Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Biocatalysis, sustainability, and industrial
applications: Show me the metrics
Pablo Domínguez de María
Biocatalysis has been traditionally considered an environment-
friendly and industrial toolbox for organic synthesis. However,
in many cases these statements are not substantiated with real
metrics (e.g. E-factor and productivities). This paper discusses
recent developments that may contribute to generate greener
biocatalytic applications with industrial interest. Namely,
aqueous-based processes with extremely high substrate
loadings; solvent-free reactions in which neither solvents nor
water are used; and finally, biogenic solvents as well as deep
eutectic solvents. From all of them, solvent-free systems are
superior both in terms of greenness and industrial use (but
they cannot be always implemented). The described systems
can be combined in flow (continuous), and with solvent and
water recycling, leading to powerful alternatives for future
sustainable processes. Overall, providing metrics is key to
honestly assess a biotransformation and to put forth further
research to improve systems.
Addresses
Sustainable Momentum, SL, Av. Ansite 3, 4-6, 35011 Las Palmas de
Gran Canaria, Canary Is, Spain
Corresponding author: Domínguez de María, Pablo (dominguez@
sustainable-momentum.net)
Current Opinion in Green and Sustainable Chemistry 2021,
31:100514
This review comes from a themed issue on Heterogeneous bio-
catalysis for sustainable chemistry
Edited by Lorena Betancor and Fernando López-Gallego
https://doi.org/10.1016/j.cogsc.2021.100514
2452-2236/© 2021 Elsevier B.V. All rights reserved.
Keywords
Biocatalysis, Green chemistry, E-factor, Solvent-free, Water, Deep
eutectic solvents.
Biocatalysis and the green chemistry
principles
Green and sustainable chemistry strategies have
emerged over the last decades, as reflected in educa-
tional programs focusing on the interdisciplinary
training of qualified professionals [1*]. Due to issues
associated with resource depletion, global warming, and
ecosystem degradation, novel paradigms that can
ameliorate these challenges must be established, and
having an educated manpower is key to success. The 12
Green Chemistry principles have served (and do serve)
as the references to be followed [1*].
www.sciencedirect.com
Green and Sustainable Chemistry
Biocatalysis has gained importance as strategy to
(industrially) produce chemicals with high selectivity,
while also being considered a sustainable alternative
[1,2]. However, the perspective of biocatalysis being
‘environmentally friendly’, or/and an ‘industrially sound
technology’ has not been always substantiated with
convincing metrics. As reported elsewhere [3,4], aca-
demic publications tend to (ab)use (of) somewhat empty
statements related to the greenness of a given bio-
catalytic process, and the same consideration often ap-
plies when it comes to industrial processes. Undoubtedly,
biocatalysis has the potential to become a strong ally for
Green Chemistry and Industrial Synthesis. But this
cannot be taken for granted, and enzymes need to earn
itdas it must be done by any other catalyst. To that end,
reliable metrics must be provided, together with a
constructive criticism reflecting the further needed im-
provements. Fortunately, the interests of Green Chem-
istry can be aligned with the industrial needs of having
intensified processes with huge substrate loadings, high
yields, and simplified downstream processing units.
Green processes can be thus developed while establish-
ing highly efficient industrial applications.
To determine the environmental impact of a given
synthetic reaction, several parameters have been pro-
posed. The Sheldon’s E-factor (kgs of waste/kg of
product) is surely the most popular one [5,6]. Its
simplicity, intuitiveness, and ease of application make it
highly recommendable to assess (bio)catalytic reactions.
The E-factor has been complemented with other alter-
natives, such as the Eþ-factor, which takes into account
the energy contribution to the waste (as CO2 emissions)
[7*,8]. As any (bio)chemical process generates wastes,
the goal should be providing greener and more circular
alternatives, to align industrial cycles with the natural
ones. Similarly, it must be noted that the environmental
impact of a (bio)catalytic reaction does not only concern
the chemical reaction itself. Substrates and catalysts
must be produced (or extracted) and transported where
the synthesis takes place. Ultimately, products must be
packaged and delivered to the final destination. Overall,
