Systematic optimization of a biocatalytic two-liquid phase oxyfunctionalization process guided by ecological and economic assessment

Article abstract of DOI:10.1039/c2gc15985f

Next to economic success, ecological considerations have become increasingly important for companies synthesizing various compounds ranging from bulk chemicals to pharmaceuticals. In this context, the economic and ecological feasibility of asymmetric biocatalytic styrene epoxidation has previously been investigated and compared to chemical alternatives. Although the biotechnological two-liquid phase approach was found to be highly interesting in economic terms, the ecological performance is restrained by the applied organic carrier solvent bis(2-ethylhexyl)phthalate, which is toxic to humans and produced from non-renewable resources. As an alternative carrier solvent, the biodiesel constituent ethyl oleate was tested. Furthermore, the switch from glucose to glycerol as a carbon and energy source was investigated, the latter being a cheap abundant resource, as it is a waste product of the biodiesel and soap industries. Both strategies slightly reduced the productivity and final product titer. An ecological and economic assessment on process level, however, revealed a superior environmental performance (by 13%) with ethyl oleate as the extractive solvent, at the expense of slightly reduced economics (by 9%), whereas glycerol use reduced the performance with respect to both aspects. Based on available data, the application of resting cells was evaluated, providing the opportunity of more efficient carbon utilization via decoupling of growth and biotransformation. Their stability is, however, yet to be improved to achieve competitiveness. In general, this study underlines the potential of ecological and economic assessments for systematic process intensification. Even if advantages of proposed changes seem obvious, their true suitability can only be judged by detailed economic and ecological analyses at the process level. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2gc15985f

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PAPER
Systematic optimization of a biocatalytic two-liquid phase
oxyfunctionalization process guided by ecological and economic assessment†
Daniel Kuhn,a Mattijs K. Julsing,a Elmar Heinzleb and Bruno Bühler*a
Received 9th August 2011, Accepted 25th November 2011
DOI: 10.1039/c2gc15985f
Next to economic success, ecological considerations have become increasingly important for companies
synthesizing various compounds ranging from bulk chemicals to pharmaceuticals. In this context, the
economic and ecological feasibility of asymmetric biocatalytic styrene epoxidation has previously been
investigated and compared to chemical alternatives. Although the biotechnological two-liquid phase
approach was found to be highly interesting in economic terms, the ecological performance is restrained
by the applied organic carrier solvent bis(2-ethylhexyl)phthalate, which is toxic to humans and produced
from non-renewable resources. As an alternative carrier solvent, the biodiesel constituent ethyl oleate was
tested. Furthermore, the switch from glucose to glycerol as a carbon and energy source was investigated,
the latter being a cheap abundant resource, as it is a waste product of the biodiesel and soap industries.
Both strategies slightly reduced the productivity and final product titer. An ecological and economic
assessment on process level, however, revealed a superior environmental performance (by 13%) with
ethyl oleate as the extractive solvent, at the expense of slightly reduced economics (by 9%), whereas
glycerol use reduced the performance with respect to both aspects. Based on available data, the
application of resting cells was evaluated, providing the opportunity of more efficient carbon utilization
via decoupling of growth and biotransformation. Their stability is, however, yet to be improved to achieve
competitiveness. In general, this study underlines the potential of ecological and economic assessments
for systematic process intensification. Even if advantages of proposed changes seem obvious, their true
suitability can only be judged by detailed economic and ecological analyses at the process level.
