Comparative study of the bioconversion process using R-(+)- and S-(-)-limonene as substrates for Fusarium oxysporum 152B

Article abstract of DOI:10.1016/j.foodchem.2014.11.059

This study compared the bioconversion process of S-(-)-limonene into limonene-1,2-diol with the already established biotransformation of R-(+)-limonene into α-terpineol using the same biocatalyst in both processes, Fusarium oxysporum 152B. The bioconversion of the S-(-)-isomer was tested on cell permeabilisation under anaerobic conditions and using a biphasic system. When submitted to permeabilisation trials, this biocatalyst has shown a relatively high resistance; still, no production of limonene-1,2-diol and a loss of activity of the biocatalyst were observed after intense cell treatment, indicating a complete loss of cell viability. Furthermore, the results showed that this process can be characterised as an aerobic system that was catalysed by limonene-1,2-epoxide hydrolase, had an intracellular nature and was cofactor-dependent because the final product was not detected by an anaerobic process. Finally, this is the first report to characterise the bioconversion of R-(+)- and S-(-)-limonene by cellular detoxification using ultra-structural analysis.

Full text of DOI:10.1016/j.foodchem.2014.11.059

Accepted Manuscript
Comparative study of the bioconversion process using R-(+)- and S-(–)-limo-
nene as substrates for Fusarium oxysporum 152B
Gustavo Molina, Murillo L. Bution, Juliano L. Bicas, Mary Anne Heidi Dolder,
Gláucia M. Pastore
PII:
S0308-8146(14)01788-9
DOI:
http://dx.doi.org/10.1016/j.foodchem.2014.11.059
FOCH 16744
Reference:
Food Chemistry
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22 June 2014
27 October 2014
10 November 2014
Please cite this article as: Molina, G., Bution, M.L., Bicas, J.L., Dolder, M.A.H., Pastore, G.M., Comparative study
of the bioconversion process using R-(+)- and S-(–)-limonene as substrates for Fusarium oxysporum 152B, Food
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.11.059
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Comparative study of the bioconversion process using R-(+)- and S-(‒)-limonene as
substrates for Fusarium oxysporum 152B
Gustavo Molina1,2,*, Murillo L. Bution1, Juliano L. Bicas1, Mary Anne Heidi Dolder3,
Gláucia M. Pastore1
1Laboratory of Bioflavors – Department of Food Science – School of Food Engineering,
University of Campinas, Campinas, São Paulo, Brazil.
2Institute of Science and Technology, Food Engineering – UFVJM, Diamantina,
Minas Gerais, Brazil.
3Department of Structural and Functional Biology – Institute of Biology – University
of Campinas, Campinas, São Paulo, Brazil
*Corresponding author:
Postal Address: Laboratory of Bioflavors, Department of Food Science, School of Food
Engineering – University of Campinas - Monteiro Lobato St., 80, CEP: 13083-862 –
Campinas, São Paulo, Brazil.
Phone number: +55 (19) 3521-4090 - Email address: gustavomolinagm@gmail.com
ABSTRACT
This study compared the bioconversion process of S-(‒)-limonene into
limonene-1,2-diol with the already established biotransformation of R-(+)-limonene into
α-terpineol using the same biocatalyst in both processes, Fusarium oxysporum 152B.
The bioconversion of the S-(–)-isomer was tested on cell permeabilisation under
anaerobic conditions and using a biphasic system. When submitted to permeabilisation
trials, this biocatalyst has shown a relatively high resistance; still, no production of
limonene-1,2-diol and a loss of activity of the biocatalyst were observed after intense
cell treatment, indicating a complete loss of cell viability. Furthermore, the results
showed that this process can be characterised as an aerobic system that was catalysed by
limonene-1,2-epoxide hydrolase, had an intracellular nature and was cofactor-dependent
because the final product was not detected by an anaerobic process. Finally, this is the
first report to characterise the bioconversion of R-(+)- and S-(‒)-limonene by cellular
detoxification using ultra-structural analysis.

