Biotransformation of aromatic aldehydes by five species of marine microalgae

Article abstract of DOI:10.1016/S0031-9422(99)00037-0

The biotransformation of a series of aromatic aldehydes such as benzaldehyde, salicy aldehyde, methoxybenzaldehydes and mono- and dichlorobenzaldehydes by five cultures of photosynthetic microalgae are reported. The microalgae, Chlorella minutissima, Nannochloris atomus, Dunaliella parva, Porphyridium purpureum and Isochrysis galbana, reduced most of the aldehydes to the corresponding primary alcohols. Substituted aromatic aldehydes were reduced with varying selectivity depending on the nature and position of the substituent.

Full text of DOI:10.1016/S0031-9422(99)00037-0

Phytochemistry 51 (1999) 621±627
Biotransformation of aromatic aldehydes by ®ve species of
marine microalgae
Ingrid L. Hook, Shane Ryan, Helen Sheridan*
Department of Pharmacognosy, School of Pharmacy, Trinity College, Dublin, Ireland
Received 19 October 1998; received in revised form 4 January 1999
Abstract
The biotransformation of a series of aromatic aldehydes such as benzaldehyde, salicy aldehyde, methoxybenzaldehydes and
mono- and dichlorobenzaldehydes by ®ve cultures of photosynthetic microalgae are reported. The microalgae, Chlorella
minutissima, Nannochloris atomus, Dunaliella parva, Porphyridium purpureum and Isochrysis galbana, reduced most of the
aldehydes to the corresponding primary alcohols. Substituted aromatic aldehydes were reduced with varying selectivity
depending on the nature and position of the substituent. # 1999 Published by Elsevier Science Ltd. All rights reserved.
Keywords: Chlorella minutissima; Nannochloris atomus; Dunaliella parva; Porphyridium purpureum; Isochrysis galbana; Microalgae; Marine bio-
technology; Biotransformation; Aromatic aldehydes; Aromatic alcohols
1
. Introduction
sima (Chlorophyta), which has been used for many
years as a research organism in the study of photosyn-
thesis (Bold, & Wynne, 1978); Nannochloris atomus
(Chlorophyta), a non-motile unicellular alga rich in
eicasopentaenoic acid (EPA) and sterols; Dunaliella
parva (Chlorophyta), a motile, unicellular chlorophyl-
The biotransformation of organic substrates by fun-
gal (Davies, Green, Kelly, & Roberts, 1990; Laskin, &
Lechevalier, 1984) and plant cell cultures (Suga, &
Hirata, 1990) has received considerable attention over
the last number of years. Limited studies into the bio-
transformation of organic substrates by photosynthetic
microalgae have been reported during this same
period. These include the use of the unicellular green
microalga Dunaliella tertiolecta in the biotransform-
ation of a series of aldehydes (Noma, Akehi, Miki, &
Asakawa, 1992) and the biotransformation of a series
of aromatic aldehydes and ketones by photohetero-
trophic cultures of Euglena gracilis (Noma, Okajima,
Takahashi, & Asakawa, 1991; Noma, Takahashi, &
Asakawa, 1991; Noma, & Asakawa, 1998). As part of
an ongoing biotechnological study we report in this
paper the results of a study into the ability of ®ve
species of marine microalgae to biotransform a series
of aromatic aldehydes. The species under investigation
include the green, non-motile, alga Chlorella minutis-
containing
organism;
Porphyridium
purpureum
(Rhodophyta), a non-motile, unicellular organism
which is a potential source of the unsaturated fatty
acids, EPA and arachidonic acids (Bold, & Wynne,
1
978; Lee, 1989); Isochrysis galbana (Chrysophyta), a
motile unicellular organism rich in the o-3 fatty acids
such as EPA and eicosahexanoic acid (Bold, &
Wynne, 1978; Lee, 1989; Ewart, & Pruder, 1981).
2. Results and discussion
Initially the optimal growth patterns of the ®ve
microalgae were established. The growth of each test
organism in static and shake cultures was compared
over a four week growth period (Fig. 1, Table 1). For
green microalgae C. minutissima, N. atomus and D.
parva growth was paralleled by a linear absorbance at
440 nm using the method of Pirroeck, Baash, and Pohl
*
Corresponding author.
0031-9422/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00037-0

6
22
I.L. Hook et al. / Phytochemistry 51 (1999) 621±627
Fig. 1. Growth of test microalgae in shaken and static cultures measured over a four week growth period.