more holistic Life Cycle Assessments are needed
[3,5,6]. For instance, for a given biocatalytic reaction,
the impact of enzyme production (e.g. wastewater
associated to fermentation), as well as the impact of
immobilizing enzymes need to be considered [9e11].
The possibility of using biogenic resources (delivered by
biorefineries) may help diminish the environmental
burdens associated to chemical syntheses.
Current Opinion in Green and Sustainable Chemistry 2021, 31:100514
2 Heterogeneous biocatalysis for sustainable chemistry
Research on sustainable (bio)catalysis should focus on
three main interconnected areas: solvents (e.g. solvent-
free, or use of biogenic, petrochemical or neoteric sol-
vents), water (and wastewater generated), and catalyst
type (free enzyme, whole-cell, immobilized or not)
[12*]. In the following sections, biocatalytic examples
providing sound metricsdenvironmental and industri-
aldwill be discussed, as they may serve as inspirational
paths for a greener enzyme catalysis.
Biocatalysis in aqueous media: metrics, not
just statements
Enzymes have evolved in aqueous environments, and
this is surely a reason why water (buffer) is typically the
medium for biocatalysis [12*]. In fact, for hydrolases
water is a substrate as well. Water is traditionally claimed
as a green, nontoxic solvent, what creates a superior
position to fulfill the Green Chemistry postulates [3e
6]. Yet, water resultant from a (bio)chemical process is
not water anymore, but actually wastewater. It contains
traces (or higher concentrations) of chemicals, organic
solvents, reagents, (by)products, salts, enzymes, etc. It
must be treated before releasing it to the milieu. Given
that many geographical areas suffer (edible) water
shortages, it appears mandatory to carefully assess water
consumption for a synthetic reaction in aqueous media.
On that basis water must be included in the E-factor,
particularly if water is the reaction medium [3e6,12*],
and this boosts the E-factor considerably. Take, for
instance, a biocatalytic reaction in buffer with condi-
tions commonly reported in the literature, producing
~1 g product L1. The contribution of water to the E-
factor would be ~1000, and such process should not be
addressed as ‘sustainable’ or ‘green’, or ‘industrially
scalable’. However, water needs to be reaction media for
fermentations, as well as for many biocatalytic processes.
Figure 1
Contribution of water to E-factor of (bio)catalysis in aqueous media, at different product concentrations, and with recycling scenarios. For simplification,
there are no losses of water between cycles (a realistic vision would probably be 10% loses per cycle).
Current Opinion in Green and Sustainable Chemistry 2021, 31:100514
Herein, environmental metrics for water must be pro-
vided. Some solutions could be establishing water
recycling loops, together with intensified processes (e.g.
huge substrate loadings) together with continuous sys-
tems [13*].
Figure 1 depicts a theoretical exercise, pinpointing the
contribution of water to E-factor in an aqueous-based
reaction and considering a broad range of production
metrics. The intervals can be considered prototypical for
fermentations (25e200 g product L1) to biocatalysis
(50e200 g product L1) and to ‘intensified’ biocatalysis
(>200 g product L1). Similarly, the improvement in E-
factor (water) by recycling the water system one time (2
cycles) or three times (4 cycles) is shown as well.
Taking 100 g product L1 d assumed as the ‘rule-of-
thumb’ threshold for an industrially attractive proc-
essdE-factor water contribution is 10 (one cycle).
Obviously, the more intensified the process is (higher
loadings), the lower the environmental impact and the
better for an industrial application. Water recycling may
play a key role, but it has been surprisingly overlooked in
(bio)catalysis. For example, the Greiner group reported
up to 20-fold reduction in E-factor (water) through a
continuous reactor with recycling [14]. Combining high
substrate loadings with smart (continuous) recycling
may align environmental and industrial thresholds.
Recent examples comprise gas-phase fermentations
(e.g. for geranic acid or for alkenes) [15,16] where
products are gases that are stripped from the fermenter
to simplify the downstream. Hence, the media recycling
(or a continuous mode) could be envisaged. Similarly, as
a biocatalytic example, the Gröger group has developed
the production of nitriles from aldoximes by means of
aldoxime dehydratases [17,18*]. The concept has been
www.sciencedirect.com