Keywords:
Monoterpenes,
Limonene-1,2-diol,
α-Terpineol,
Anaerobic
biotransformation, Cell permeabilisation.
1. Introduction
The methods for obtaining flavour compounds include direct extraction from
nature, chemical transformations and biotechnological transformations, such as
microbial and enzymatic biotransformations, de novo synthesis and the use of genetic
engineering tools (Berger, 2007). The scientific literature contains many examples of
reviews that address the chemical reactions of terpenes to produce flavours (Swift,
2004) and the biotransformation of volatile terpenes for aroma production (Bicas,
Dionísio, & Pastore, 2009; De Carvalho & Da Fonseca, 2006).
Despite the great number of industrial applications of aroma compounds
produced via chemical synthesis, which is still responsible for a large portion of the
market due to satisfactory yields, bioprocesses possess a number of inherent advantages
compared with the classical chemical processing because they occur under mild
conditions, present high regio- and enantio-selectivity, do not generate toxic waste and
result in products that may be labelled “natural” (Bicas et al., 2009). Additionally,
biotechnological processes usually involve less damaging processing conditions for the
environment and yield desirable enantiomeric flavour compounds. Thus, bioflavours
appeal to many sectors and have a high market value (Krings & Berger, 1998).
The biocatalytic conversion of a structurally related precursor molecule
(bioconversion or biotransformation processes), is often a more adequate strategy that
allows the significantly enhanced accumulation of a desired flavour product. As a
prerequisite for this strategy, the precursor must be present in nature, and it must be
easily feasible to isolate it in sufficient amounts from the natural source in an
economically viable fashion (e.g., the monoterpenes limonene and α-pinene). Among
the most targeted substrates for biotransformation/bioconversion approaches are the
monoterpenes (Krings & Berger, 1998).
Several reports in the literature have addressed the biotransformation of
limonene to many oxygenated derivatives; these have been reviewed by Maróstica Jr.
and Pastore (2007a), and Duetz, Bouwmeester, Van Beilen, and Witholt (2003).
Research and development on the degradation of limonene has continued to draw much
attention, due to a wide variety of conversion products, such as perillic compounds,

carveol and carvone at significant amounts, which could be more valuable in the fields
of cosmetics, food ingredients, drug and chemical synthesis.
One interesting product obtained from the biotransformation of limonene is α-
terpineol. This product is a stable alcohol that is widely distributed in nature and is
typically used in household products, cosmetics, pesticide and flavour preparations.
Additionally, it is one of the most commonly used perfume chemicals (Bauer, Garbe, &
Surburg, 2001). The production of this monoterpene alcohol has been described using a
wide range of microorganisms as catalysts (Tan, Day, & Cadwallader, 1998; Adams,
Demyttenaere, & De Kimpe, 2003; Bicas, Barros, Wagner, Godoy, & Pastore, 2008a;
Bicas, Fontanille, Pastore, & Larroche, 2008b; Molina, Pinheiro, Pimentel, Santos, &
Pastore, 2013a; Molina, Pimentel, & Pastore, 2013b). Another product obtained from
the bioconversion of the monoterpenic substrate is limonene-1,2-diol, which has been
described as possessing a cool minty aroma and is consumed mainly in flavours used in
mint preparation, alcoholic and nonalcoholic beverages, chewing gum,
gelatins/puddings and other food products (Burdock & Fenaroli, 2010).
A few reports have studied the bioconversion of both enantiomers of limonene,
R-(+)- and S-(–)-limonene, including those of Demyttenaere, Van Belleghem, and De
Kimpe (2001), Adams et al. (2003), and Bicas et al. (2008b), which aimed to compare
the results, metabolic pathways and characterise their metabolism.
Thus, the main objective of this work was to characterise the biotransformation
of S-(‒)-limonene into limonene-1,2-diol, studying the improvements to this process
using cell permeabilisation under anaerobic conditions and with the adoption of a
biphasic system. In addition, these results were compared with the literature recently
produced on the same microorganism, using R-(+)-limonene as the substrate for the
production of α-terpineol. Furthermore, this study aimed to characterise and compare
the bioconversion of R-(+)- and S-(‒)-limonene using ultra-structural analysis.
2. Material and methods
2.1. Microorganism and chemicals
The microbial strain employed in this study was identified as Fusarium
oxysporum 152B (Prazeres, Cruz, & Pastore, 2006). The fungal strain was maintained
on yeast malt (YM) agar (in g L‒1: agar = 20; glucose = 10; peptone = 5; yeast extract =
3; malt extract = 3, pH ~ 6.7) and stored at 4 °C.