limited information are required,9,12 such as the calculation of
Introduction
environmental indices on the basis of ABC classification.9
Biocatalysis offers new opportunities in the synthesis of indus-
trial goods, especially when the target products are difficult to
obtain by traditional chemical means.1–4 In order to profit from
the great promises of biocatalysis, it is essential to develop these
ideas and have them implemented in industrial processes,5,6
which are still scarce compared to the respective scientific publi-
cation activity. In this respect, ecological and economic assess-
ment methodologies7–10 can support decision-making and
knowledge-based engineering strategies.9,11 For early develop-
ment phases, simple and flexible methodologies coping with
The latter assessment tool has been used for the ecological
and economic evaluation of biocatalytic oxyfunctionalization
using whole microbial cells in a two-liquid phase system, includ-
ing a comparison against chemical process alternatives.13
Specifically, the biocatalytic approach was based on recombinant
Escherichia coli, harboring the styrene monooxygenase StyAB
of the Pseudomonas sp. strain VLB120 and catalyzing the asym-
metric epoxidation of styrene to enantiopure (S)-styrene oxide
(e.e. >99.5%).14–17 Oxygenase catalysis in whole cells is gener-
ally preferred over isolated enzyme catalysis due to the cofactor
(e.g., NADH) dependency, low stability in isolated form, and
often multicomponent nature of these enzymes.18–20 The toxicity
of hydrophobic substrates and products can be alleviated by the
use of a second immiscible organic carrier solvent, which serves
as a substrate reservoir and product sink.21–23 For asymmetric
styrene epoxidation, this biotechnological setup proved to be
more cost efficient than three chemical alternatives.13 From an
environmental point of view, however, the biotechnological
process was inferior to one of the investigated chemical alterna-
tives, mainly due to the environmental burden imposed by the
solvent bis(2-ethylhexyl)phthalate (BEHP), which is toxic to
humans and is produced from petroleum-based resources. The
aLaboratory of Chemical Biotechnology, Department of Biochemical
and Chemical Engineering, Technische Universität Dortmund,
Emil-Figge-Str. 66, 44221 Dortmund, Germany.
E-mail: bruno.buehler@bci.tu-dortmund.de; Fax: +49 231 7557382;
Tel: +49 231 7557384
bBiochemical Engineering Institute, Saarland University, Campus A 1.5,
66123 Saarbrücken, Germany
†Electronic supplementary information (ESI) available: Figure A: Flow-
sheet for the biocatalytic (S)-styrene oxide production. Table A: Detailed
mass-balances of resource consumption and waste generation; Table B:
Detailed environmental indices of the growing cell processes; Table C:
Detailed environmental indices of the resting cell processes; Table D:
Criteria for ABC classification. See DOI: 10.1039/c2gc15985f
This journal is © The Royal Society of Chemistry 2012
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Table 1 Partition coefficients of styrene, styrene oxide, and
2-phenylethanol during two-liquid phase biotransformations
solvent is thus the obvious target for improving the ecological
performance of biotechnological two-liquid phase processes.
Next to the organic phase, the carbon and energy source (e.g.,
glucose) was found to be critical with regard to production costs.
Finding either cost-saving alternatives or improving the product
yield on the energy source will reduce related expenses. The
latter can be achieved by the timely separation of growth and
biotransformation phases, e.g., by applying the biocatalyst in a
resting state.24–26 Thereby, the cells remain metabolically active,
but are not able to produce biomass due to a nutrient limitation,
such as lack of nitrogen sources in the medium. Hence, energy
metabolism can be maximally exploited for the biotransform-
ation (i.e., cofactor regeneration). Indeed, application of the bio-
catalyst in a resting state led to a 3.3 and 1.9-fold improvement
in average product yields on glucose and maximum specific
activities, respectively, confirming the attractiveness of this
approach for asymmetric styrene epoxidation.26
The aim of this study was the optimization of the ecological
and economic performance of the biocatalytic asymmetric
styrene epoxidation. Replacement of the organic solvent BEHP
by the renewable biodiesel component ethyl oleate (EO) was
therefore investigated, as well as glycerol as a promising carbon
source, which is in abundance as it is the waste product of soap
and biodiesel industries. The suitability of these reaction engi-
neering approaches was economically and ecologically assessed
in detail. Moreover, biotransformations based on resting cells
were included in the assessment. To date, such case studies
systematically assessing the advantages and challenges during
(bio-)process development are limited in number despite their
potential for the implementation of bio-based reactions in the
chemical industry.5,11 Finally, this paper shows the potential of
the systematic assessment of lab-scale optimization studies for
the implementation of bioprocesses on an industrial scale.
BEHP27
EO
Styrene
Styrene oxide
2-Phenylethanol
2990 200
170 20
6.0 0.5
2600 490
138 13
5.9 1.1
As compared to biotransformations with BEHP, maximal
epoxidation activities were about 20% lower using EO.
Final product titers, however, accounted for 445 mM (53.5 g
Lorg−1), which is 159 mM lower than with BEHP. This lower
product titer is likely to be associated with the lower partition
coefficient of EO for styrene oxide (Table 1), resulting in
higher aqueous concentrations at identical overall concentrations
in the two-liquid phase system. As a consequence, the bio-
catalyst was toxified and thus inactivated at lower overall styrene
oxide titers. At the end of the biotransformation, cellular
metabolism broke down before the styrene was completely
converted.