successful for the synthesis of adiponitrile [17] and may
serve as an alternative for current petrochemical routes
[18*]. A recent impressive case reports the production
of aliphatic nitriles in aqueous media, with substrate
loadings of up to 1.4 kg L1 [19**] (Scheme 1).
The process used E. coli whole cells overexpressing the
aldoxime dehydratase from Bacillus sp. Ethanol (10% v/v)
was used as a cosolvent to assist aldoxime dissolution,
and the substrate was added stepwise (up to 9 times).
Given the remarkable high substrate loadings, the
contribution of water to the E-factor resulted in the
range of 1. A continuous process with water recycling
might even improve the reported figures. Overall,
identifying enzymes and variants that can accept these
severe conditions appears as an interesting research path
to align sustainability and industrial applications.
Biocatalysis in solvent free: the ‘Holy Grail’
of sustainability and industrial applications
The classic statement is ‘the best solvent is no solvent at
all’. If every chemical process could be efficiently con-
ducted in neat substrates, there would not be any
incentive for developing other process systems. Solvent-
free (neat substrates) reactions enable the highest
substrate loadings together with a largely diminished E-
factor, as neither solvents nor water are employed.
Despite these advantages, the development of solvent-
free approaches remains underexplored, except for hy-
drolases and hydroxynitrile lyases [3]. Lipases in
nonaqueous media (namely organic solvents or even
solvent-free) are known for decades now [3]. Solvent-
free (continuous) esterifications enable highly inte-
grated and environment-friendly strategies [13*,20].
Lipases evolved to catalyze hydrolysis at the interphase
between aqueous media and hydrophobic media. Thus,
their evolution has developed biochemical features in
the protein scaffold (salt bridges, interaction of amino
acidic networks, etc.) [21,22], conferring them special
robustness. However, when it comes to other enzymes,
examples in solvent free are scarce. Despite some ex-
amples showcased in the 80e90s with dehydrogenases, it
was not until more recently that a renewed interest in
solvent-free media emerged, with some examples re-
ported for oxidoreductases [23e25]. To overcome the
enzyme instability in such media (unlike lipases), some
Scheme 1
Aldoxime dehydratase (E. coli whole-cells)
OH
N
Buffer - Ethanol (90:10 v/v); 30 ºC; 24 h
Stepwise addition (9 times)
Up to 1.4 kg L-1
Biocatalytic synthesis of aliphatic nitriles using aldoxime dehydratases and high substrate loadings in aqueous media [19**].
www.sciencedirect.com
Biocatalysis, sustainability, and industry Domínguez de María 3
(nonbulk) water is added, and whole cells are used, as
they protect the enzyme from the severe conditions,
enabling an efficient biotransformation. Excellent pro-
ductivities (hundreds of grams of product per liter) are
reached with low overall E-factors (<5) [3]. Solvent-free
has been used in continuous for the enantioselective
reduction of 2-butanone to (R)- or (S)-2-butanol, with
outstanding productivities (>2200 g product L1 d1)
[26]. Once again, with these approaches both environ-
mental goals and industrial specs are reached.
Very recently, solvent-free media has been reported for
other enzyme types, such as aldoxime dehydratases
[27**] and peroxygenases [28*,29**]. For aldoxime
dehydratases, E. coli cells overexpressing the enzyme
were immobilized in ‘super-absorbers’, namely poly-
acrylic acid with high water-binding affinity. That
immobilization led to an ‘immobilized aqueous phase’,
what enabled the efficient use of the biocatalyst in
solvent-free, and under flow conditions [27**]. For
peroxygenases, the first proof-of-concept reported the
stepwise addition of tBuOOH as oxidant over neat al-
kenes for asymmetric epoxidations. The unspecific
peroxygenase from Agrocybe aegerita (AaeUPO) was used,
and product concentrations reached up to 360 mM,
leading to low E-factors [28**]. The same enzyme was
combined with a photobiocatalyst to produce H2O2 in
situ (out of LED light and O2), to conduct the enzymatic
hydroxylation of (neat) cyclohexane. Herein, to rein-
force the stability of the enzyme, immobilization in
alginate beads was established [29**]. The combination
of some (nonbulk) water addition, together with whole
cells and immobilization seem to be key to build bio-
catalytic solvent-free systems with high production and
low(er) environmental impact.
Biocatalysis in organic and neoteric
solvents
Previous sections have shown how aqueous media or a
solvent-free can be established, leading to integrated
systems in which industrial attractiveness and promising
environmental metrics are provided. However, not all
enzymes or not all syntheses will afford solvent-free
media, and other options must be explored. Biogenic
media seem an alternative to (try to) combine solvents
with acceptable environmental impact. Applications of
enzymes in 2-methyltetrahydrofuran (2-MeTHF) [30], as
N
93 % conversion
(Water contribution to E factor ~ 1)
Current Opinion in Green and Sustainable Chemistry
Current Opinion in Green and Sustainable Chemistry 2021, 31:100514
4 Heterogeneous biocatalysis for sustainable chemistry