The chemical standards used as substrates in this study were R-(+)-limonene
(98% purity, Sigma-Aldrich) and S-(–)-limonene (96% purity, Sigma-Aldrich). The
reagents, n-hexadecane (99% purity, Sigma-Aldrich) and cyclohexanol (99%, Sigma-
Aldrich), were kept at 4 °C. Ethyl acetate and n-decane were used as the solvent and
internal standard, respectively, and were of the best commercial grade.
2.2. Inoculum preparation
Agar (approximately 1.5 cm2) with a pre-grown culture of F. oxysporum 152B
(72-h old) was transferred to a 250-mL conical flask filled with 50 mL of the agro-
industrial residue known as cassava wastewater (or manipueira) medium. The material
was homogenised under sterile conditions with an Ultra-Turrax® T18 (Ika, Wilmington,
NC) until the solid matter had been completely disrupted. After incubation at 30 °C and
200 rpm for 48 h, the biomass was recovered by vacuum filtration using a Buchner
funnel and paper filter Whatman o1 (Bicas et al., 2008a).
2.3. Comparative study of the bioconversion process using R-(+)- and S-(‒)-
limonene
The bioconversion process of S-(–)-limonene into limonene-1,2-diol by
Fusarium oxysporum 152B was characterised following the methodologies described
below, and the results were compared with specific references to the biotransformation
of R-(+)-limonene into α-terpineol, as recently published by Bicas et al. (2008a) and
Bicas, Quadros, Neri-Numa, and Pastore (2010) using the same fungal strain, Fusarium
oxysporum 152B. All of the experiments were performed at least in triplicate.
2.3.1. Biotransformation procedure under optimised conditions
The biomass was weighed, and 3 g were distributed amongst 250-mL conical
flask filled with 50 mL of mineral (MM) medium (in g L‒1: MgSO4.7H2O = 0.5; NaNO3
= 3; K2HPO4 = 1; KCl = 0.5 and Fe2SO4 = 0.001). Subsequently, 5 g L‒1 of R-(+)- and
S-(-)-limonene were added to each flask. The flasks were incubated under optimised
conditions for each substrate. For the R-(+)-isomer, the conditions were at 30 °C and
200 rpm for agitation (Bicas et al., 2010a), whereas, for the S-(–)-limonene, the
conditions were pH 6.5, 28 °C and 250 rpm for agitation. Periodically, 1-mL samples
were taken to monitor the consumption of the substrate and the production of the

volatile compounds until 96 h by gas chromatography with flame ionisation detection
(GC-FID).
2.3.2. Influence of cell permeabilisation
The influence of cell permeabilisation on the biotransformation yield was
evaluated following different protocols. The biomass obtained (Section 2.2) was treated
o
as follows: i) for 1 h at 30 C and 150 rpm with 6% (v/v) diethyl ether/chloroform
mixture; ii) successive cycles of freezing (4 h at freezing temperature and 30 minutes at
30 °C, repeated three times); iii) sonication cycles (1, 5 and 10 min repeated three times,
keeping the system at low temperature, approximately 5 °C); and iv) lyophilisation in a
freeze-drier (model LS 3000; Equipamentos Terroni, São Paulo, Brazil) (adapted from
Fontanille and Larroche (2003) and Bicas et al. (2010a)).
2.3.3. Biotransformation in anaerobic conditions
For the anaerobic biotransformation, 10 g of the fresh biomass (Section 2.2),
0.5% v/v of substrate S-(‒)-limonene and 200 mL of mineral medium were transferred
into a conical flask with two inlets (one at the base and another at the neck) plugged
with a rubber stopper. At the beginning of the conversion, as well as after each
sampling, the medium was flushed by bubbling N2 from the base inlet for 5 min to
eliminate the oxygen dissolved in both the medium and headspace. The flasks were
incubated at the same conditions as described above for 96 h (adapted from Bicas et al.,
2010a).
2.3.4. Biotransformation in biphasic system
For the biphasic biotransformation system, the biomass produced following the
protocol described in section 2.2 was distributed within a 250-mL conical flask filled
with 25 mL of aqueous phase (consisting of MM) and 25 mL of organic phase. For this
study, the organic phases selected were hexadecane and cyclohexanol. R-(+)- and S-(–)-
limonene were added to reach 5 g L‒1 of the organic phase. The flasks were incubated at
30 °C and at 200 rpm. Periodically, 1 µL of the organic phase was directly injected into
the GC-FID to monitor the consumption of the substrate and the production of
limonene-1,2-diol (adapted from Bicas et al., 2008b).
2.4. Determination and quantification of the volatile compounds

Samples were extracted (40 s in vortex) using the same volume of ethyl acetate.
After phase separation, the organic fraction was dried over sodium sulfate and 1 µL of
this phase was injected in split mode (split ratio of 1:10) into a gas chromatograph with
a flame ionisation detector (GC–FID) HP-7890 (Agilent Technologies, Santa Clara,
CA,) coupled to an HP-5 column (30 m length × 0.25 mm i.d. × 0.25 µm film
thickness). Helium was used as the carrier gas (1.0 mL min‒1), and the oven temperature
was kept at 80 °C for 3 min, raised at 20 °C min‒1 until 200 °C and held for 4 min. The
temperatures of the injector and detector were kept at 250 °C. Substrates and products
were quantified by a calibration curve using n-decane as an internal standard. All
experiments were performed in triplicate.
2.5. Ultra-structural analysis
2.5.1. Scanning electron microscopy (SEM)
The fungal strain was fixed for 24 hours in 4% glutaraldehyde in 0.1 M sodium
cacodylate buffer (pH 7.2), dehydrated in ascending alcohol series (70–100%),
subjected to two acetone 100% baths of 15 min each and dried at the critical point
(Balzers CPD 030). After dehydration, the material was placed on aluminium supports
attached with double-sided tape and sputter-coated with gold (in sputtering Balzers SD
050). The fungi were examined with a Jeol P15 SEM and photographed.
2.5.2. Transmission electron microscopy (TEM)
The microorganism was fixed for 4 hours in 2.5% glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.2). Post-fixation was carried out in 1% osmium
tetroxide in the same buffer. En bloc staining with 2% uranyl acetate was followed by a
series of ethanol dehydration steps. The microorganism was then embedded in Eppon
resin and sectioned with a Leica ultramicrotome. Ultra-thin sections were stained with
uranyl acetate and lead and then photographed with a CM 100 Philips TEM at 80 kV.
3. Results and discussion
3.1. Comparison of bioconversion process using different isomers of limonene
In previous studies, Bicas et al. (2008a) described the optimisation of the ten
main process variables involved in the biotransformation of R-(+)-limonene to R-(+)-α-
terpineol by Fusarium oxysporum 152B through a Plackett-Burman matrix with 16
assays, including the effects of the medium composition, substrate concentration,