Next to the carrier solvent, the carbon source was found to be
an important raw material cost factor for styrene epoxidation.13
With respect to carbon source selection, the ability of E. coli to
grow on glycerol as the sole energy and carbon source is an
interesting feature. Glycerol bears a considerable economic and
ecological potential as it is an inevitable by-product of the bio-
diesel and soap industries.31–33 Additionally, the high reduction
degree of the carbon atoms in glycerol compared to common
sugars may foster the regeneration of reducing equivalents,34
which was found to restrict styrene epoxidation in growing
cells.35 Therefore, glycerol was investigated as the sole source
for carbon and energy during styrene epoxidation (Fig. 1C,
Table 2) The performance of the BEHP-based biotransformation
was lower with glycerol as compared to glucose, with lower final
product titers and lower specific activities at similar cell concen-
trations. Towards the end of the biotransformation, the activities
decreased and ceased due to long exposure to high product con-
centrations, resulting in incomplete styrene conversion.
Results and discussion
Investigation of different organic carrier solvents and carbon
sources for biocatalytic styrene epoxidation in a two-liquid phase
system
The combination of EO as the extractive solvent and glycerol
as the carbon source also allowed productive biotransformation,
but resulted in the lowest activities and final product titer after a
slightly prolonged biotransformation (Fig. 1D, Table 2). The
slower styrene oxide accumulation was disadvantageous for the
final product titer, because the cells were exposed to high con-
centrations for a longer time.
The glucose/BEHP setup showed the best performance in
terms of productivities and final product titers (Table 2).
Although both changes, from BEHP to EO and from glucose to
glycerol, had a negative effect on biotransformation perform-
ance, styrene epoxidation at lab-scale using EO and/or glycerol
still appeared to achieve considerable productivities. Therefore,
the environmental and economic impact of these changes on
the process level was investigated in detail and compared to the
original setup using BEHP and glucose. Furthermore, a recently
published resting cell approach, using a similar setup with
BEHP and enabling a high styrene oxide yield on glucose, was
included in the assessment. With respect to the enantioselectiv-
ity, e.e. values of 99.5, 99.4, and 99.7% have been found for the
In consideration of the recent finding that BEHP compromises
the ecological viability of the biocatalytic two-liquid phase
process for styrene epoxidation, the selection of the organic
solvent was revisited in this study. So far, BEHP was the best
performing organic phase in terms of efficient product extraction,
low foaming tendency, and non-toxicity for microbes. Neverthe-
less, the maximum achievable (S)-styrene oxide titer was still
restricted by the toxic effects of styrene oxide and possibly 2-
phenylethanol.13,27 This stresses the importance of a high extrac-
tion efficiency by the organic solvent for these products in order
to keep their aqueous concentration low. The fatty acid ester EO
was identified as a promising alternative for BEHP, because of
its favorable partition coefficients (comparable to BEHP,
Table 1). EO is the major component of canola-based biodiesel
(55–65 wt%)28 and is considered as an attractive solvent due to
its biodegradability, non-toxic nature, and the production from
renewable feed stocks.28–30 EO was tested as an organic solvent
in an identical setup and compared to the BEHP-based process
(Fig. 1A,B, Table 2).
646 | Green Chem., 2012, 14, 645–653
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Table 2 Process parameters for all biotransformations
BEHP
Glucose
EO
Glucose
BEHP
Glycerol
EO
Glycerol
Resting cellsa
10 gCDW L−1
Resting cellsa
30 gCDW L−1
Biotransformation time
Initial styrene
Final styrene
[h]
8
695
21
604
69
5.4
39.3
113.7
91.0
7.4
8
8
9
10
7
[mM]
[mM]
[mM]
[mM]
718
149
445
40
721
114
490
49
695
165
412
36
729
315
325
31
—
9.8
—
20.0
6.5
1.0
1.95
3.91
104
4.34
672
173
412
33
—
30.3
—
39.3
13.7
2.4
3.53
8.70
80
Final (S)-styrene oxide
Final 2-phenylethanol
Cell concentration after batch
Max. cell concentration
C-source fed (fed-batch, biotransf.)
C-source fed (biotransf.)