I.L. Hook et al. / Phytochemistry 51 (1999) 621±627
623
Fig. 1 (continued)

6
24
I.L. Hook et al. / Phytochemistry 51 (1999) 621±627
Fig. 1 (continued)
(
1984). This method was not suited to measuring the
cultures of P. purpureum also tended to grow as
clumps of cells due to the presence of a thick sulphated
polysaccharide which encapsulates the cells of all red
microalgae (Fattarusso, & Piatelli, 1980; South, &
Whittick, 1987). When cultures of P. purpureum are
shaken the clumps of cells break up increasing the
e€ective surface area available for nutrient uptake
thereby increasing cell growth. In contrast with the
other test organisms growth of I. galbana was favoured
in static culture. Shaking cultures of I. galbana may
interfere with normal motility and hence the organisms
complex reproductive cycle (Bold, & Wynne, 1978;
Lee, 1989; Borowitzka, 1988).
When static and shake patterns were compared for
all ®ve species (Fig. 1) it was decided that biotrans-
formation studies for I. galbana would be carried out
under static conditions whilst biotransformation by
cultures of C. minutissima, N. atomus, D. parva and P.
purpureum would be examined under shake conditions.
growth of either P. purpureum or I. galbana where a
haemocytometer was used (Kirsop, & Doyle, 1991).
Results indicate that shake cultures of N. atomus, D.
parva and P. purpureum led to an increase in growth
relative to static cultures. Growth of C. minutissima
increased dramatically in both shaken and static cul-
tures between 2 and 3 weeks.
After four weeks, growth within static cultures was
greater than within the corresponding shaken cultures.
D. parva exhibited the greatest di€erence in growth on
shaking, with this di€erence most dramatic after four
weeks. Steady growth was observed in cultures of N.
atomus over the four week period, after which time the
best growth was observed in shaken cultures. The
growth of P. purpureum in shake cultures was consist-
ently higher than growth in corresponding static cul-
tures. This observation may be attributed to increased
uptake of nutrients on shaking. In static conditions
Table 1
Comparison in relative growth patterns between shaken and static cultures after four weeks growth
Algal culture
C. minutissima
N. atomus
D. parva
P. purpureum
I. galbana
Static (%)
Shake (%)
100
96
100
150
100
419
100
245
100
5