cultivation conditions and inoculum size and then the central composite design
methodology. Thus, the authors improved significantly the production of R-(+)-α-
terpineol, from the 450 mg L‒1 previously described by Maróstica Jr. and Pastore
(2007b) up to 2.44 g L‒1. Next, another study conducted by the same group proposed an
integrated process for the co-production of lipase and R-(+)-α-terpineol, in which the
concentration of this product was re-quantified with another technique to reach
approximately 4 g L‒1 after a 48-h processing period. The product remained stable until
96 h (Bicas et al., 2010a).
However, as previously observed in our experiments (data not shown) the
biotransformation of S-(–)-limonene did not result in the same product. The compound
obtained was identified by mass spectrometry as limonene-1,2-diol (90% similarity),
which was confirmed by the mass spectrum and retention index of the commercial
standard (Sigma-Aldrich).
This bioconversion process results from the ring double bond epoxidation of
limonene, followed by the corresponding diol formation. However, as proven
elsewhere, the production of the intermediate limonene-1,2-epoxide was not detected,
indicating that the reaction proceeded mainly due to the diol production. When the
microorganism was evaluated by its potential to convert limonene-1,2-epoxide, the
accumulation of limonene-1,2-diol seemed to suggest that Fusarium oxysporum might
be implicated in another pathway for limonene, similar to that described by Van der
Werf, Swarts, and De Bont (1999).
Thus, the bioconversion processes of R-(+)- and S-(‒)-limonene were performed
according to the optimised processing conditions for the production of both α-terpineol
(as published recently by Bicas et al. (2008b)) and limonene-1,2-diol by Fusarium
oxysporum 152B. It was decided to repeat the experiment using R-(+)-limonene as a
substrate to ensure an experimental control and to improve the parameters for a suitable
process comparison.
Figure 1A graphically represents a comparison of the profiles of the two
bioconversion processes. It is possible to observe that, in this process, the results
obtained for the biotransformation of R-(+)-limonene reached approximately 3.9 g L‒1
of α-terpineol under the conditions presented by Bicas et al. (2010a). The difference
observed when compared with the reported value (~ 4 g L‒1) by the authors may be
related to potential small variations in the concentrations, rounding or calibration curves
of the analyser. Some slight variations in the bioconversion profiles of different isomers

of limonene can also be observed. Table 1 summarises all of the comparisons observed
in the R-(+)- and S-(–)-limonene bioconversion processes.
Assays were carried out using a mixture of both substrates at the same
concentration, which were added according to the same procedure (reaching a final
concentration of 5 g L‒1 of substrate) to show that the rate of conversion was much
higher for α-terpineol. On the other hand, limonene-1,2-diol was found only in small
concentrations, indicating that the reaction proceeds favourably for the production of α-
terpineol and that the activity obtained for the R-(+)-isomer was at least three times
higher than that for S-(–)-limonene (data not shown). Braddock and Cadwallader (1995)
observed that a Pseudomonas gladioli was capable to convert R-(+)- and S-(–)-
limonene, and the activity obtained for the R-(+)-isomer was more than 10 times
greater. Additionally, as can be seen in Table 1, the production of α-terpineol reached a
maximum concentration at 48 h, while limonene-1,2-diol only peaked at 72 h using
Fusarium oxysporum 152B.
Braddock and Cadwallader (1995) observed the enzyme from the bacterium
Pseudomonas gladioli responsible for converting limonene into α-terpineol was
enantiospecific and enantioselective for the conversion of R-(+)-limonene into R-(+)-α-
terpineol. On the other hand, a Sphingobium sp. strain showed lower selectivity, as it
converted both R-(+)- and S-(–)-isomers specifically into R-(+)- and S-(–)-α-terpineol,
respectively, with virtually the same efficiency (Bicas, Fontanille, Pastore, & Larroche,
2010b). In this case, the authors found traces of limonene-1,2-diol as a by-product of the
biotransformation of S-(–)-limonene.
It is important to observe the high capacity of this fungal strain in the
bioconversion of limonene considering that the concentrations observed for both
products are very promising. Demyttenaere et al. (2001) evaluated the ability of 60
fungal strains to biotransform R-(+)- and S-(–)-limonene, revealing the potential of
Penicillium digitatum and Corynespora cassiicola for the production of α-terpineol and
limonene-1,2-diol, from both limonene isomers, respectively; while the
−)
biotransformation of R-(+)-limonene into R-(+)-α-terpineol and S-( -limonene into a
mixture of products was also investigated by Penicillium digitatum (Adams et al.,
2003).
Other processes on bioconversion of terpenes by fungal biocatalysts or their
spores have been reported in the literature, such as reported by Demyttenaere and De
Kimpe (2001) for the biotransformation of geraniol and nerol by sporulated surface

