Intensification and economic and ecological assessment of a biocatalytic oxyfunctionalization process

Article abstract of DOI:10.1039/b921896c

Bio-based processes are commonly accepted to be environmentally preferable to chemical alternatives. Reasons include high selectivities, the avoidance of heavy metals, and mild reaction conditions. However, ecological benefits and economic viability have to be verified in each case. Oxygenases are a very attractive enzyme class, allowing selective oxyfunctionalization by introduction of molecular oxygen into hydrocarbons at optical purities unparalleled by traditional chemical methods. Here, styrene monooxygenase from Pseudomonas sp. strain VLB120 was used in recombinant Escherichia coli for the production of enantiopure (S)-styrene oxide from styrene. Substrate and product toxicity was attenuated using a two-liquid phase system with bis(2-ethylhexyl)phthalate as organic carrier solvent. By maintaining previously reported productivities for a longer time period, product concentrations were doubled to 36.3 g L _tot^-1 making it one of the most productive biocatalytic oxyfunctionalization processes. This biotransformation was incorporated along with an appropriate upstream and downstream processing into a complete process scheme and compared with chemical process alternatives. Ecological assessment showed the bioprocess to be superior to two chemical process alternatives and slightly inferior to the third one, with solvent use being the most critical factor. However, the bioprocess performed best in terms of production costs ($10.2 kg^-1). This study underlines the importance of a detailed ecological and economic assessment of bioprocesses to verify their sustainability and to identify weak points with respect to environmental and/or economical sustainability.

Full text of DOI:10.1039/b921896c

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Intensification and economic and ecological assessment of a biocatalytic
oxyfunctionalization process†
a
b
b
a
a,c
Daniel Kuhn, Muhammad Abdul Kholiq, Elmar Heinzle, Bruno B u¨ hler* and Andreas Schmid
Received 22nd October 2009, Accepted 12th March 2010
First published as an Advance Article on the web 9th April 2010
DOI: 10.1039/b921896c
Bio-based processes are commonly accepted to be environmentally preferable to chemical
alternatives. Reasons include high selectivities, the avoidance of heavy metals, and mild reaction
conditions. However, ecological benefits and economic viability have to be verified in each case.
Oxygenases are a very attractive enzyme class, allowing selective oxyfunctionalization by
introduction of molecular oxygen into hydrocarbons at optical purities unparalleled by traditional
chemical methods. Here, styrene monooxygenase from Pseudomonas sp. strain VLB120 was used
in recombinant Escherichia coli for the production of enantiopure (S)-styrene oxide from styrene.
Substrate and product toxicity was attenuated using a two-liquid phase system with
bis(2-ethylhexyl)phthalate as organic carrier solvent. By maintaining previously reported
productivities for a longer time period, product concentrations were doubled to 36.3 g Ltot
making it one of the most productive biocatalytic oxyfunctionalization processes. This
-
1
biotransformation was incorporated along with an appropriate upstream and downstream
processing into a complete process scheme and compared with chemical process alternatives.
Ecological assessment showed the bioprocess to be superior to two chemical process alternatives
and slightly inferior to the third one, with solvent use being the most critical factor. However, the
-
1
bioprocess performed best in terms of production costs ($10.2 kg ). This study underlines the
importance of a detailed ecological and economic assessment of bioprocesses to verify their
sustainability and to identify weak points with respect to environmental and/or economical
sustainability.
Introduction
include cofactor dependence, uncoupling, substrate and product
toxicities, substrate uptake, and instability of the biocatalyst.
6
Biocatalysis is emerging more and more in the fine chemical
industry due to the great versatility and specificity biocatalysts
All these issues make technology transfer from a laboratory to
an industrial scale a demanding and time-consuming task.
A systematic assessment supports the scale-up procedure and
also shortens the development time by the early identification
1–3
can offer. Although the number of bio-based processes run-
ning on a commercial scale is steadily increasing, the majority
1
of applications are restricted to a few enzyme classes so far.
The implementation of oxygenases, for example, is restricted
8,9
of potential bottle-necks and disadvantages. At early stages
of the planning phase, the design is still flexible and changes
are thus less cost and time intensive than later on. Nevertheless,
4,5
to only a few large scale processes, despite their widely-
recognized attractiveness as highly regio- and enantiospecific
there are only a few detailed studies assessing the economic and
6,7
catalysts. Such selective oxyfunctionalizations of C–H bonds
are difficult to achieve by traditional chemical means. The
reasons for the confined utilization of oxygenases are divers, but
10–12
the ecological potential of oxygenase-based processes.
Next
to economic parameters, environmental considerations gained
attention and are of equal relevance due to the increasing public
3,13
awareness and stricter legislation. For the quantification of
the impact of processes on the environment, a large number of
a
Laboratory of Chemical Biotechnology, Department of Biochemical and
14–18
assessment tools have been developed over the past decades.
Chemical Engineering, Technische Universit a¨ t Dortmund,
Emil-Figge-Str. 66, 44221, Dortmund, Germany.
E-mail: bruno.buehler@bci.tu-dortmund.de; Fax: +49 231 7557382;
Tel: +49 231 7557384
Simple and flexible methodologies are required for the char-
acterization of processes at early development stages, as, for
example, the quantification of the environmental burden by so
b
Technische Biochemie, Universit a¨ t des Saarlandes, Campus A 1.5,
16,19–21
called environmental indices.
6
6123, Saarbr u¨ cken, Germany
As an example for an efficient oxyfunctionalization bio-
catalyst, styrene monooxygenase containing recombinant Es-
cherichia coli has been extensively investigated (Fig. 1). This
enzyme originates from Pseudomonas sp. strain VLB120 and
proved to be a promising biotechnological tool for the selective
epoxidation of styrene derivatives to (S)-epoxides with enan-
c
Leibniz-Institut f u¨ r Analytische Wissenschaften – ISAS, Otto-Hahn-Str.
6
b, 44227, Dortmund, Germany
†
Electronic supplementary information (ESI) available: Supplementary
Table A: Criteria for ABC classification; Supplementary Table B: ABC
classification of all compounds; Supplementary Table C: Detailed mass-
balances for all (S)-styrene oxide production processes; Supplementary
Table D: Environmental indices for all (S)-styrene oxide production
processes. See DOI: 10.1039/b921896c
22,23
tiomeric excesses over 98% (99.5% for (S)-styrene oxide).
This journal is © The Royal Society of Chemistry 2010
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Table 1 Process performance during biotransformation
33
Parameter
Unit
Park et al.