C-source remaining
Acetate formed
−1
−1
[g
L
L
]
]
6.6
5.5
6.7
CDW
[g
32.8
109.1
85.6
2.3
34.9
93.6
63.4
1.0
37.6
123.6
93.3
1.1
CDW
[g]
[g]
−1
[g Laq
[g Laq
]
]
−1
3.6
6.0
5.3
4.7
Ave. productivity
Max. productivity
[g Ltot−1 h−1
]
]
4.54
6.57
56
3.34
4.63
44
3.68
4.71
40
2.75
3.87
36
[g Ltot−1 h−1
−1
Max. spec. epoxidation rate
[U gCDW ]
Product yieldb
[mol mol−1
]
1.30
0.96
0.72
0.41
2.89
Cell mass is given in gram of cell dry weight (gCDW). a Data source: resting cell biotransfromations presented in Fig. 1 in Julsing et al.26 b Average
product yield on carbon source is calculated considering the amount of carbon source consumed during biotransformation.
Fig. 1 Styrene epoxidation by E. coli JM101 (pSPZ10) during two-liquid phase biotransformation with different carbon sources and organic carrier
solvents. (A) BEHP/glucose setup as published before,13 (B) EO/glucose; (C) BEHP/glycerol, (D) EO/glycerol. At 0 h, the biotransformation was
initiated by the addition of 1 L of the respective carrier solvent containing 80 mL styrene (∼695 mM) and 10 mL octane to induce styAB expression.
During the biotransformation, the carbon source feed rate was kept constant and was not reduced until the carbon source started to accumulate in the
aqueous phase at the end of the biotransformation.
Process schemes for styrene epoxidation catalyzed by
growing cells
BEHP/glucose, BEHP/glycerol, and EO/glucose setups, respect-
ively, indicating that these changes in process setup did not
influence the enantioselectivity of the biocatalyst. This confirms
earlier findings that variations in biocatalyst formulation (isolated
StyAB, resting cells, growing cells) and in reaction conditions
(cofactor regeneration system, presence of a solvent, phase ratio,
scale) do not affect the e.e. of styrene epoxidation catalyzed by
StyAB.14–16,36–40
For the economic and ecological assessment of the different bio-
transformation setups with growing cells (Fig. 1; Table 2),
detailed mass balances including upstream and downstream
operations are required. These were obtained by the software
SuperPro Designer, which allows the design of manufacturing
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Fig. 2 Mass-balances of resource consumption (A) and waste generation (B). Abbreviation: resting, resting cell biotransformation with biomass con-
centration indicated below.
plants on the basis of flow sheets.41 The flow sheet used in this
study was described before13 and is provided in the supplemen-
tary information.† Details of the individual unit operations are
described in the experimental part and the paper indicated above.
The downstream processing with EO as an organic phase was
identical to the one with BEHP, which is due to the comparable
distillation properties of these solvents. The production scale was
set at 1000 tons per year, which is typical for the synthesis of
comparable chiral compounds.42,43
Aside from the described deviations, the unit operations for the
resting cell plant were identical to the growing cell setup. In the
following section, all the biocatalytic (S)-styrene oxide process
alternatives are ecologically and economically assessed and their
performances compared and discussed in detail.
Process assessment - resource consumption and waste generation
The calculation of mass-balances is the first step in the evalu-
ation of a process and often a first indication for its economic
and ecological performance. Fig. 2 summarizes the substrate
consumption (input) and the waste generation (output) of the
investigated (S)-styrene oxide production processes.