I.L. Hook et al. / Phytochemistry 51 (1999) 621±627
625
The compounds chosen for study were a series of aro-
matic aldehydes including the parent aldehyde benzal-
dehyde (1), the 2-, 3- and 4-monochlorobenzaldehydes
alcohol within 24 h (Noma et al., 1992) and Euglena
which carried out the same biotransformation but less
eciently (49%) (Noma et al., 1991; Noma, &
Asakawa, 1998).
(
7
2±4), the 2,3-, 2,4- and 3,4-dichlorobenzaldehydes (5±
), the 2-, 3- and 4-methoxybenzaldehydes (8±10) and
3-Chlorobenzaldehyde (3) was biotransformed to
some degree by all the microalgal species. P. purpur-
eum gave the best result with 99% of substrate bio-
transformed by the fourth day. N. atomus (83%) and
D. parva (70%) reached their maxima after 5 days. In
contrast cultures of I. galbana did not commence bio-
transformation of substrate until the third day after in-
oculation reaching a maximum biotransformation of
vanillin (11). Using these compounds the relationship
between structure and biotransformation ability of the
test organisms was studied.
An initial experiment using various concentrations
of organic substrates indicated that substrate toxicity
increased with time and that after ®ve days exposure
to aromatic substrates all cultures showed a marked
decrease in cell viability. In general substrates were
most toxic to I. galbana and P. purpureum. The most
susceptible green alga was D. parva. Substrate concen-
trations >200 ppm were too toxic so that biotrans-
formation studies were carried out at concentrations of
6
8% of 3 by the ®fth day. C. minutissima was the least
e€ective organism in the biotransformation of 3 with
maximum yields of 10% being achieved after ®ve days.
Maximum biotransformation of 4-chlorobenzalde-
hyde (4) occurred after ®ve days for all microalgal
species Table 2 with D. parva giving the highest yield
100 ppm.
The biotransformation of aromatic aldehydes by the
(
95%) of product after this time. However less than
% of p-chlorobenzylalcohol was produced by I. gal-
test microalgae is summarised in Table 2.
Benzaldehyde (1) was readily reduced to benzyl alcohol
by all species. This transformation was completed by
C. minutissima, N. atomus and P. purpureum within a
1
bana. We feel that this low level of transformation
may be explained by the fact that 4 was more toxic to
I. galbana than to other species.
24 h period. Cultures of I. galbana e€ected the bio-
A series of dichlorinated aldehydes were also incu-
bated with the ®ve algal cultures Table 2. Less than
transformation of 1 by the third day after inoculation
while D. parva took four days to completely biotrans-
form 1. These results are in keeping with those
observed by Noma et al. for the biotransformation of
benzaldehyde by D. tertiolecta (Noma et al., 1992) and
E. gracilis (Noma et al., 1991; Noma, & Asakawa,
1
% of 2,3-dichlorobenzaldehyde (5) was biotrans-
formed to the corresponding alcohol by all species.
Results are in keeping with the low levels of biotrans-
formation observed for 2-chlorobenzaldehyde (2) by
all species. In contrast, 2,4-dichlorobenzaldehyde (6)
was biotransformed to a greater extent than 5 Table 2.
Maximum reduction of 6 was carried out by D. parva
1998). However for both species reduction was com-
pleted within 24 h. Although most of the substituted
aldehydes were reduced to the corresponding primary
alcohols not all of the chlorinated substrates were
reduced by all of the microalgae.
After the ®ve day period 2-chlorobenzaldehyde (2)
was partially biotransformed by only two species, C.
minutissima (9%) and N. atomus (1%). Selectivity in
the transformation of 2 may be attributed to the proxi-
mity of the chlorine atom to the aldehyde group.
Results are in contrast to those reported for D. tertio-
lecta which totally biotransformed 2 its corresponding
(
12%) and N. atomus (7%).
Only partial reduction of 3,4-dichlorobenzaldehyde
7) was also observed within the test period. Maximum
(
conversion was carried out by D. parva (27%) and P.
purpureum (22%). I. galbana metabolised less than 1%
of substrate within the ®ve day period Table 2. When
the biotransformation of 5, 6 and 7 is compared with
that of 2-chlorobenzylaldehyde (2), 3-chlorobenzylalde-
hyde (3) and 4-chlorobenzaldehyde (4), a signi®cant
Table 2
Relative % biotransformation of aromatic aldehydes (1±11) by all algal species after ®ve days ( after nine days)
Ã
Culture
Substrate
1
2
3
4
5
6
7
8
9
10
11
C. minutissima
N. atomus
100
100
100
100
100
100
100
9
1
0
0
0
10
83
70
99
68
80
89
95
76
1
<1
1
7
6
3
100
100
94
97
100
97
0
0
0
0
0
<1
<1
<1
<1
100
98
D. parva
P. purpureum
12
4
27
22
1
97
100
100
100
100
58
96
100
100
100
100
I. galbana
D. Tertiolacta (Noma et al., 1992)
E. Gracilis (Noma et al., 1991; Noma, & Asakawa, 1998)
1
Ã
100
100
100
100
100
100
±
±
100
±
100
100
100
25
100
100