This study
Biotransformation time
Initial styrene concentration
Final (S)-styrene oxide
concentration
Final 2-phenylethanol
concentration
Cell concentration after batch
Maximal cell concentration
Glucose fed
h
mM
mM
4.5
340
314
8
695
604
mM
69
-
1
1
a
g
g
g
g
CDW
L
L
aq
aq
9
5.4
-
CDW
32
69
10
39.3
113.7
3.6
4.54
6.57
1821
56
a
a
Acetate formed
g Ltot-
1
h
-1
-1
4.19
6.49
1800
60
Average productivity
Max. productivity
-1
g Ltot 1h
Max. volumetric productivity U Laq-
Max. specific epoxidation rate U gCDW-
1
Fig. 1 Styrene epoxidation by recombinant E. coli in a two-liquid
phase system. Styrene monooxygenase (SMO) from Pseudomonas sp.
strain VLB120 selectively oxidizes styrene to (S)-styrene oxide. This
reaction depends on the cofactor NADH, which is regenerated by cell
metabolism. Bis(2-ethylhexyl)phthalate (BEHP) was used as extraction
solvent alleviating substrate and product toxicity.
a
Values estimated from figure.
studies will help to characterize the benefits and challenges
of biotechnology and ultimately facilitate and accelerate the
implementation of sustainable biocatalytic processes in fine
chemical and pharmaceutical syntheses.
Enantiopure epoxides are attractive, high value intermediates,
because of their potential and versatility as building blocks
for the synthesis of many biologically active compounds in
2
4,25
Results and discussion
optically pure form.
(S)-Styrene oxide, for instance, can serve
26
as a precursor for the synthesis of the nematocide levamisole,
Intensification of a biocatalytic styrene epoxidation process
based on recombinant E. coli
27,28
29
analgestics,
or the antiarrhythmic drug mexiletine.
The dependence of monooxygenases on reduction equiva-
lents, typically in the form of NAD(P)H, and their limited
stability outside of microbial cells favor the use of whole
The highest (S)-styrene oxide concentration achieved so far in
biocatalytic two-liquid phase biotransformations with BEHP
30
-1
33
cells as biocatalysts over isolated enzymes. Thus, recombinant
E. coli expressing the styrene monooxygenase genes styAB have
been developed and used showing promising rates for (S)-
as extractive phase was 36.7 g Lorg (Table 1). In order
to fully exploit the economic and ecological potential of a
biocatalytic process, it is necessary to identify limiting factors
on the reaction level and to optimize the process performance to
a maximum. To investigate possible constraining effects such as
substrate limitation and product inhibition, the initial substrate
concentration and the styrene feeding profile were varied. In the
above mentioned biotransformation, 340 mM of styrene were
added to the organic phase, an amount which led to complete
substrate depletion and substrate limitation at the end of the
biotransformation. In order to avoid such a confinement by
styrene availability, its addition was increased stepwise in several
experiments (results not shown). It was found that the most
favorable initial styrene concentration was 700 mM, since this
was the highest quantity still completely converted. Fig. 2 shows
the typical course of the optimized biotransformation.
31,32
styrene oxide formation.
Substrate and product toxicities
were alleviated by in situ product extraction and substrate
supply via a second liquid phase of bis(2-ethylhexyl)phthalate
3
1
-1
(
BEHP). Thereby, final product concentrations of 36.7 g Lorg
-
1
-1
and volumetric productivities of 4.19 g Ltot
achieved during fed-batch cultivation outperforming contin-
uous cultivation by reduced medium requirements.
h
have been
33
34,35
The
33
scalability of this process was positively evaluated at an early
stage by producing 388 g of (S)-styrene oxide at a 30 L
36
pilot scale. Although promising results have been achieved
by using isolated styrene monooxygenase in combination with
3
7
38,39
formate dehydrogenase-based or reductive electrochemical
cofactor regeneration, the process based on growing recom-
33
binant E. coli proved to be superior in terms of produc-
tivity (≥9-fold higher) and achieved product concentration
After addition of the organic phase, the expression of the
styrene monooxygenase genes was induced by octane and
epoxidation activity was already detected within the first hour.
The specific epoxidation activity approached a maximum of
(
≥3-fold higher).
In the present study, this process was intensified with respect
-
1
to product concentration and its economic and ecological
feasibility was evaluated and compared to chemical alternatives.
The latter consisted of three different unselective epoxidation
56 U gCDW after 3 h. These activities are in accordance
33
with previous biotransformations. The maximal activity was
independent of the initial styrene concentration, indicating
the absence of substrate inhibition. This was confirmed by
experiments wherein styrene was fed stepwise, which did not
improve the process performance (results not shown). During
continuous cultivation at lower styrene concentrations the
activities of growing E. coli JM101 (pSPZ10) have been shown
40–42
43,44
reactions
combined with hydrolytic kinetic resolution.
The direct chemical epoxidation leads to unsatisfying enan-
45,46
tiomeric excesses.
This study contributes to the field of quantitative economic
and ecological evaluations comparing biocatalytic and chem-
47,48
34
ical process alternatives.
A greater number of such case
to be limited by the intracellular NADH availability. This can
8
16 | Green Chem., 2010, 12, 815–827
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Fig. 2 Styrene epoxidation by E. coli JM101 (pSPZ10) during two-liquid phase biotransformation. After the batch phase was finished (time -2.83 h),
glucose feed was started. At 0 h, the biotransformation was initiated by the addition of 1 L of BEHP containing 80 ml styrene and 10 ml octane. The
-1
phase ratio between the organic and the aqueous phase was 1 : 1. During the biotransformation, the feed rate was kept constant at 12.38 gglucose
and was not reduced until glucose was detected in the aqueous phase at the end of the biotransformation.
h
also be expected for the biotransformation during fed-batch
cultivation. 2-Phenylethanol accumulated as a by-product at
approximately 10% of the rate of styrene oxide formation as
be feasible and mature for an in-depth ecological and economic
assessment.
22,36
seen in previous studies.
Hydrolysis of (S)-styrene oxide was
Biotechnological (S)-styrene oxide production process
virtually prevented by its extraction to the organic phase to a
level of ~0.1% of the (S)-styrene oxide produced, which is in
accordance to literature.
An economic and ecological assessment relies on the knowledge
of detailed mass-balances over the entire process (for details, see
Experimental part). Therefore, the biotransformation of Fig. 2
has to be combined with suitable upstream and downstream op-
erations. The biocatalytic (S)-styrene oxide production plant was
designed and dimensioned by means of the engineering software
33,37
The specific activity of the cells was maintained above 40 U
-
1
g
CDW , until growth stopped after 5 to 6 h of biotransformation.
At this stage, glucose started to accumulate in the aqueous phase,
despite reducing the glucose feed rate after glucose detection
in the fermentation broth. This, together with the increasing
dissolved oxygen concentration, indicated a reduction of the
cellular metabolic activity, finally leading to cessation of growth
and epoxidation. Styrene limitation can be excluded as a cause
of the activity decrease, since the styrene concentration was still
8
SuperPro Designer version 6.0. This simulation tool allows the
calculation of overall mass-balances by the generation of flow-
sheets. The process scheme of the biotechnological production
process is shown in Fig. 3. On the basis of the production scale
of comparable chiral compounds, the production scale of such
a (S)-styrene oxide production plant was estimated to be 1000
well above the apparent K value of 12.7 ± 1.9 mM in the organic
s
34
phase, when the activities were declining between 5 and 7 h of
biotransformation. The activity loss can be ascribed to the rising
aqueous styrene oxide concentrations, reaching 2.2 mM and
1,2
tons per year. Subsequently the implemented unit operations
are addressed in detail.
