 1.7-
fold and 4.3-fold higher yields, respectively, than a solu-
tion-based refolding procedure.
Economics
A study comparing economics of EBA-based processes
with traditional approaches coupling centrifugation and/
or filtration with packed bed chromatography was under-
taken using IgG as model protein [56]. The study clearly
revealed an economic advantage of about 50% when
using EBA technology. It was, however, pointed out that
already existing conventional large-scale equipment
might lead to a considerable decrease of this advantage.
The major cost factor that determines process economy is
found in the cost and lifetime of resins used.
The cost and lifetime of resins was also topic of a second
study that took reduced resin capacity as a result of cell/
adsorbent interactions and reduced resin lifetime due to
harsher CIP (cleaning in place) conditions into considera-
tion [57]. In this study, a minimal economical threshold of
resin capacity and lifetime compared with packed bed
resins could be identified.
Membrane-based separations
Integration of cell separation and protein isolation from
mammalian cell cultures was demonstrated using a mem-
brane with lysine as an affinity ligand combined with a
rotating disc device to prevent membrane fouling [58]. In
this study, tissue plasminogen activator could be recov-
ered from CHO cell cultures as particular free solution
yielding 86% of the target protein while at the same time
causing 95% reduction of the bulk protein.
Aqueous two-phase systems
Extraction of proteins from crude feedstocks is another
approach combining solid/liquid separation, product con-
centration and a modest purification in one step. Aqueous
two-phase systems provide a suitable environment to
maintain biological activity and protein solubility [59].
Extraction has advantages at high biomass concentration
[60] and for the ease of scale up. Recent work focused on
the application of thermoseparating systems exploiting
either the phase behaviour of certain nonionic detergents
[61] or by substitution of PEG with EOPO in combina-
tion with a second hydrophilic polymer [62,63]. The
extraction of modified cutinase and a hydrophobin-endo-
Current Opinion in Microbiology 2005, 8:294–300
298 Ecology and industrial microbiology
Figure 2
E. coli
Tris buffer pH8, EDTA
NaOH,SDS
Potassium phosphate
buffer pH7
PEG
Alkaline lysis
Neutralization
Integrated purification process for the recovery of pDNA from E. coli based on aqueous two phase separations as presented by Frerix et al. [68]. The
SDS PAGE gel shows the obtained separation of pDNA and RNA into bottom and top phase.
glucanase fusion was optimised and scale up demon-
strated to 500 l and 1200 l, respectively [63,64]. Deter-
gent-based systems offer the advantage that a single
auxiliary chemical is needed at a rather low concentration
to achieve phase formation. The approach requires a
certain hydrophobicity of the target protein to give high
yields in a single step extraction. This may be engineered
with a fusion partner [65] or it exploits a given property in
case of membrane-associated proteins [66].
PEG in combination with potassium phosphate showed
to be a suitable system for selective extraction of plas-
mid DNA [67,68]. Phase systems with composition of
15% PEG 800 and 20% potassium phosphate at pH 7.0
showed a strong partitioning of plasmid DNA to the
bottom phase linked to a clear reduction of open circular
pDNA, whereas proteins, gDNA and RNA remain in
the top or interphase. A striking advantage of the
current process can be seen in Figure 2, namely that
the complete procedure of lysis, precipitation, clarifica-
tion and extraction can be done in one vessel [68]. In
various other studies that exploit PEG/salt systems, it
was shown that recombinant cutinase furnished with a
tryptophan–proline peptide tag could be directly recov-
ered from E. coli homogenate using a PEG-phosphate
system [69,70]. In combination with hydrophobic inter-
action chromatography a PEG-free phase with an overall
yield of 83% and final purity of 95% could be obtained
[70].
Conclusions
The productivity, yield and economy of bioprocesses can
be considerably improved by process integration. Besides
these advantages, integrated processes still wait for broad
industrial application because of their higher complexity,
and thus there is a need for detailed process knowledge of
the applicant. A solution to this could lie in an increase of
process robustness, easy strategies for process develop-
ment and trouble shooting and better education of
employees. Besides the use of integrated processes, a
Current Opinion in Microbiology 2005, 8:294–300
pDNA
RNA
Extraction
directed process development towards a higher degree of
parameter optimisation at reduced development time
could lead to better process performance. This approach
could be simplified by the introduction of high-through-
put screening (HTS) techniques as a tool in early process
development. This approach promises a high level of
acceptance in industry on the condition that tools for
an automated screening are widely available, the majority
of unit operations in downstream processing can be
described by finite bath or simple flow-through experi-
ments and a much larger experimental basis for process
development is obtained.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
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