−)
cultures of Penicillium digitatum, and the biotransformation of R-(+)- and S-( -
citronellol by sporulated surface cultures of Aspergillus niger into cis- and trans-rose
oxides and nerol oxide (Demyttenaere, Vanoverschelde, & De Kimpe, 2004). Also,
Demyttenaere et al. (2000) described the biotransformation of geraniol, nerol and citral
by Aspergillus niger in submerged liquid, sporulated surface cultures and spore
suspensions. Among the major bioconversion products obtained from geraniol and nerol
by liquid cultures were linalool and α-terpineol, respectively.
Additionally, several processes studied the biotechnological production of α-
terpineol, and the production of this monoterpene alcohol is one of the highest already
described, and the concentrations reported are higher only for the distinct process
developed by Bicas et al. (2008b) and Bicas et al. (2010b), using Sphingobium sp. as
biocatalyst.
Meanwhile, the production of limonene-1,2-diol has been less explored in recent
years, and the yield obtained from this process is also among the highest concentrations
ever described. Abraham, Hoffmann, Kieslich, Reng, and Stumpf (1985) outlined a well
distinguished process for recovering good yields of 1S,2S,4R-limonene-1,2-diol from R-
(+)-limonene with continuous substrate feeding in a 100-L bioreactor filled with 70 L of
culture medium. When 1.3 kg of substrate were used, 900 g of 1S,2S,4R-limonene-1,2-
diol and small amounts of the 1R,2R,4R-diasteroisomer were recovered after a 96-h
process, thus constituting an economic way of preparing diols. In addition, an
Aspergillus sp., i.e., A. cellulosae, was capable of converting both enantiomers and the
racemate of limonene into limonene-trans-1,2-diol as the main product (Noma,
Yamasaki, & Asakawa, 1992). In fact, some authors concluded that diols are common
intermediates in the monoterpene metabolism of fungi (Mukherjee, Kraidman, & Hill,
1973).
3.2. Influence of biomass permeabilisation treatment on biotransformation
The microbial cell membrane acts as an osmotic barrier; frequently, the
precursors and products involved in biotransformation processes move across this
structure by passive diffusional transport. When intact microbial cells are used in
biocatalysis, therefore, the cell membrane may severely hamper a bioconversion from
working optimally, as the substrate influx and product efflux by diffusion can be slow
with regard to the maximal reaction rate (Van der Werf, Hartmans, & Van den Tweel,
1995).

For these reasons, the cells used in commercial bioconversions are often
permeabilised to improve the rate of exchange between intra- and extra-cellular media.
Several methods have been described for this purpose (Felix, 1982). Microbial cells, for
example, can also be effectively broken by physical means. Sonication is convenient
and effective at the laboratory scale, while high-pressure extrusion equipment is
currently the method of choice for industry (Cabral, 2001).
In this sense, the purpose of the research presented here has been to determine
the effects of certain factors upon fungal biocatalysts. These factors include successive
freezings and length of storage between each successive freezing, solvent
permeabilisation and the effect of lyophilisation in the fungal biomass. In this sense,
bioconversion experiments were carried out with either concentrated fresh cells or crude
enzymatic extracts. The latter could be considered as a form of the biocatalyst that is
unable to perform cofactor-dependent reactions (Bicas et al., 2008b).
It can be noted in Figure 1B that, in the case of a single freezing‒thawing,
Fusarium oxysporum manifests a percentage survival that is constant and independent
of the initial cell concentration, providing good resistance when treated with this initial
technique. When submitted to successive cycles (see Methods), it is also possible to
observe a considerable decrease in this capacity when applied during the bioconversion
of the S-(–)-limonene process, in which the activity is less than 50%.
The positive effect of the freezing‒thawing procedure on the bioconversion
efficiency was demonstrated in a recent study, in which the authors showed that this
treatment led to a large increase in both the reaction rate and yield of isonovalal
production (Fontanille, Le Flèche, & Larroche, 2002). This process lead to a semi-
permeabilisation technique that causes cell ruptures to release some of the intracellular
enzymes (Fontanille & Larroche, 2003). Bicas et al. (2010b) used this technique to
show that freeze-thawed cells can accelerate the process for the biotransformation of R-
(+)-limonene into R-(+)-α-terpineol, indicating the release of the enzyme responsible for
this conversion and confirming its intracellular nature. When solvent treatment and
successive cycles of freezing‒thawing were combined, no production of limonene-1,2-
diol and a loss of activity of the biocatalyst were noted, indicating complete loss of cell
viability.
Lyophilisation/freeze-drying is defined as a stabilising process in which the
substances are first frozen, followed by a sublimation (primary drying) and desorption
(secondary drying) step to reduce the water content to levels that will no longer support