The upstream processing comprises the preparation and the
sterilization of the growth medium and precultivation in a seed
fermenter. Sterilization is an important issue in bioprocesses
using genetically modified organisms (GMOs), because con-
tamination by other microbes and release of the GMOs have
to be prevented or minimized, respectively. Prior to sterilization,
the following cleaning-in-place (CIP) procedure is implemented
3
.1 mM after 5 and 7 h, respectively. These concentrations have
33
been shown to heavily affect E. coli growth and metabolism.
The toxification by the product restricts the maximal product
concentration to around 600 mM (S)-styrene oxide. However,
-
1
-1
the productivity was maintained above 4 g Ltot h for 4 h before
toxification, and therefore the final product concentration was
-
1
7
2.6 g Lorg (= 604 mM) and thus nearly doubled compared
in the process scheme. CIP included washing steps with water
to previous experiments (Table 1). This improvement halves the
medium requirements and equipment size.
◦
(
5 min), 0.5 M sodium hydroxide (80 C, 15 min) and again with
water (5 min). Afterwards, the clean fermenters are sterilized in
The high genetic stability of the recombinant strain is
emphasized by the stable biocatalyst performance in the absence
of antibiotics during four successive runs each involving batch
and fed-batch cultivation including biotransformation (results
not shown). Bioprocessing without antibiotics greatly improves
its economic and ecological sustainability.
The avoidance of substrate limitation and the absence of
substrate inhibition allowed the improvement of the biocatalytic
process to an upper limit imposed by product toxicity. This
optimized process meets the performance of the most powerful
oxygenase-based processes described, and thus is considered to
◦
place by steam at 150 C for 15 min.
Prior to the fermentations, the Riesenberg cultivation medium
is heat-sterilized and afterwards complemented with a separately
filtered solution containing glucose, magnesium sulfate, trace
elements and thiamine. 90% of the medium is directly channeled
to the main fermenters and 10% is used for precultivation in the
3
seed fermenter (5.25 m ). The growth of E. coli JM101 (pSPZ10)
is described by the empirical reaction eqn (1).
0.39 C H O + 1.30 O + 0.20 NH → 1.00 CH O N
6
12
6
2
3
1.8 0.5
0.2
+
1.35 CO + 1.75 H O
2
2
(1)
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Fig. 3 Flow-sheet for biotechnological (S)-styrene oxide production. The flow-sheet was generated in the SuperPro Designer software. For details,
see Experimental part. Framed circles describe input mass streams and framed squares output or waste streams.
Precultivation takes 12 h and stops after the depletion of the
carbon source glucose at a cell concentration of approximately
rate is of high importance, since oxygen is not only required for
growth, but also for the epoxidation reaction. Oxygen transfer
-
1
3
5.4 gCDW L . In the main fermenters, the batch phase is started
rates were shown to be sufficient even for 100 m fermenters
10
by the transfer of the preculture from the seed fermenter.
The growth reaction is identical to the above described eqn
for a similar two-liquid phase process. The optimal reactor
size for the two main fermenters in terms of investment costs
3
(
8
1), but glucose is expected to be completely consumed after
h of cultivation. After depletion of the carbon source, a
and capacity utilization is 56.4 m , wherein the oxygen transfer
achieved by aeration and stirring is expected to satisfy the oxygen
demand of the biocatalyst.
feed, consisting of a separately filtered glucose and magnesium
sulfate solution, is initiated. The biotransformation is started
by organic phase addition after 4 h of fed-batch cultivation.
The reaction stoichiometry during fed-batch cultivation and
biotransformation is focusing on (S)-styrene oxide and the
biomass is regarded as a by-product (eqn (2)).
The feasibility of downstream processing consisting of phase
separation by centrifugation and distillation was demonstrated
36
previously by Panke et al. On industrial scales a continuous
downstream processing is preferred. Thus, the setup shown in
Fig. 3 includes a storage tank, which enables a continuous flow
towards a disk-stack centrifuge, separating the broth into a solid
0
.98 C
6
H
N
H
12
O
6
+ 3.77 O
2
+ 0.46 NH
+ 0.10 C
10O + 0.04 C
3
+ 1.15 C
8
H
8
→ 2.28
(cells), an aqueous, and an organic liquid phase. Legislation
CH1.8
O
0.5
0.2 + 3.39 CO
O + 0.11 C
2
2
H
4
O
2
+ 4.18 H
2
O + 1.00
requires sterilization and complete inactivation of genetically
engineered cells before disposal. Subsequent use, e.g. as fertilizer,
is therefore not straight forward. Disposal costs for the cell
C
8
8
8
H
8
H
8
evaporated
(2)
Eqn (2) is based on the data shown in Table 1 and the values
for oxygen, carbon dioxide, and water were calculated by atom
-
1
paste and the aqueous phase were set to $0.15 and $0.0025 kg ,
respectively.
balancing. Evaporation of styrene (C
proportion of styrene that was neither converted to styrene oxide
O) nor to 2-phenylethanol (C
The scalability of the reaction setup was previously shown by
a two-liquid phase styrene epoxidation process at a pilot-scale,
8
H
8
) was considered as the
Work-up of the organic phase is done by continuous distil-
lation. Due to obvious ecological and economic reasons, the
recycling of the organic carrier solvent, in this case BEHP, is
crucial for two-liquid phase processes on an industrial scale. This
is done in the first distillation column. The quality of recycled
BEHP was ensured by a purge flow of 5%. Volatile impurities
such as remaining traces of styrene and octane are separated
(
C
8
H
8
8
H10O).
36
reaching a similar process performance as at a laboratory scale.
The crucial parameters for up-scaling are mass transfer rates and
the avoidance of inhomogeneities. Especially, the oxygen transfer
8
18 | Green Chem., 2010, 12, 815–827
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from the product stream in the second distillation step, before
S)-styrene oxide is separated from 2-phenylethanol in the last
column. Due to its rose-like odor, 2-phenylethanol is considered
to be industrially relevant in the food and flavor industries. The
quality of this output stream would allow additional marketing,
which was not considered here.
The process model as represented by the flow-sheet in
Fig. 3 sets the basis for an in-depth ecological and economic
assessment of the feasibility of biocatalytic (S)-styrene oxide
production on industrial scale.
Therefore, a recycling stream with a recovery of 90% is imple-
mented in the process models (Fig. 4).
(
Separation of (S)-styrene oxide from (R)-1-phenyl-1,2-
49
44
ethanediol was achieved by vacuum distillation. The enantiop-
ure (R)-1-phenyl-1,2-ethanediol constitutes a potential build-
ing block for the synthesis of (R)-mandelic acid, b-lactam
55
antibiotics, and analytical reagents. As in the case of 2-
phenylethanol, separate marketing was not considered.