Process schemes for styrene epoxidation catalyzed by resting
cells
The use of resources was most efficient in the BEHP/glucose
setup with growing cells, where 21.1 kg of substrates (15.9 kg
water) were used for the production of 1 kg of (S)-styrene oxide
(kgSO). In correlation with the lower productivities obtained, the
growing cell setups based on BEHP/glycerol, EO/glucose, and
EO/glycerol consumed 13, 32, and 42% more substrates per
kgSO produced, respectively. Both biotransformations with
resting cells were clearly inferior to the equivalent growing cell
counterpart (BEHP/glucose) with a 42–52% higher resource
demand. This is due to the relatively high resource demand for
biomass synthesis overriding benefits from the excellent product
yields on glucose of resting cells during biotransformation. The
low catalytic stability of resting cells, resulting in lower overall
productivities and product titers,26 is an important factor causing
With respect to the product yield on glucose during styrene
epoxidation, resting cells have been proven to be clearly superior
to growing cells.26 Whereas growing E. coli JM101 (pSPZ10)
−1 13
exhibited an average yield of 1.30 mol molglucose
,
the same
−1
strain reached an average yield of 4.34 mol molglucose in a
resting state.26 This efficient carbon source utilization makes
resting cell biotransformations worth considering for ecological
and economic assessment. The reaction stoichiometries for
resting cell bioconversions were based on data derived from
Julsing et al. Two experimental data sets with E. coli JM101
(pSPZ10) as biocatalyst were taken into consideration, which
differed in the applied biomass concentration, 10 and 30 gCDW
L−1 (CDW: cell dry weight). Since BEHP and glucose were
used as organic solvent and carbon source, respectively,
upstream and downstream processing is identical to the respect-
ive growing cell setup. The operation of the main fermenter
differs and can be divided in to a batch (8 h), an induction (4 h),
and a biotransformation phase (10 and 7 h with 10 and 30 gCDW
L−1, respectively). For the 30 gCDW L−1 setup, a fed-batch phase
additionally is introduced between the batch and the induction
phase, in order to reach the biomass indicated. The switch from
the induction to the biotransformation phase requires a nutrient
limitation in the medium to establish the resting cell state. On
the lab-scale, cells were harvested by centrifugation, washed,
and resuspended in fresh medium lacking a nitrogen source.26
On the industrial scale such a procedure is not feasible.44
However, the cells do not need to be separated from the growth
medium to achieve the resting state. For process evaluation, a
process scheme was assumed, in which the resting cell state is
induced by depletion of nitrogen within the growth medium.44
this high resource demand. Reduction of the biomass concen-
−1
tration from 30 to 10 g
L
decreases the growth-related and
CDW
thus the total glucose demand, but affects the productivity and
the product titers to such an extent that the overall mass balance
is even worse.
From a mass balance perspective, the process based on BEHP,
glucose, and growing cells performs best. This, however, does
not necessarily reflect the ecological and economical impact of
the investigated changes. In the following, this impact is quan-
tified by means of weighing environmental burdens and costs
caused by the different substrate and waste streams.
Process assessment - environmental impact potential
The environmental impact potential of a process is not only
defined by the amount of resources consumed and waste formed
648 | Green Chem., 2012, 14, 645–653
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inputs already during early process development phases. With
increasing detail level, other indicators may be included, such as
the utilization of fertilizer, the probability and severity of acci-
dents, emission by vehicles during farming and transpor-
tation.7,48 With respect to land use, glucose as well as glycerol
are regarded as environmentally friendly by the ABC classifi-
cation system.49 As a consequence, the contribution of the
carbon source to the total environmental indices for styrene
epoxidation is relatively small. The higher substrate demand of
glycerol-grown cells increases the respective environmental
indices. Hence, the switching of the carbon source is not
beneficial for the overall ecological performance. However, it
should be noted that the use of glycerol derived as a by-product
of another production process reduces the environmental burden
of the preceding process and might therefore be ecologically
attractive.
The application of resting cells is inferior in terms of ecologi-
cal aspects compared to the growing cell setups. In accordance
to the mass balances, the 10 gCDW L−1 setup suffers more from
the lower productivity during the biotransformation than it
Fig. 3 Environmental indices for the biocatalytic styrene epoxidation
processes. Abbreviation: resting, resting cell biotransformations with
biomass concentration indicated below.
profits from saving growth-related substrates. The application of
−1
but also by the individual impact of the materials involved.
Various ecological categories are characterized using an ABC
classification system that is providing a basis to estimate ecologi-
cal weighting factors that are used together with the material
balance to estimate an overall environmental index (see exper-
imental for details).9,45,46 The results achieved with this assess-
ment method are presented in Fig. 3.
a higher cell density of 30 g
L
improves the environmental
CDW
performance. The environmental index, however, was 1.4 times
higher compared to the equivalent with growing cells, correlating
with the higher resource demand (Fig. 2).
In summary, using EO instead of BEHP as extractive organic
phase significantly improved the ecological performance of bio-
catalytic styrene epoxidation, whereas the application of glycerol
and resting cells was neutral or negative with respect to ecologi-
cal efficiency.