biological growth or chemical reactions (Jennings, 1999). The lyophilisation process
was tested due to great interest from a technical point of view because it standardises
and simplifies inoculation while enabling biocatalyst storage (Bicas et al., 2010a,b).
When the concentrated biomass was lyophilised and tested in the bioconversion process,
the production of limonene-1,2-diol could not be detected due to the possible
inactivation of the biocatalyst, which showed no activity. Hence, it is supposed that,
somehow, the lyophilisation process damages the enzyme, reducing its activity. Despite
this, the Fusarium oxysporum 152B strain employed in this study has shown a relatively
high resistance to cell permeabilisation.
The suspension resulting from the permeabilisation operation was membrane-
filtered (0.45 µm) to remove the remaining viable cells and tested in the bioconversion
process. The results obtained with the crude enzymatic extracts of Fusarium oxysporum
for the biotransformation of S-(‒)-limonene into limonene-1,2-diol have shown that this
reaction was catalysed by a cofactor-dependent enzyme. Conversely, the enzyme
responsible for the biotransformation of R-(+)-limonene into α-terpineol by the same
fungal strain seem to be cofactor-independent (Maróstica Jr. & Pastore, 2007b; Bicas et
al., 2010a), such as the α-terpineol dehydratase from P. gladioli (Cadwallader,
Braddock, & Parish, 1992).
Furthermore, membrane permeabilisation did not improve the biotransformation
rate processes because it has been proven that cofactors are needed for this
bioconversion process; the use of resting cells without respiratory activity was not
successful.
3.3. Biotransformation in anaerobic conditions
Biotransformation in anaerobic conditions is one of the tools used to characterise
the enzyme involved in the process, especially when related to required cofactors (Bicas
et al., 2008b). From a technical point of view, this is also an important piece of
information because it establishes the need for the system aeration (Bicas et al., 2010b).
The conduction of the bioconversion process of S-(–)-limonene in anaerobic conditions
demonstrated a need for oxygen in its activity. In this approach, limonene-1,2-diol
production was not detected in the culture medium, as would characterise an aerobic
system; thus, the process was considered cofactor-dependent (see Table 1). This finding
is in agreement with those published by Bicas et al. (2008b), which showed that
limonene-1,2-epoxide hydrolase, the first enzyme in this metabolic pathway, was

cofactor-independent, but the following steps did need cofactors. Thus, the entire
pathway could be completed only in aerobically operated whole cells. In addition, Bicas
et al. (2010a) showed that the biotransformation of R-(+)-limonene into R-(+)-α-
terpineol, although it occurred at a lower rate, was catalysed by an oxygen-independent
enzyme and, consequently, was independent of cofactors. The independence of
cofactors is also related to the increase in the biotransformation rate of a freeze‒thawed
biomass, as observed by these authors, because only cofactor-independent enzymes
would act in the extracellular medium.
In summary, these observations reinforce the hypothesis that Fusarium
oxysporum 152B presents two parallel metabolisms for isomers of limonene, depending
on the substrate. The bioconversion of S-(–)-limonene passes through limonene-1,2-
oxide and limonene-1,2-diol in an aerobic process leading to energy production, while
the R-(+)-limonene bioconversion to α-terpineol, was oxygen-independent and could be
viewed as a process for the detoxification of the medium (Griffin, Whyllie, Markham, &
Leach, 1999).
3.4. Biotransformation in biphasic system
In microbial cultivation process, particularly in high-cell-density cultures,
productivity is often limited by the transport of a substrate, of which oxygen is one
important example (Puthli, Rathod, & Pandit, 2005). From this perspective, there is a
great concern about the disadvantages associated with the high aeration and agitation
rates necessary to maintain high levels of oxygen solubility in the medium due to the
cell’s sensitivity to hydrodynamic stress, the high power consumption and the increases
in operation costs (Gomes, Aguedo, Teixeira, & Belo, 2007).
Thus, some authors discuss the possibility of adding an organic solvent to the
system, using a water-immiscible phase with a higher solubility for oxygen to improve
oxygen transfer rate (Gomes et al., 2007; Mehrnia, Towfighi, Bonakdarpour, &
Akbarnejad, 2005). The addition of an appropriate phase can reduce the problems
associated with the low solubility of substrate and product, even while minimising their
toxic effects for microorganisms (Bicas, Maróstica Jr., Barros, Molina, & Pastore,
2013). One practical example of this technique is the well-developed process for the
production of α-terpineol through the biotransformation of limonene, from which
concentrations up to 120‒130 g L–1 of the product were obtained using a Sphingobium
sp. (Bicas et al., 2010b).