For the epoxidation of double bonds, the ferric phenanthro-
40
line catalyst of Dubois et al. is promising (Fig. 4A). This
catalyst efficiently converts a wide range of alkenes including
terminal alkenes such as styrene. The oxidant is peracetic
acid, which has to be added in excess (2 equimolar). The
selectivity of the reaction is 63%, because low pH and the
presence of acetic acid cause the formation of the by-product
phenyl acetaldehyde (37%). Two distillation columns assure the
quality of the acetonitrile recycling stream and separate the
volatile acids from the product stream, respectively. The second
column, where acetic acid and peracetic acid are evaporated
and collected in the distillate, is critical with respect to safety
and corrosion. The third column separates styrene oxide from
phenyl acetaldehyde. Separation of these two components is
difficult, because their chemical characteristics, including their
boiling points, are highly similar. Thus, isolation of the racemic
styrene oxide to high purity might be difficult to realize on a large
scale.
Alternative chemical processes for (S)-styrene oxide production
In order to compare the biocatalytic epoxidation and chemical
process alternatives with respect to economic and environmental
performance, chemical routes towards (S)-styrene oxide with an
enantiomeric excess of >98% were analyzed.
The chemical equivalent to the styrene monooxygenase cat-
alyzed reaction is the selective epoxidation by means of salen
transition metal catalysts discovered by the Jacobsen group,
46
often referred to as Jacobsen catalysts. However, the direct
epoxidation of terminal double bonds, as in styrene, suffers
50
from insufficient selectivities (e.e. of around 36%) and/or
◦
51
impractical reaction temperatures of -78 C (e.e. of 86%).
Hydrolytic kinetic resolution of racemic styrene oxide is
an attractive alternative leading to e.e. values comparable
43
to the styrene monooxygenase catalyzed epoxidation. The
dependence on racemic styrene oxide as a substrate requires
a preceding unselective epoxidation of styrene. For this reaction
step, three different methods with high productivities have been
Titanium silicate catalysts (TS-1) were used for the epoxida-
41,56–58
tion of terminal double bonds by several groups (Fig. 4B).
This catalyst often leads to the formation of by-products such
as benzaldehyde or phenyl acetaldehyde. Rode et al. almost
completely avoided by-product formation by maintaining the
40
published. These are based on a ferric phenanthroline,
a
41
52
41
titanium silicate and a manganese sulfate catalyst. For the
design of the chemical (S)-styrene oxide production processes,
these unselective epoxidation reactions were coupled to the
hydrolytic kinetic resolution to obtain the product with high
optical purity (e.e. 98%) from styrene. In the following, the
hydrolytic kinetic resolution and then the three epoxidation
methods are discussed in the context of process modeling and
their flow-sheets (Fig. 4).
pH at 7.5–8.0. Styrene is epoxidized by hydrogen peroxide to
styrene oxide (selectivity: 96.5%), while benzaldehyde is only
41
formed to a small extent as a by-product (selectivity: 3.1%).
The heterogeneous nature of the titanium silicate catalyst allows
41
catalyst recovery by simple filtration of the reaction mixture.
The synthesis of the catalyst is rather simple, but requires
59
expensive tetra-tert-butyl orthotitanate. Since titanium silicate
structures are prone to substantial deactivation due to titanium
leaking from active sites and deterioration of the catalyst surface
The hydrolytic kinetic resolution reported by Tokunaga et al.
includes the selective conversion of one enantiomer of racemic
styrene oxide to the corresponding 1-phenyl-1,2-ethanediol
60
in the presence of aqueous hydrogen peroxide and methanol,
its reuse does not make sense making recycling obsolete. The
solvents for the epoxidation, methanol and acetonitrile, can
easily be separated from the product stream by distillation. Their
consumption is minimized by recycling. The quality is assured by
replacing 5% of this stream by fresh solvents per cycle. A smaller
column is used to separate benzaldehyde from styrene oxide,
before channeling the product stream to the kinetic resolution.
In a simple method published by Lane et al., manganese salts,
such as manganese sulfate, in combination with bicarbonate
catalyze the epoxidation of alkenes using aqueous hydrogen
43
by the attack of water. In the procedure described, (S)-
styrene oxide is isolated in an e.e. of 98% and a yield of
III
3
8% by applying 0.008 equivalents of (S,S)-(salen)Co (OAc)
catalyst and 0.7 equivalents of water. Water-miscible solvents
such as tetrahydrofuran would enhance the epoxide yield to
around 44%, but simultaneously increase the complexity of the
44
process.
S,S)-(salen)Co (OAc) is inactivated during the reaction, but
III
(
II
the inactive (S,S)-(salen)Co can be completely regenerated by
dissolving the catalyst in an appropriate solvent, e.g. toluene,
followed by treatment with acetic acid in the presence of
61
peroxide as oxidant (Fig. 4C). Buffering of the reaction
42
system by urea improves the reaction. The epoxidation tank
contains a two-phase reaction mixture consisting of an organic
styrene phase and an aqueous phase containing urea, sodium
bicarbonate, manganese sulfate, and hydrogen peroxide. After
the reaction, the two phases are separated by a decanter
centrifuge and the organic phase is distilled to generate a purified
styrene oxide stream to the hydrolytic kinetic resolution tank.
44
oxygen.
Due to its complex synthesis
recycling of the chiral catalyst is desirable and also technically
44,53
and hence its high price,
54
feasible, as it can be recovered by filtration or in the retentate
43
of a distillation column. No reduction of the performance
43,44
was observed with reused catalyst for three consecutive runs.
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Fig. 4 Flow-charts of the chemical epoxidation processes. The flow-sheets were generated by means of the SuperPro Designer software. Processes
based on ferric phenanthroline (a), titanium silicate (b), and manganese sulfate (c) catalysts are depicted. Framed circles describe input mass streams
and framed squares output or waste streams. Abbreviations: acetonitrile, ACN; ferric phenanthroline catalyst, Fe-catalyst; titanium silicate catalyst,
TS-1 catalyst.
Inexpensive manganese sulfate is used in small amounts. Thus,
in contrast to the previously presented catalysts, its reuse is
economically irrelevant and its recycling is not considered in
this study. Unfortunately, the by-products formed to 5% during
the reaction have not been identified. Here, it is assumed that
these impurities can be removed via the volatile fraction of a
distillation column as in the cases of benzaldehyde and phenyl
acetaldehyde.