Scheme 2

O OH
Horse liver ADH (HLADH) 2
H
2
Cinnamyl alcohol
75 g L-1 d-1
2 NADH + 2 H + 2 NAD+

N O
N OH
Cl- O +
Cl- OH
OH Choline Chloride: 1,4-Butanediol (1:4 mol:mol) OH
OH
OH DES - Buffer (80-20 v/v)

Current Opinion in Green and Sustainable Chemistry

ADH-catalyzed alcohol reduction in DES-water media, using 1,4-butanediol as smart substrate to shift the equilibrium to product formation [38**].

well as in cyclopentyl methyl ether (CPME) [31] have
been reported. For instance, the Rother group has
described the use of imine reductases in CPME, enabling
imine reduction in water-free media (to avoid sponta-
neous hydrolysis) [32]. More recently, other biogenic
solvents like (cellulose-derived) dihydrolevoglucosenone
(CyreneÔ) have been assessed for biocatalysis for lipases
[33] and oxidoreductases [34]. Research is needed to
identify benign synthetic routes for these solvents, as well
as their potential toxicology profiles. Screening of novel
enzymes should consider these solvents, as this might
lead to the identification of useful catalysts for biogenic
media.
The last decade witnessed the use of deep eutectic
solvents (DES) in biocatalysis. Given their ease of
preparation and biodegradability, DES are considered
alternatives for future (bio)syntheses (albeit DES
components are not always biogenic). A recent strategy
in biocatalysis is using DES as reaction media and as
substrate, thus enhancing the loadings significantly.
Lipase-catalyzed ‘solvent-free’ esterifications of
menthol in DES using it as DES component have been
implemented [35,36]. The process reached almost full
conversion (>95%) when ()-menthol was employed
[35] and could also serve as kinetic resolution when the
DES was designed with racemic menthol [36]. In
another area, the DES containing cofactor regeneration
substrates has been developed for oxidoreductases.
Thus, DES-containing glucose (combined with buffer)
was used as ancillary substrate for the enantioselective
reduction of ketones to afford chiral alcohols, with
excellent conversions (99%) and enantioselectivities
(>99%) [37**]. Likewise, the Kara group has reported
the production of cinnamyl alcohol through enzymatic
reduction of cinnamaldehyde was conducted in a DES-
containing 1,4-butanediol as component and as smart
co-substrate. With that approach, a promising produc-
tivity of cinnamyl alcohol of 75 g L1 d1 were reached
under nonoptimized conditions [38**] (Scheme 2).
Many DES often exert a high viscosity, further
enhanced with high substrate loadings. To circumvent
this, water ‘as cosolvent’ (up to 20% v/v) can be added
and DES remains as nonconventional media (as
observed by full conversions in lipase-catalyzed ester-
ifications). DES-water media displays a significantly
reduced viscosity for efficient biotransformations
[39] and enables biocatalytic flow processes. The
Guajardo group has reported the continuous lipase-
catalyzed esterification of benzoic acid with glycerol
in a DES containing choline chloride and glycerol as
component and substrate [40**]. The combination of
low viscous (biodegradable) nonconventional media
with flow systems may certainly enable powerful syn-
ergies for future biotransformations [13*]. To reinforce
the robustness of lipases, their immobilization in cross-
linking aggregates leads to highly stable derivatives to
perform reactions in DES [41].
Conclusions
Biocatalysis can actually become a fundamental actor to
develop green chemistry with industrial interest. To
reach that, metrics are needed, and the E-factor is an
excellent rapid-to-use parameter to assess enzymatic
reactions. Processes in water, solvent-free or in biogenic
and neoteric solvents display potential, but the final
environmental impact must be decided case by case
(from E-factor to more holistic Life Cycle Assessment
iterations). The assessment must cover not only the
process but also other aspects such as the environmental
contribution of the enzyme production, immobilization,
etc. [42e44], as well as aspects related to downstream
processing. In a general sense, solvent-free systems are
superior, but they cannot be always implemented. The
quest for enzymes that can efficiently perform reactions
in highly intensified concepts, as well as the setup of
flow continuous processes appear as future promising
tools to align green(er) metrics with industrial
applications.