Despite the advantages provided by the adoption of this type of system for some
processes, in this study, it was observed that the limonene-1,2-diol produced from the
bioconversion of S-(–)-limonene was partitioned preferentially within the aqueous
phase. This result is in agreement with the results published by De Carvalho, Van
Keulen, and Da Fonseca (2000), who conducted partition studies with limonene-1,2-diol
and found this compound primarily dissolved in the aqueous phase (0–20 mM),
compared with the limonene-1,2-oxide in the organic phase. The consequence of this
phenomenon was the production of low concentrations of limonene-1,2-diol (less than 1
g L‒1 of organic phase), clearly affecting the biocatalyst as it accumulated in the
aqueous phase.
In this work, the organic phase was changed to cyclohexanol in the hope that this
phase would have a greater chance of success within the experiment to solubilise both
the substrate and product. However, the results showed that, even though a higher diol
concentration was attained using this organic phase, the results were not satisfactory,
reaching less than 2 g L‒1 of organic phase but with more than 75% of the product being
detected in the aqueous phase.
It is important to emphasise that the accumulated product in the aqueous phase
can play a significant inhibitory role within the biocatalyst cells, potentially explaining,
at least in part, the observed results. Although the results collected with this second
solvent were better, the production of limonene-1,2-diol through the biotransformation
of S-(–)-limonene was not possible. Similarly, Bicas et al. (2010a) reported a similar
inability to perform the process for the production of α-terpineol from the
biotransformation of R-(+)-limonene.
3.5. Ultra-morphological and ultra-structural analysis
The ultra-morphological analysis was performed using scanning electron
microscopy (SEM) and aimed to detect the similarities and differences between the
ascomycete fungus Fusarium oxysporum 152B grown in absence of terpenes (glucose
as carbon source). This analysis also compared the biomasses that were in contact with
either the R-(+)- or S-(–)-limonene terpenes that were used as substrates during the
bioconversion process.
The ultra-morphological comparative analysis of the samples studied showed no
significance differences between them (Figures 2 and 3). These results indicate that,
although isomers of limonene were present in two of the culture media analysed in this

study, these terpenes had no toxic activity that could promote morphological changes in
this biocatalyst. The ultra-morphological and ultra-structural studies performed with
Trichophyton mentagrophytes, a dermatophyte fungus, reflect the morphological
changes in their hyphae, which had collapsed and deformed after the microorganism
was challenged with different concentrations of the terpenes citral, eugenol, nerolidol
and α-terpineol (Park et al., 2009). In the referenced study, the toxicity of the terpene
tested showed clear effects on the microorganism.
To the detriment of the ultra-morphological analyses that did not show changes,
the present ultra-structural analysis showed conspicuous differences between fungi
grown in the absence of terpenes (Figure 4) and fungi grown in the presence of the
terpenes, R-(+)- and S-(–)-limonene (Figure 5). In the latter cases, the presence of
abundant smooth endoplasmic reticulum (Ser), numerous vacuoles and vesicles were
noted, some of which are seen as Ser product (Figure 5). Therefore, these results
constitute evidence of the intense action of this microorganism in cellular detoxification,
as may occur through the hydroxylation, isomerisation and bioconversion of xenobiotics
by the cytochrome P450 enzymes that are present in the membranes of the smooth
endoplasmic reticulum (Bernhardt, 2006; Isin & Guengerich, 2007).
The electron micrograph of F. oxysporum grown without the presence of
terpenes clearly shows a cytoplasm rich in ribosomes, in addition to several
mitochondria (Figure 4). These features demonstrate a state of high metabolic activity,
in which the intense protein synthesis of cells can be observed. However, the electron
micrographs of cultured fungi with both R-(+)- and S-(–)-limonene showed a different
morphological and physiological profile. In this case, the cytoplasm is mostly occupied
by the smooth endoplasmic reticulum and vesicles that characterise cells in
detoxification state (Figure 5).
Bicas et al. (2008b) observed that traces of limonene-1,2-diol were present
throughout the growth phase (< 38 h) of Pseudomonas fluorescens and that α-terpineol
accumulated at low levels between 38 h to 44 h (end of exponential phase- beginning of
the stationary phase). As discussed above, these observations support the hypothesis
that Fusarium oxysporum has two parallel metabolisms to limonene isomers: one that
occurs through the primary metabolism to produce energy and another that passes
through the secondary metabolism, which is dedicated to the limonene detoxification.
The latter could be confirmed by the analysis of electron micrographs, which are the