For the two-liquid phase styrene epoxidation with growing
cells, the organic solvent BEHP already has been identified as
the most critical factor from an ecological perspective.13 This
was verified in this study as the environmental indices of all
BEHP-based processes were dominated by the organic phase,
contributing to approximately 40% of the total environmental
indices. A switch to the biodiesel component EO reduces the
impact of the organic phase significantly from 26.2 to 2.1 and
31.7 to 2.2 for the glucose- and glycerol-based setups, respect-
ively. EO is not toxic and its production from renewable
resources requires only moderate land use (‘B’-classification),
whereas BEHP requires more steps for its synthesis and requires
fossil raw materials (‘B’-classification) and is toxic (‘A’-classifi-
cation). Thus, the environmental compatibility of the total
process was enhanced as displayed by the reduction of the total
environmental indices. Opening the scope for the application of
biodiesel provides new opportunities towards more environmen-
tally friendly processes by replacing harmful solvents.28,29,47
The applicability of EO in bioprocesses is affirmed by the
absence of any radical polymerization during biotransformations
with intense aeration (results not shown). Such broader appli-
cation may support economics of biodiesel production in general
by accessing new markets, beside the usage as fuel.47
Process assessment - economic potential
Equally important to the environmental potential is the economic
feasibility of the different styrene epoxidation setups under
investigation, as the profitability of a process should not signifi-
cantly be compromised by operational changes. Annual pro-
duction costs are a key parameter and are represented in Fig. 4
and Table 3 for a 1000 t a−1 scale.
With growing cells, the annual production costs range
between 10.2 and 11.7 $ kgSO−1. The differences are mainly pro-
ductivity-related. With decreasing productivities, facility-depen-
dent costs, installments, and raw material costs rise due to
increasing equipment size and increasing resource demands. The
contribution of EO to the raw material expenses is slightly lower
than that of BEHP, due to the lower density at a similar price per
kg. However, as an effect of the lower productivities in the EO-
based setups, the overall raw material costs rise above the
expenses of the BEHP-based processes, although the difference
between the two different setups is relatively small.
The impact of the carbon source on the operational costs
appeared to be more important. It has to be considered that
market prices for glucose and glycerol vary depending on econ-
omic developments and the location of the production site. This
is especially true for the glycerol price and availability, which
vary depending on the biodiesel industry being supported by
governmental subsidies and tax benefits to variable extents.47
Following the market price development for refined glycerol
described amongst others by Pagliaro et al.,50 equal prices for
A major difference between glycerol and glucose is their
different production routes. The assessment of the agricultural
production of glucose illustrates the challenge in judging sub-
strates from differing origins. Agriculture involves numerous
grey inputs, which are environmental burdens arising during the
preparation of the input compounds, before they enter the exam-
ined process.46 Here, complexity of synthesis, raw material avail-
ability, and land use were considered for assessing the grey
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Table 3 Production costs for (S)-styrene oxide
BEHP
glucose
k$
BEHP
glycerol
k$
EO
glucose
k$
EO
glycerol
k$
Resting cells 10 gCDW
Resting cells 30 gCDW
L−1
k$
L−1
k$
Facility dependent costs
Depreciation
Insurance
3179
1808
190
3269
1865
196
3426
1964
207
3503
2012
212
3761
2174
229
3612
2081
219
Local taxes
Factory expenses
Maintenance
381
300
500
393
300
515
413
300
542
424
300
555
458
300
600
438
300
574
Raw materials
Ammonium
Carbon source
Organic phase
Octane
2862
17
862
1170
42
3265
19
934
1413
52
3227
19
1168
1094
57
3652
23
1469
1187
62
3818
7
618
2183
78
3938
17
1291
1705
62
Riesenberg salts
Styrene
Thiamine
117
632
5
140
680
5
153
703
6
166
707
6
210
699
8
166
665
7
Trace elements
Magnesium sulfate
Sodium hydroxide
1
14
2
1
19
2
1
23
3
1
28
3
1
11
3
1
21
3
Labor
2801
420
251
259
437
2793
419
218
298
506
2787
418
367
314
576
2766
415
341
354
633
2731
410
171
353
771
2745
412
358
356
640
Laboratory/QC
Consumables
Waste treatment
Utilities
Total unit production
costs
10 209
10 768
11115
11 664
12 015
12 061
Average installmenta
3716
3838
4036
4141
4468
4282
Annual production costs
13 925
14 606
15 151
15 805
16 483
16 343
a Average annual installment payment over 10 years includes installment of direct fixed capital (interest rate: 9% over a period of 10 years) and
installment of working capital (interest rate: 12%; period: 6 years).