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20 | Green Chem., 2010, 12, 815–827
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Table 2 Mass-balances of resource consumption and waste generation
Ferric phenanthroline Titanium silicate
Manganese sulfate
Biotechnol. process
Input/kg
Output/kg
kgSO
Input/kg
kgSO
Output/kg
kgSO
Input/kg
kgSO
Output/kg
kgSO
Input/kg
kgSO
Output/kg
kgSO
kgSO-
1
-1
-1
-1
-1
-1
-1
-1
(
(
5
R)-1-Phenyl-1,2-ethanediol
S)-Styrene oxide
0
0
0
0.01
0
4.03
0
2.76
0
8.72
0.64
16.15
1.08
1.00
0
1.60
0
2.01
1.18
0
0
0
0.31
0.01
0
0
0
1.89
0
0.48
0.35
3.04
1.13
1.00
0.05
0.01
0
0
0
0.09
0
0.33
0.44
3.04
0
0
1.33
0.01
0
0
0
1.85
2.83
5.92
0.87
12.80
1.12
1.00
0.76
0.01
0
0
0
0.11
2.83
6.07
0.90
0
0
0
0
0
1.00
0
0.05
0
0
0
0.03
0
16.65
3.34
21.07
0% Hydrogen peroxide
Acetic acid
Glucose
Peracetic acid
Phenyl acetaldehyde
Styrene
Urea
Water
1.69
0
0
1.00
0
15.92
2.46
21.07
0
8.57
0.71
Miscellaneous
Sum
16.15
12.80
-1
Substances contributing less than 1 kg kg(S)-styrene oxide to the mass-balances are summarized in the category “miscellaneous”.
Fig. 5 Mass-balances of resource consumption and waste generation. Resource consumption is displayed in (a), whereas product and waste
-1
formation is shown in (b). Substances contributing less than 1 kg kg(S)-styrene oxide to the mass-balances are summarized in the category “miscellaneous”.
Abbreviations: Fe-cat, ferric phenanthroline; TS-1, titanium silicate; MnSO
4
, manganese sulfate; Biol, biotechnological process.
The incorporation of the presented process models in the Su-
perPro Designer software allowed the economic and ecological
assessment including the calculations of the mass-balances.
oxidants is a disadvantage of these reactions. Furthermore, the
oxidants are employed in aqueous solutions, hydrogen peroxide
in a 50% and peracetic acid in a 32% solution, increasing the
mass balances by the contained water fraction, which, however,
is only of minor ecological and economic significance. Apart
from the ecological impact of the oxidants, their handling may
cause additional safety costs, which were not included in the
economical assessment.
Process assessment: resource consumption and waste generation
Balancing all of the involved chemical compounds is typically
the starting point of any economic and ecological assessment.
Quantification of resource consumption and waste generation
serves as the basis for the assessment procedure and allows
first insights in the sustainability of a process. Lower input
streams reduce the environmental burden by definition, since the
resources are utilized more effectively. Resource consumption
and waste generation of the aforementioned process variants in
terms of total mass are displayed in Fig. 5 and Table 2.
The titanium silicate process excels in the low resource
demand and waste generation, consuming only 3.04 kg re-
sources for the production of 1 kg of (S)-styrene oxide (sub-
sequently kgSO). The recycling of the epoxidation solvents and
the efficient use of hydrogen peroxide are the main reasons for
this outstanding performance.
As expected from the typically high water requirement of
biocatalytic processes (here 76%), the biotechnological setup
-
1
requires the highest amount of resources (21.07 kg kgSO ). The
cleaning-in-place procedures for the fermenters adding another
4.91 kg of water and 0.04 kg of sodium hydroxide for the
production of 1 kg (S)-styrene oxide are not included. The
reuse of the medium is hampered by toxic concentrations of
(S)-styrene oxide and 2-phenylethanol and the accumulation
of acetic acid. Other substances added in prominent amounts
are the carbon source glucose and the organic phase BEHP.
Since the styrene monooxygenase is highly enantioselective,
the lowest amount of styrene needs to be employed and the
only by-product, 2-phenylethanol, is formed to a minor extent
-
1
The manganese sulfate and the ferric phenanthroline pro-
cesses are inferior with larger resource demands of 12.80 and
(116 g kgSO ).
E-factors, defined as the mass ratio of waste to desired
product, are an environmental metric concept addressing the
-
1
1
6.15 kg kgSO , respectively. The dependence on an excess of
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Table 3 Environmental indices for the input and the output side of the processes and overall environmental indices
Biotechnol.
process
Ferric phenanthroline
Titanium silicate
Manganese sulfate
input
output
total
input
output
total
input
output
total
input
output
total
(
5
S)-styrene oxide
0% hydrogen peroxide
0
0
46.2
0
29.2
0
58.1
0
0
8.6
21.8
163.9
27.7
0
17.6
0
89.5
24.1
0
8.6
15.3
182.9
0
46.2
1.2
0.1
0
0
1.9
0
0.3
11.6
61.4
27.7
5.1
0.1
0
0
17.7
0
0.4
8.9
59.9
0
46.2
19.2
0.1
0
27.7
29.9
0.1
0
0
17.5
17.6
6.0
5.8
104.7
0
0
0
31.1
0
21.8
0
15.9
9.7
78.6
46.2
0
0.9
22.9
0
0.6
0
16.6
14.6
102.0
27.7
0
0.5
26.2
0
9.1
0
16.4
12.7
92.6
10.9
0.2
0
0
41.4
0
0.5
4.9
57.7
46.0
0.2
0
acetic acid
BEHP
peracetic acid
styrene
urea
water
0.1
0
136.6
60.3
0
8.7
5.7
211.5
0
0
40.3
2.8
5.9
2.3
97.5
2.2
27.5
6.1
8.2
109.5
miscellaneous
sum
Input and output indices were added in a 40 : 60 ratio to obtain the overall environmental indices (referred to as total index in the text). Substances
contributing to a minor extent are summarized in the category “miscellaneous”.
Fig. 6 Environmental indices for the input (a) and the output (b) side of the process and overall environmental indices (c). Input and output indices
were added in a 40 : 60 ratio to obtain the overall environmental indices (referred to as total index in the text). Substances contributing to a minor
extent are summarized in the category “miscellaneous”. Abbreviations: Fe-cat, ferric phenanthroline; TS-1, titanium silicate; MnSO
4
, manganese
sulfate; Biol, biotechnological process.
14,62
problem of waste generation in synthetic industry.
All
Process assessment: environmental impact potential
involved compounds are considered with the sole exception of
water. This neglect of water utilization uprates the biotech-
nological setup, which has with 3.42 a lower E-factor than
the ferric phenanthroline (6.58) and manganese sulfate (5.72)
based processes. Titanium silicate remains the most favorable
epoxidation catalyst regarding the waste generation (E-factor:
The environmental burden of a process is not only defined by
the amount of resources consumed and waste formed, but also
by their harmfulness. It is self-explanatory that 1 kg of heavy
metal or solvent waste imposes a bigger ecological problem than
1 kg of water. Quantification and balancing of the multi-faceted
impacts on the environment was performed by the assignment of
1
.71). It is noteworthy that all presented processes are be-
16,20,21
low or at the lower limit of the E-factors of 5–50 typically
so-called environmental indices to all compounds involved.
The results are presented in Fig. 6 and Table 3.
-
1
encountered in fine chemical processes of 100–10 000 t a
scale.
62
In terms of sustainability, the ferric phenanthroline process
clearly performed the worst (total environmental index: 183).
This poor performance is mainly due to safety and corrosion
risks by high amounts of peracetic and acetic acids. Further-
more, significant by-product formation leads to a high consump-
tion of all resources and contributes to the environmental index
on the output side (acetic acid, phenyl acetaldehyde, and (R)-1-
phenyl-1,2-ethanediol).