6
26
I.L. Hook et al. / Phytochemistry 51 (1999) 621±627
decrease in the eciency of transformation of the
dichlorinated substrates is observed.
reduced to a lesser extent than the 3- and 4-mono-
chlorinated counterparts. Methoxyaldehydes were all
reduced with the exception of vanillin a result which is
consistent with literature reports (Noma et al., 1992).
Of the organisms under investigation D. parva
exhibited the greatest ability to biotransform mono-
and dichlorobenzaldehydes while N. atomus was best
at biotransforming methoxyaldehydes. The organisms
under investigation in this study exhibit greater selec-
tivity in the reduction of aromatic aldehydes than D.
tertiolecta or E. gracilis. The ability of the test organ-
isms to biotransform substrate appears to be depen-
dent on the nature and position of the substituent.
The present method of study is useful and the
workup is easy, however a long interval is required for
reaction to occur. The ability of the test organisms to
selectively biotransform a range of natural and syn-
thetic products is in progress.
The biotransformation of a series of monomethoxy-
lated aldehydes was also investigated in this study. The
reduction of 2-methoxybenzaldehyde (8) was com-
pleted within ®ve days by all of the test microalgae
with the exception of D. parva which achieved a maxi-
mum biotransformation of (94%) within this time
frame. I. galbana had completed the total biotrans-
formation of this substrate after three days. In contrast
D. tertiolecta and E. gracilis totally biotransformed
this substrate within 24 h (Noma et al., 1992; Noma et
al., 1991; Noma, Takahashi, & Asakawa, 1991; Noma,
&
Asakawa, 1998).
A more selective reduction of 3-methoxybenzalde-
hyde (9) was catalysed by P. purpureum with only 58%
of substrate being transformed in the ®ve day period.
In fact this maximum was observed after four days
and did not alter after this time. The other organisms
reduced the substrate as e€ectively (>95%) as the lit-
erature examples.
3. Experimental
Almost total biotransformation of 4-methoxy ben-
zaldehyde (10) occurred within the ®ve day period
Table 2. The biotransformation was more rapid than
that of 8 and 9 with 75% being transformed by C.
minutissima, I. galbana and P. purpureum within the
3.1. General experimental procedures
Tlc was carried out on plastic sheets pre-coated (0.2
mm) with Si gel 60F254 (Merck). GC analyses were
carried out using a Perkin Elmer Sigma-3 GC, FID
glass column (2 m; 1.75 mm i.d.) packed with 15%
Carbowax 20 M on Gas Chrom. WAW (80±100
®
rst three days. N. atomus and D. parva reached this
level on day four. The results for the monomethoxy-
lated substrates contrast with those published for D.
tertiolecta which gives the same primary alcohols when
incubated with 8, 9 and 10 once again the biotrans-
formations are total and occur within a 24 h period.
The biotransformation of vanillin (4-hydroxy-3-
methoxybenzaldehyde, 11) was also investigated in this
study but it was not metabolised by any of the algal
species under investigation. This may be due to the
deactivating e€ect of the 4-hydroxyl group previously
reported by Noma et al. (1992) who have observed
that substrates with hydroxy and nitro substituents at
the 2- and 4-positions were dicult to transform or
were not transformed at all. Some biotransformation
of 11 has been reported for D. tertiolecta, where the
only product observed was the primary alcohol.
However this biotransformation was less ecient than
for the other substrates with a maximum biotrans-
formation (25%) being observed after nine days
mesh), operated at various temperatures. N ¯ow rate
2
30 ml/min. For dichlorinated benzaldehydes GC ana-
lyses were carried out using a Perkin Elmer Gas
Chromatograph 8700 ®tted with a stainless steel-glass
1
lined column: 1 Â 6 ft GLT: in; mm i.d., packed with
8
Carbowax 20M (10%) on Chromosorb WAW (100±
120 mesh). Injector temp: 2208C, oven temp.: 1958C,
detector temp: 1958C. N ¯ow rate 30 ml/min.
2
3.2. Algal cultures
The algae used in this experiment were Chlorella
a
minutissima Fott and Novakova ; Nannochloris atomus
a
Butcher ; Dunaliella parva Lerche Isochrysis galbana
b
a
b
(T-Isochrysis ) ; Porphyridium purpureum ( Regional
a
b
Technical College, Galway, Ireland,
Carolina
Biological Supply Company, USA). The stock cultures
were maintained in an arti®cial sea water (ASW) cul-
ture medium and cultured for 30 days (Mockler,
1992). The algae were grown at a constant temperature
of 258C, and illuminated by cool white ¯uorescent
tubes kept on a six/eighteen hour dark/light cycle at
(
Noma et al., 1992).
All the algal species investigated demonstrate the re-
duction of the aromatic aldehydes to their correspond-
ing alcohols to some extent. Substituted aromatic
aldehydes were reduced with varying eciency depend-
ing on the nature and position of the substituent. The
chlorinated benzaldehydes were reduced less eciently
than their methoxylated counterparts. Of the dichlori-
nated substrates 3,4-dichlorobenzaldehyde (7) was bio-
transformed to the greatest extent, however it was
�
2
light intensity of 20 W m . At subculture 1:10 or 1:5
dilutions were made of algal broth into sucient fresh
ASW to give a 40% ¯ask ®ll. All procedures were car-
ried out using strict aseptic protocol. All determi-
nations were carried out in triplicate. Algal dry