Current Opinion in Green and Sustainable Chemistry 2021, 31:100514 www.sciencedirect.com


Funding
This research did not receive any specific grant from
funding agencies in the public, commercial or not-for-
profit sectors.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.
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Current Opinion in Green and Sustainable Chemistry 2021, 31:100514
Use of glucose as component for DES, as well as ancillary substrate
for oxidoreductases. Excellent conversions and enantioselectivities
were reached, showing for the first time the conceptual approach of
using sugars as DES components for a cheap solvent with
applications.
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First proof-of-concept of using smart cosubstrates as DES
component (solvent) as well as substrate for shifting enzymatic
oxidoreductions to high productivities. As exemplary substrate,
the production of cinnamyl aldohol – a compound with industrial
interest – is reported in promising figures in a water-free (non-
bulk) media.
39. Guajardo N, Domínguez de María P, Ahumada K,
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40. Guajardo N, Schrebler R, Domínguez de María P: From batch to
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catalyzed esterifications in low viscous deep eutectic sol-
vents with buffer as cosolvent. Bioresour Technol 2019, 273:
320–325.
Biocatalysis in continuous systems using DES as media and as sub-
strate (glycerol) for lipase-catalyzed esterifications. Combination of
water and DES leads to a low viscous non-conventional solvent, that
enables the pumping for a continuous device, while enabling efficient
esterifications until full conversion.
41. Guajardo N, Ahumada K, Domínguez de María P: Immobilized
lipase-CLEA aggregates encapsulated in Lentikats® as
robust biocatalysts for continuous processes in deep
eutectic solvents. J Biotechnol 2020, 310:97–102.
42. Ripoll M, Velasco-Lozano S, Jackson E, Diamanti E, Betancor L,
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serinol catalyzed by biocatalytic composites made of whole
cells and immobilized enzymes. Green Chem 2021, 23:
1140–1146.
43. Grajales-Hernández D, Armendáriz-Ruiz M, Velasco-
Lozano S, López-Gallego F, Mateos-Díaz JC: Chitosan-
based CLEAs from Aspergillus Niger type A feruloyl
esterase: high-productivity biocatalyst for alkyl ferulate
synthesis. Appl Microbiol Biotechnol 2020, 104:
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44. Velasco-Lozano S, da Silva ES, Llop J, López-Gallego F: Sus-
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