The titanium silicate process exhibits the lowest environmen-
tal index (60) due to its minimal resource demand as exemplified
by the almost 100% utilization of hydrogen peroxide as oxidant.
In contrast, the manganese sulfate process requires an excess
of hydrogen peroxide, which, together with the high amount of
urea waste, leads to an intermediate environmental index (105).
In conclusion, the titanium silicate catalyst based process is
clearly the most favorable procedure regarding resource demand
-
1
(
3.04 kg kgSO ), followed by the manganese sulfate and the
iron phenanthroline catalyst based processes with resource
-
1
demands of 12.80 and 16.15 kg kgSO , respectively. Although
-
1
the biocatalytic setup depends on 21.07 kg kgSO , it can be
considered economically and environmentally interesting, as the
-
1
dominating proportion is water with 15.92 kg kgSO , which
is cheap and environmentally harmless. In order to take the
differing environmental impact of the involved chemicals into
account, environmental indices are introducedinthe next section
for the quantitative assessment of the overall impact of the
processes.
8
22 | Green Chem., 2010, 12, 815–827
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The biotechnological process performs second in terms of sus-
tainability (total environmental index: 93), despite showing the
highest input and output mass-balances. The biotechnological
approach profits from the avoidance of harmful substances, e.g.
by using molecular oxygen from air as oxidant. The organic
solvent BEHP is the most problematic factor and is negatively
judged due to its origin from fossil resources and its toxicity
to humans (environmental index: 26). Although replacement of
the organic phase is obviously favored from an environmental
perspective, it is difficult to find an alternative solvent with
similar extraction and superior ecological characteristics. BEHP
was chosen as it provides a high organic/aqueous partition
coefficient for styrene oxide, considering also other important
criteria for solvent selection such as low water solubility,
low toxicity to microbes, chemical and thermal stability, non-
biodegradability, low foam formation in bioreactors, and avail-
biocontainment entail extra measures. In the evaluated example,
the seed and main fermenters make up 55% of the equipment
costs, also because the downstream processing is straightforward
and relatively cost saving. The purchase costs of the fermenters
might be reduced by modulating and rebuilding second-hand
65
fermenters or leasing equipment. In the latter case, recurring
costs have to be weighed with investment costs. The comparably
dilute product concentration in the fermenter is another reason
for the high investment costs. As a consequence, the equipment
has to be designed larger as compared to chemical plants to
10,47,66
achieve similar product amounts.
This fact stresses the
importance of the process intensification presented in the first
part of this study.
In contrast to the investment costs, the biotechnological
process outperforms all chemical processes with respect to
operational costs (Table 5).
63,64
ability at low prices.
A lower extraction efficiency for styrene
The discrepancy among the different processes is mainly
caused by differing raw material costs. As expected, the biocat-
alytic process benefits from the absence of expensive chemical
catalysts, which dominate the material costs of all chemical
processes. The integration of biocatalytic synthesis into the (S)-
styrene oxide production plant is advantageous, because the
catalyst does not have to be purchased or produced in a separate
process. The medium costs for the growth of the biocatalyst
oxide would increase its aqueous concentration, resulting in an
earlier inactivation of the biocatalyst and finally in a reduced
final product concentration.
From an ecological perspective, this study clearly identifies
the carrier solvent to be the most critical factor in two-liquid
phase biotransformations. Thus, environmental sustainability
is proposed to be taken up as a key parameter for solvent
selection.
-
1
($1.0 kgSO ) contribute only to a minor extent to the overall
costs, although a high amount of biomass is formed during the
fed-batch cultivation. Thus, the total expenses for raw materials,
Process assessment: economic potential
-
1
i.e. $2.9 kgSO , play a subordinate role (28% of the operational
-
1
Economic success and profitability are the driving forces for the
development of all new processes in industry. A process can be
beneficial in terms of ecology, but will never be implemented
if the economic parameters are unfavorable. Therefore the
economic viability of the four processes was evaluated on the
basis of the SuperPro Designer flow-sheets as they contain
all necessary equipment, material streams, and labor-requiring
operations for a reliable estimation of the fixed and variable
production costs. The approach for the economic assessment is
described in detail in the Experimental section. The resulting
key economic parameters for plants designed for an annual
production of 1000 t (S)-styrene oxide are summarized in
Table 4.
costs) for the bioprocess, with BEHP ($1.2 kgSO ), the growth
-
1
-1
medium ($1.0 kgSO ) and styrene ($0.6 kgSO ) as major factors.
This contrasts the significant costs of the epoxidation cata-
-
1
lysts ferric phenanthroline ($23.3 kgSO ) and titanium silicate
($8.4 kgSO ). The inexpensive manganese sulfate is the exception
profiting from its simplicity ($0.01 kgSO ).
-
1
-
1
For the bioprocess, the factory-related fixed costs are higher
than the raw material costs. Especially, the depreciation of the
expensive equipment has a pronounced impact. In this study,
the depreciation period was assumed to be 10 years for all
equipment. In the case of the fermenters, one can argue for
10,67
longer depreciation periods of 12 or 15 years.
This will,
however, only slightly reduce the depreciation costs by $0.09
-
1
The investment costs for the biotechnological plant were
found to be 1.4 to 2.1 times higher as compared to the
chemical alternatives. Biocatalytic processes typically are capital
intensive, because large and complex fermenters are required.
Main reasons for this are usually dilute reaction systems and
complex requirements for process control to guarantee optimal
and $0.18 kgSO , respectively.
Labor-related expenses belong to the major cost factors in
the biocatalytic plant. Compared to the chemical processes,
especially the upstream processing of the biocatalytic setup
is more complex and more labor-intensive. Furthermore, the
cultivation and biotransformation steps have to be monitored
carefully. Thus, less operational effort is required in all chemical
10,65
growth of microorganisms.
Additionally, sterilization and
Table 4 Economic parameters for (S)-styrene oxide production plants
Parameter
Unit
Ferric phenanthroline
Titanium silicate
Manganese sulfate
Biotechnol. process
Capital investment
Direct fixed capital
Working capital
Start-up costs
Annual operating costs
million $
million $
million $
million $
million $ a-
15.1
10.9
3.6
0.6
41.6
10.1
8.4
1.4
0.4
16.9
9.6
8.2
1.0
0.4
12.8
20.4
18.9
0.6
0.9
10.2
1
Data sources are listed in the assessment methods part.
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1
0
Table 5 Production costs for 1 kg of (S)-styrene oxide
Fe-cat. TS-1 MnSO
accounted for 70% of the production costs, whereas (S)-styrene
-
1
oxide production at a 1000 t a scale was dominated by fixed
costs accounting for 63% of the total costs.
4
Biol.
-1
-1
-1
-1
Category
[$ kg ] [$ kg ] [$ kg ] [$ kg ]
From an economic perspective, only the manganese sulfate-
based plant can compete with the biotechnological process. This
is visualized in Fig. 7 showing the net profit and the payback
time as a function of the selling price.