I.L. Hook et al. / Phytochemistry 51 (1999) 621±627
627
weights were determined from 25 ml aliquots of cul-
tures ®ltered through a 1.2 mm membrane ®lter and
dried in a fan assisted oven <408C. Growth of the
algae was assessed on day 30 of the growth cycle.
Growth of all green algae was monitored by measuring
the absorbance of whole cells at 440 nm. Growth of
cultures of P. purpureum and I. galbana was deter-
mined using a haemocytometer with Neubauer rulings
tered and evaporated to dryness in vacuo. The residue
was re-dissolved in Et O (1 ml) and analysed under
2
standard GC conditions. The three replicates for a
given data point of the time course experiment for
each alga were combined. The components of the
diethylether extracts were separated either by column
chromatography on silica gel (Si gel 60F254, E. Merck,
Darmstadt) or by prep. tlc. Products were visualised
by UV light (254 nm, as dark quenching bands) or
using 1% vanillin in conc. H SO (1008C for 5 min) or
3
in a 1 mm chamber. Each algal type (1:5 dilution)
was set up in conical ¯asks (six, 100 ml) (8 ml of algal
broth in 32 ml ASW). Growth under standard static
and shaking cultures was measured after 7, 14, 21 and
2
4
freshly prepared iodoplatinate spray.
28 days. Shaking cultures were placed on an orbital
shaker set at 90 rpm.
References
3.3. Toxicity measurement
Bold, H. C., & Wynne, M. J. (1978). Introduction to the algae, struc-
ture and reproduction. NJ: Prentice Hall Inc.
Borowitzka, M. (1988). In M. A. Borowitzka, & L. J. Borowitzka,
Microalgal biotechnology (pp. 154±196). Cambridge University
Press.
Each algal type (1:5 dilution) was set up in 10 ml
pyrex test tubes (1 ml algal broth in 4 ml ASW).
Growth under standard static conditions was allowed
for seven days. Substrates were dissolved in methanol
and 1 ml of this solution was added to the algae to
bring ®nal substrate concentrations to 100 ppm. In ad-
dition two types of control were used. Control 1:
algae+ASW only; and control 2: algae+ASW+1 ml
methanol (i.e. substrate free). All concentrations and
controls were set up in triplicate. To monitor toxicity,
aliquots (0.5 ml) were removed aseptically from the
culture tubes at intervals of 24, 48, 72, 96 and 120 h.
Survival of all the green algae was monitored by
measuring the absorbance of whole cells at 440 nm.
Survival of Isochrysis and Porphyridium was deter-
mined by cell count using a haemocytometer.
Davies, D. G., Green, R. H., Kelly, D. R., & Roberts, S. M. (1990).
Biotechnology, 10, 2.
Ewart, J. W., & Pruder, G. D. J. (1981). World Mariculture Society,
12(1), 333±339.
Fattarusso, E., & Piatelli, M. (1980). In P. J. (Ed). Scheuer, Marine
natural products (pp. 95±139). New York: Academic Press.
Kirsop, B. E., & Doyle, A. (eds). (1991). Maintainance of microor-
ganisms and cultured cells ± A manual of laboratory methods (2nd
ed.). London: Academic Press.
Laskin, A. I., & Lechevalier, H. A. (1984) Microbial transformations
In CRC handbook of microbiology (Vol. 7, 2nd ed.). Boca
Raton: CRC Press.
Lee, R. Z. (1989). In R. E. Lee, Phycology (pp. 3±32). Cambridge
Univ. Press.
Mockler, U., M.Sc. thesis 1992. Department of Pharmacognosy,
Trinity College Dublin.
Noma, Y., & Asakawa, Y. (1998). Biotechnology in agriculture and
forestry. In Y. P. S. Baji, Medicinal and aromatic plants X, Vol 41
3.4. Biotransformations
(
pp. 194±237). Berlin: Springer.
The culture conditions are as outlined above.
Dilutions of microalgae (1:5) were made in conical
asks (250 ml) to provide a 40% ¯ask ®ll (i.e. 20 ml
algal broth into 80 ml ASW). Algae were ®rst allowed
to grow statically for seven days at which stage a sol-
ution of substrate in methanol (1 ml) was added.
Aliquots (10 ml) were taken from culture ¯asks in tri-
plicate at intervals of 24, 48, 72, 96 and 120 h. These
Noma, Y., Okajima, Y., Takahashi, H., & Asakawa, Y. (1991).
Phytochemistry, 30(9), 2969±2972.
¯
Noma, Y., Takahashi, H., & Asakawa, Y. (1991). Phytochemistry,
30(4), 1147±1151.
Noma, Y., Akehi, E., Miki, N.,
Phytochemistry, 31(2), 515±517.
&
Asakawa, Y. (1992).
Pirroeck, M., Baash, K. H., & Pohl, P. (1984). Phytochemistry,
3(2), 207±216.
2
South, G. R., & Whittick, A. (1987). Introduction to phycology (pp.
1±18). Oxford: Blackwell Scienti®c.
were extracted with equal volumes of distilled Et O.
2
The ether layer was separated, dried over Na SO , ®l-
2
Suga, T., & Hirata, T. (1990). Phytochemistry, 29(8), 2393±2460.
4