Fig. 4 Annual production costs at current carbon source prices (A) and in dependence of the glycerol price (B). For the economic assessment, a pur-
chase price of 0.5 $ kg−1 was assumed for glucose and glycerol. Costs for the carbon source and organic phase are displayed separately and are not
included in the cost of raw materials.
glucose and glycerol (0.5 $ kg−1) were assumed to be realistic
(status: 2009). On this basis, glucose is the preferred growth sub-
strate for styrene epoxidation (Fig. 4B). In order to evaluate the
effect of the glycerol price on annual production costs, a sensi-
tivity analysis was performed showing that, depending on the
solvent used, glycerol becomes cost-competitive below prices of
0.20 or 0.31 $ kg−1 (Fig. 4B). Glycerol costs may be lowered to
these dimensions by coupling the styrene oxide plant to another
process, in which glycerol accumulates as a waste product.
However, the crude glycerol formed as a by-product is normally
of low grade. For example, during biodiesel production, glycerol
streams are typically contaminated by high amounts of short-
chain alcohols, free fatty acids, and inorganic salts used during
transesterification.50 This may affect costs for downstream pro-
cessing while still sustaining biocatalyst production and activity.
For a final judgment of the achievable profitability with the two
650 | Green Chem., 2012, 14, 645–653
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carbon sources, the purification costs have to be weighed against
the purchase price of glucose and the savings with respect to gly-
cerol disposal costs of the preceding process.
plasmid pSPZ10 contains the styrene monooxygenase genes
styAB from the Pseudomonas sp. strain VLB120 under the
control of the alk regulatory system from Pseudomonas putida
GPo1.16
From an economical point of view, the application of resting
cells for styrene epoxidation is inferior to the use of growing
cells (Table 3). In correlation to the generally lower productiv-
ities and final product titers, the investment for larger equipment
and the higher resource consumption increase facility-dependent
expenses, installments, and raw material costs. The setup with
lower cell concentrations economically benefits from the lower
growth-related substrate demand. As consequence, waiving high
biomass concentrations is able to compensate for the lower pro-
ductivities in economic terms, resulting in nearly identical pro-
duction costs for both investigated resting cell setups. Reuse of
resting cells for several cycles represents a possibility to reduce
time and resources consumed for biomass production. Princi-
pally, recycling of the biocatalyst is possible, as inhibition rather
than permanent deactivation of the biocatalyst was observed
with resting cells.26 In order to avoid tedious cell harvesting after
each cycle, cell immobilization (e.g., in biofilms51,52) or cell
retention in membrane reactors53,54 (problematic with stable
emulsions) may facilitate biocatalyst recovery.
Transformants were selected on lysogeny broth (LB) agar
plates containing 50 μg mL−1 kanamycin.56 A single colony was
picked and used to inoculate 5 ml liquid LB medium. After
reaching the stationary growth phase, 1 mL was diluted in
99 mL M9 medium56 complemented with 1 mL L−1 US* trace
elements,57 1 mL L−1 1% (w/v) thiamine solution, and 2 mL
L−1 1 M magnesium sulfate (MgSO4) solution. After overnight
growth, this culture was used to inoculate a KLF 2000 reactor
(Bioengineering, Wald, Switzerland) containing 900 mL
modified Riesenberg medium.58 Either 15 g L−1 glucose or 20 g
L−1 glycerol were used as sole carbon and energy source. The
pH was maintained at 7.20 by adding 30% phosphoric acid and
25% NH4OH solutions. The latter additionally served as a nitro-
gen feed. The batch phase was run overnight. Aeration and stir-
ring rate were maintained constant at 1 L min−1 and 1500 rpm,
respectively. After depletion of the carbon source, a feed consist-
ing of either 730 g L−1 glucose and 19.6 g L−1 MgSO4 × 7H2O
or 600 g L−1 glycerol and 16.1 g L−1 MgSO4 × 7H2O was
started and increased until a biomass concentration of approxi-
mately 18 g L−1 was reached. Then, the biotransformation was
started by the addition of the organic phase consisting of
910 mL carrier solvent, either BEHP or EO, 80 mL styrene, and
10 mL octane serving as the inducer of styAB expression. The
dissolved oxygen concentration was kept above 20% of satur-
ation by increasing the stirring speed (to a maximum of 2800
rpm) and the aeration rate (up to 2.5 L min−1). Antifoam A
(Sigma–Aldrich Chemie Gmbh, Steinheim, Germany) was
added only in case of excessive foaming. The biotransformation
was monitored by taking samples every hour as described pre-
viously.57 For two-liquid phase biotransformations, volumetric
rates and concentrations are given per liter of aqueous phase
(Laq), organic phase (Lorg), or total volume (Ltot).