Facility dependent costs
depreciation
insurance
local taxes (property)
factory expenses
maintenance
1.96
1.04
0.11
0.22
0.30
0.30
1.57
0.79
0.08
0.17
0.30
0.23
1.55
0.78
0.08
0.16
0.30
0.22
3.16
1.80
0.19
0.38
0.30
0.50
While the net profit only depends on the revenues and the
operational costs, the calculation of the payback time also
considers the capital investment. At low selling prices, the
biotechnological process outcompetes the chemical alternatives
Raw materials
34.95
2.48
—
0.01
0.74
—
13.02
2.48
0.69
0.01
0.18
—
—
—
—
—
0.03
—
—
—
—
1.22
—
8.40
0.01
—
7.77
2.48
2.96
0.01
—
—
—
—
—
0.01
—
—
—
—
0.47
1.21
—
2.86
—
—
—
—
0.02
1.17
—
0.86
—
—
0.04
—
0.12
—
0.63
0.01
—
—
—
II
(
salen)Co catalyst
-
1
5
0% hydrogen peroxide
in both categories. Above prices of $15.4 kgSO , the payback time
favors the manganese sulfate process due to low investment costs.
The best process method from a environmental point of view, the
titanium silicate based process, reaches similar payback times
acetic acid
acetonitrile
ammonium
bis(2-ethylhexyl)phthalate
ferric phenanthroline catalyst 23.25
glucose
manganese sulfate
methanol
octane
peracetic acid
riesenberg medium salts
sodium bicarbonate
styrene
thiamine hydrochloride
titanium silicate catalyst
toluene
—
-
1
only at a relatively high selling price of $24 kgSO . The ferric
phenanthroline process is not competitive with unit production
—
—
—
—
6.59
—
—
1.87
—
—
-
1
costs of $41 kgSO
.
Borole et al. performed a similar economic analysis of
an enzymatic (S)-styrene oxide production plant based on
chloroperoxidase of the fungus Caldariomyces fumago (scale:
-
1
11
2
00 tSO a ). The high costs for the enzyme resulted in non-
-
1
competitive (S)-styrene oxide production costs of 1 153 $ kgSO .
—
This is clearly inferior to all epoxidation processes evaluated
here, but might be improved by gene expression and enzyme
production in recombinant Aspergillus niger as proposed in
the respective study. According to the authors, a selling price
0.01
—
—
0.01
0.63
—
urea
trace elements
—
0.00
Total labor expenses
Laboratory/quality control
Consumables
Waste treatment
Utilities
Total unit production costs
1.62
0.24
—
2.31
0.50
41.59
1.66
0.25
0.12
0.15
0.11
16.89
1.56
0.23
—
1.70
0.04
12.85
2.86
0.43
0.25
0.26
0.37
10.19
-1
of $25 kg could be assumed for enantiopure styrene oxide.
Except for the ferric phenanthroline process, all evaluated
processes are highly profitable at this selling price. However, with
increasing amounts of (S)-styrene oxide produced, the market
price might decrease favoring processes already profitable at
lower selling prices as the styrene monooxygenase- and the
manganese sulfate-based reaction setups.
Data sources are listed in the assessment method part.
plants despite the fact that two reaction steps have to be
employed.
Conclusion
The high fixed and the low variable production costs of the
biocatalytic process would clearlyfavor larger annual production
volumes, as the contribution of the fixed costs with respect to
total costs decreases with increasing scale. For example, in an
economic study on a comparable two-liquid phase process for
the annual production of 10 000 t 1-octanol, variable expenses
The process intensification presented in the first part of this
study shows that two-liquid phase biotransformations allow
the production of toxic compounds such as (S)-styrene oxide
from cheap, but toxic, substrates such as styrene by whole-cell
biocatalysis to high final product concentrations (72.6 g Lorg ).
The process performance is comparable to the most powerful
-
1
Fig. 7 Net profit and payback time as a function of the selling price. Net profits (a) and payback time (b) are shown for selling prices between $8
-1
and $30 kg .
8
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Table 6 Summary of the ecological and environmental assessment
Parameter
Unit
Ferric phenanthroline
Titanium silicate
Manganese sulfate
Biotechnol. process
1
Resource consumption
Environmental index
Capital investment
kg kgSO-
16.15
182.9
15.1
41.6
44.3
3.04
59.9
10.1
16.9
18.7
12.80
104.7
9.6
12.8
14.6
21.07
92.6
20.4
10.2
13.9
million $
$ kgSO-
$ kgSO-
1
Production costs
1
Production costs including
a
instalment payment
a
Average annual instalment payment over 10 years include instalment of direct fixed capital (interest rate: 9% over a period of 10 years) and instalment
of working capital (interest rate: 12%; period: 6 years).
oxygenase-based processes in terms of product concentration
pH was maintained at 7.20 by adding 30% phosphoric acid
and 25% NH OH solutions. The latter additionally served as
-
1
-1
and average productivity (4.19 g Ltot h ), proving a basis for
the industrial feasibility of this remarkably attractive enzyme
4
a nitrogen feed. The batch phase lasted approximately 12 h
(overnight). Aeration and stirring rate were maintained constant
6,7
class. In an in-depth environmental and economic assessment
-
1
-1
of this optimized process for a 1000 t a scale, the organic solvent
BEHP was identified as the most critical factor. Its origin from
fossil resources and its human toxicity were the main reasons
for the negative judgment resulting in a contribution of 28%
to the overall environmental index of the bioprocess. Thus, the
environmental compatibility of the organic extractive phase has
to be addressed foremost in the bioprocess to become ecologi-
cally superior to the best chemical (S)-styrene oxide production
method, which is based on titanium silicate as epoxidation
catalyst (Table 6). Nevertheless, the biotechnological approach
was found to be highly interesting, because it outcompeted all
evaluated chemical alternatives in terms of economics (Table 6).
Whereas the production costs for 1 kg (S)-styrene oxide by
the environmentally friendliest method amount to $16.9, the
biocatalytic process produced the same quantity for $10.2 or
on 1 L min and 1500 rpm, respectively. After depletion of
-
1
the carbon source, a feed consisting of 730 g L glucose and
-
1
19.6 g L MgSO
4
·7 H
2
O was started and increased stepwise until
-
1
a biomass concentration of approximately 18 g L was reached.
Then, the biotransformation was started by the addition of the
organic phase consisting of 910 ml BEHP, 80 ml styrene, and
10 ml octane serving as the inducer of styAB expression. The
phase ratio of the organic to the aqueous phase was 1 : 1 during
the biotransformation. The dissolved oxygen concentration was
kept above 20% by increasing the stirring speed (to 2800 rpm)
and the aeration rate (up to 2.5 L min ). Antifoam A (Sigma-
Aldrich Chemie Gmbh, Steinheim, Germany) was added only
in cases of excessive foaming. The biotransformation was moni-
tored by taking samples every hour. Sampling was performed as
-
1
70
described previously. For two-liquid phase biotransformations,
volumetric rates and concentrations are given per litre of
aqueous phase (Laq), organic phase (Lorg), or total volume (Ltot).