first ultra-structural reports to characterise the bioconversion of terpenes by cellular
detoxification.
4. Conclusion
The results collected so far with Fusarium oxysporum 152B encourage further
studies on this biocatalyst to better characterise the involvement of its enzyme system in
the bioconversion of R-(+)-limonene into α-terpineol and S-(‒)-limonene into limonene-
1,2-diol, particularly considering the concentration of the products here achieved, 4.0
and 3.7 g L‒1, respectively. In this sense, the present study represents an extensive
comparative investigation into the bioconversion processes leading to these products. It
was observed that the enzyme related to the production of limonene-1,2-diol from S-(‒)-
limonene, limonene-1,2-epoxide hydrolase, has an intracellular nature, is highly
influenced by an aerobic system and seems to be cofactor-dependent, as it was not
detected under anaerobic processes. Meanwhile, the results recently published for R-(+)-
limonene prove that the enzyme responsible for the production of α-terpineol, a
limonene hydratase, by the biotransformation of R-(+)-limonene has an intracellular
nature and acts under anaerobic conditions. Furthermore, an ultra-morphological and
ultra-structural analysis was conducted to compare Fusarium oxysporum grown on
glucose (as control) and that of the biomass resulting in the biotransformation of R-(+)-
and S-(‒)-limonene, during which the cytoplasm was mostly occupied by the smooth
endoplasmic reticulum, vesicles and vacuoles that characterise cells in a detoxification
state. This is the first report that characterises the bioconversion of R-(+)- and S-(‒)-
limonene by cellular detoxification using ultra-structural analysis.
5. Acknowledgments
The authors acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nivel
Superior (CAPES), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)
and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for the
financial support.
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A)
B)
Figure 1. A) Comparison of the profile between the biotransformation of R-(+)-
■) into α ●) into
limonene ( -terpineol (--□--) and the bioconversion of S-(–)-limonene (
limonene-1,2-diol (--○--) by Fusarium oxysporum 152B. B) Influence of cell
■). The
permeabilisation in the production of limonene-1,2-diol (control experiment:
techniques employed were: one cycle of freezing (●), successive cycles of freezing–
thawing (▲), solvent permeabilisation (♦) and solvent permeabilisation combined with
successive cycles of freezing‒thawing (-x-).

Figure 2. Sections A-E: Scanning electron microscopy (SEM) showing the ultra-
morphology of Fusarium oxysporum, a filamentous fungus, grown without the presence
of terpenes (control) using glucose as carbon source. Hy = hyphae.

Figure 3. Sections A-C: Scanning electron microscopy (SEM) showing the ultra-
morphology of Fusarium oxysporum grown in the presence of R-(+)-limonene as
substrate during the biotransformation process. Sections D-F: SEM showing the ultra-
morphology of Fusarium oxysporum grown in the presence of S-(‒)-limonene as
substrate. Hy = hyphae.

Figure 4. Sections A-C: Electron micrograph showing cross-sections of Fusarium
oxysporum grown without the presence of terpenes (control) using glucose as carbon
source. Ne = nuclear envelope; Pm = plasma membrane, Mi= mitochondrion; Nu =
nucleus; Cw= cell wall; Ve = vesicle.

Figure 5. Sections A-B: Electron micrograph showing cross sections of Fusarium
oxysporum in the presence of R-(+)-limonene) as substrate during the biotransformation
process. Sections C-D: Electron micrograph showing cross-sections of Fusarium
oxysporum in the presence of S-(‒)-limonene as substrate. Ne = nuclear envelope; Pm =
plasma membrane, Mi= mitochondrion; Nu = nucleus; Cw = cell wall; Ser = smooth
endoplasmic reticulum; Ve = vesicle.

Table 1 – Comparative study of the bioconversion of R-(+)- and S-(–)-limonene
performed by Fusarium oxysporum 152B.
substrates
variables
R-(+)-limonenea
α-terpineol
4 g L-1
48 h
32.6%
hydratase
non inducible
intracellular
anaerobic
detoxification
S-(‒)-limonene
limonene-1,2-diol
3.7 g L-1
72 h
42.2%
limonene-1,2-epoxide hydrolase
non inducible
intracellular
aerobic
product
concentration
time
extraction efficiency
enzyme involved
inducibility
nature
conditions
metabolism
energy
aData obtained from Bicas et al. (2008a) and Bicas et al. (2010a) using the same
biocatalyst used in this process, Fusarium oxysporum 152B, with R-(+)-limonene as
substrate for the biotransformation process.

Highlights:
1. Complete process comparison of R-(+)- and S-(‒)-limonene fungal bioconversion
2. Characterization of the bioconversion of S-(‒)-limonene into limonene-1,2-diol
3. One of the highest concentrations obtained of α-terpineol and limonene-1,2-diol
4. First report to characterize the bioconversion using ultra-structural analysis