At the present state, growing cells are economically more
attractive for asymmetric styrene epoxidation than resting cells,
whereas the use of EO and glycerol may be economically inter-
esting, depending on the company-specific weighing of environ-
mental factors and the development of raw material prices.
Conclusion
In conclusion, styrene epoxidation by growing cells was both
ecologically and economically superior to resting cells, mainly
due to the higher stability of the biocatalyst. The assessment also
showed that the glucose amount required for the production of
the resting cell biocatalyst is an important factor for overall per-
formance evaluation. However, potential recycling of resting cell
biocatalysts opens the opportunity to capitalize on their excellent
yield on glucose during the biotransformation.
In this study, we were able to optimize the ecological perform-
ance of a whole-cell two-liquid phase biotransformation by
exchanging the organic phase. EO was shown to be an environ-
mentally attractive and cost-competitive solvent. Application of
biodiesel provides new opportunities to improve solvent-based
processes with respect to ecological aspects. Furthermore, it was
shown that glycerol can be used as a carbon source for styrene
epoxidation, giving better flexibility in terms of carbon source
selection. However, the process performance suffered from lower
productivities.
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-5 ms
column (Varian, Inc., Palo Alto, CA, USA). Analytical con-
ditions were identical as published before.13 Specific epoxidation
activities are given in units (U) per gram cell dry weight (CDW),
whereby 1 U is defined as the activity forming 1 μmol product
per 1 min.
The enantiomeric excess of (S)-styrene oxide was determined
by separation of the two enantiomers on a chiral RT-BetaDEXsm
column (Restek Corporation, Bellefonte, PA, USA). After main-
taining the temperature at 100 °C for 1 min, the oven temperature
was raised to 105 °C at a heating rate of 0.5 °C min−1, followed
by heating at 30 °C min−1 to 230 °C, which was maintained for
3.8 min. The enantiomeric excess (e.e.) was calculated according
This study affirms the capability of ecological and economic
assessments to identify critical process parameters and to
enable systematic process development towards industrial
implementation.
Experimental
to the following equation: e.e. [%] = 100 × |(S − R)| (S + R)−1
Glucose, glycerol, 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
.
Strain, cultivation and two-liquid-phase biotransformation
E. coli JM101 (supE thi-1 Δ(lac-proAB) F′ [ traD36 proAB+
lacIq lacZΔM15]) was used as the host strain.55 The expression
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America, Inc., Pleasanton, CA, USA), after extraction of
aqueous styrene, styrene oxide, and 2-phenylethanol by BEHP.
The flow rate of 5 mM sulfuric acid as mobile phase was set to
1.0 ml min−1. The column temperature was kept constant at
40 °C. Detection of the analytes was done by a L-2420 UV-Vis
and a L-2490 refractive index module.
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.
Acknowledgements
Andreas Schmid (TU Dortmund) is acknowledged for fruitful
collaboration and mentoring. Furthermore, we thank Rudolf
Krumbholz and Peter Lembke (K.D.-Pharma GmbH, Germany),
Michael Breuer and Peter Saling (BASF SE, Germany), Muham-
mad Abdul Kholiq (Environmental Technology Center – BPPT,
Indonesia), and Jin Byung Park (Ewha Womans University,
Korea) for helpful discussions. This work was supported by the
Deutsche Bundesstiftung Umwelt (DBU; project AZ13145) and
by the Ministry of Innovation, Science, Research and Technol-
ogy of North Rhine-Westphalia (Bio.NRW, Technology Platform
Biocatalysis, RedoxCell).
Cell concentrations were determined spectrophotometrically
on a Libra S11 spectrophotometer (Biochrom Ltd., Cambridge,
UK) at a wavelength of 450 nm.59
Assessment method
Economic and ecological process efficiencies were assessed by
means of a method designed for utilization at early development
stages of fine chemical processes.9,41,45,46 Mass-balances were
generated on the basis of process flow-sheets by SuperPro
Designer version 6.0 (Intelligen, Inc., Scotch Plains, NY, United
States).
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 cat-
egories are land use, raw material availability, complexity of syn-
thesis, 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), whereby “A”
stands for high, “B” for medium, and “C” for low risk in the
respective category. The criteria for the classification have been
published elsewhere.13,49 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
40 : 60 ratio.
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