6
0% of the expenses. Solving the problem of the environmental
burden of the organic phase would ultimately guarantee whole-
cell two-liquid phase biotransformations and oxygenase-based
biocatalysis an important role in the development of greener
syntheses on industrial scale.
Analytics
Concentrations of styrene, styrene oxide, and 2-phenylethanol
were measured by a TRACE GC Ultra (Thermo Fisher Scientific
Inc., Waltham, MA, USA) equipped with a FactorFour VF-
Experimental
5
ms column (Varian, Inc., Palo Alto, CA, USA). The oven
Strain, cultivation and two-liquid-phase biotransformation
◦
◦
temperature was raised from 50 C to 140 C at a heating rate
of 15 C min , followed by heating at a rate of 100 C min
+
q
◦
-1
◦
-1
E. coli JM101 (supE thi-1 D(lac-proAB) F’[traD36 proAB lacI
◦
lacZDM15]), a derivative of E. coli K-12, was used as recom-
to 300 C, which was maintained for 3.5 min. Detection
was achieved by a flame ionization detector (FID). Specific
epoxidation activities are given in units (U), whereby 1 U is
defined as 1 mmol product formed in 1 min.
Glucose and acetate were separated on an Aminex HPX-
87-H column (Bio-Rad Laboratories, Hercules, CA, USA) in
a LaChrom Elite HPLC system (Hitachi High Technologies
America, Inc., Pleasanton, CA, USA), after extraction of
aqueous styrene by BEHP. The flow rate of 2.5 mM sulfuric
acid as mobile phase was set to 1.0 ml min . The temperature
was kept constant at 60 C. Detection of the analytes was done
by a L-2420 UV-Vis and a L-2490 refractive index module.
Cell concentrations were determined spectrometrically on a
Libra S11 spectrophotometer (Biochrom Ltd., Cambridge, UK)
at a wavelength of 450 nm (OD450), whereby one OD450 unit
68
binant host strain. The expression plasmid pSPZ10 is based
on the pBR322 vector and contains the styrene monooxygenase
genes styAB from Pseudomonas sp. strain VLB120, under the
control of the alk regulatory system from Pseudomonas putida
31
GPo1.
Transformants were selected on Luria Bertani (LB) agar plates
-
1
69
containing 50 mg ml kanamycin. A single colony was picked
and used to inoculate 5 ml liquid LB medium. After reaching the
69
-1
stationary growth phase, 1 ml was diluted in 96 ml M9 medium
-
1
70
-1
◦
complemented with 1 ml L US* trace elements, 1 ml L
% (w/v) thiamine solution, and 2 ml L-1 1 M magnesium
sulfate (MgSO ) solution. After overnight growth, this culture
was used to inoculate a KLF 2000 reactor (Bioengineering,
1
4
71
Wald, Switzerland) containing 900 ml Riesenberg medium.
1
-1 72
1
5 g L- glucose were used as carbon and energy source. The
corresponded to a cell dry weight (CDW) of 0.166 g L .
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Assessment method
the Deutsche Bundesstiftung Umwelt (DBU; project AZ13145)
is gratefully acknowledged.
Economic and ecological process efficiencies were assessed by
means of a method designed for utilization at early development
stages of fine chemical processes.
8,16,20,21
References
Mass-balances were
generated on the basis of process flow-sheets created with the
simulation tool, SuperPro Designer version 6.0 (Intelligen, Inc.,
Scotch Plains, NY, United States).
1
2
3
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For the calculation of the environmental indices, all sub-
stances are characterized in different categories according to
their impact on human health and on the environment. These
categories are land use, raw material availability, complexity of
synthesis, thermal risk, acute toxicity, chronic toxicity, biological
risk, ecotoxicity, global warming potential, ozone depletion
potential, photochemical ozone creation potential, odor, and
eutrophication potential. For each category, the substances
are classified in three rating levels (ABC analysis), where “A”
stands for high, “B” for medium and “C” for low risk in the
respective category. The criteria for the classification have been
4
5
6
7
8
9
E. Heinzle and K. Hungerb u¨ hler, Chimia, 1997, 51, 176–183.
19
published elsewhere. Weighing factors are used to account for
the differing significance of the impact categories, allowing the
calculation of an environmental index for the input and the
output materials. The total environmental index of a process
is obtained by balancing the input and the output indices in a
1
0 R. G. Mathys, A. Schmid and B. Witholt, Biotechnol. Bioeng., 1999,
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Kingdom, 2008, pp. 200–227.
1
1
40 : 60 ratio.
SuperPro Designer comprises comprehensive databases for
the calculation of numerous economic parameters. These values
were used for the economic assessment unless otherwise stated.
Purchase prices for the equipment were taken from the SuperPro
Designer database. The direct investment costs are the sum of the
equipment purchase costs (PC) and charges for the installation
1
3 S. Hellweg, U. Fischer, M. Scheringer and K. Hungerb u¨ hler, Green
Chem., 2004, 6, 418–427.
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1
5 P. Saling, A. Kicherer, B. Dittrich-Kr a¨ mer, R. Wittlinger, W. Zombik,
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1
(
40% of PC), the instrumentation (15% of PC), piping (46% of
3
407.
PC), electronic facilities (10% of PC), buildings (16% of PC),
yard improvements (14% of PC), and service facilities 60% of
1
1
7 A. D. Curzons, D. J. C. Constable, D. N. Mortimer and V. L.
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73
PC) as suggested by Roffler et al. Additionally, indirect costs
such as expenses for engineering and the contractor’s fee (16%
of direct costs) and construction (10% of direct costs) have to be
taken into account for the total capital investment. Raw material
costs were complemented with industrial market prices from the
ICIS webpage (www.ICIS.com) or, if necessary, by divison of the
lab-scale prices from Sigma Aldrich (www.sigmaaldrich.com)
by a factor of 10. The depreciation period was assumed to be
2
2
5
Verduyn and K. Hungerb
413.
3
2
2 S. Panke, B. Witholt, A. Schmid and M. G. Wubbolts, Appl. Environ.
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1
0 years with a salvage value of 5%. Insurance and local taxes
23 A. Schmid, K. Hofstetter, H. J. Feiten, F. Hollmann and B. Witholt,
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were estimated to account for 1% and 2% of the investment
2
2
4 P. Besse and H. Veschambre, Tetrahedron, 1994, 50, 8885–8927.
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costs, respectively, whereas the factory expenses were fixed to
1
$
300 000 a- in all processes. For the maintenance costs, unit-
specific default values of SuperPro Designer were used. Basic
labor costs (BLC) are estimated from the labor requirements of
each operational step in the factory and assuming a standard
wage of $30 h- for an operator. The total labor expenses
additionally include charges for fringe benefits (40% of BLC),
supervision (20% of BLC), operational supplies (10% of BLC),
and administration (60% of BLC). Laboratory costs for quality
checks were 15% of the total labor costs.
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9 C. Franchini, A. Carocci, A. Catalano, M. M. Cavalluzzi, F. Corbo,
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8
26 | Green Chem., 2010, 12, 815–827
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