World Journal of Microbiology and Biotechnology

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World Journal of Microbiology and Biotechnology
Volume 21, Number 2

Biotransformation of biphenyl by the filamentous fungus
Talaromyces helicus
(101 - 106)
Maria C. Romero, Elke Hammer, Renate Hanschke, Angelica M. Arambarri, Frieder
Schauer
DOI: 10.1007/s11274-004-2779-y
Solid-state fermentation production of tetracycline by Streptomyces
strains using some agricultural wastes as substrate
(107 - 114)
Agnes E. Asagbra, Abiodun I. Sanni, Olusola B. Oyewole
DOI: 10.1007/s11274-004-2778-z
Influence of glucose and oxygen on the production of ethyl acetate
and isoamyl acetate by a Saccharomyces cerevisiae strain during
alcoholic fermentation
(115 - 121)
C. Plata, J. C. Mauricio, C. Millán, J. M. Ortega
DOI: 10.1007/s11274-004-2780-5
Rapid screening of Aspergillus terreus mutants for overproduction
of lovastatin
(123 - 125)
M. A. Vilches Ferrón, J. L. Casas López, J. A. Sánchez Pérez, J. M. Fernández Sevilla, Y.
Chisti
DOI: 10.1007/s11274-004-3045-z
Impact of balanced substrate flux on the metabolic process
employing fuzzy logic during the cultivation of Bacillus thuringiensis
var. Galleriae
(127 - 134)
R. K. I. Anderson and Kunthala Jayaraman
DOI: 10.1007/s11274-004-3043-1
Degradation and corrosive activities of fungi in a diesel–mild steel–
aqueous system
(135 - 142)
Fátima Menezes Bento, Iwona Boguslava Beech, Christine Claire Gaylarde, Gelsa Edith
Englert, Iduvirges Lourdes Muller
DOI: 10.1007/s11274-004-3042-2
Changes in lignocellulolytic enzyme activites in six Pleurotus spp.
strains cultivated on coffee pulp in confrontation with Trichoderma
spp.
(143 - 150)
G. Mata, D. M. Murrieta Hernández, L. G. Iglesias Andreu
DOI: 10.1007/s11274-004-3041-3
Milk-clotting protease production by Nocardiopsis sp. in an
inexpensive medium
(151 - 154)
M. T. H. Cavalcanti, C. R. Martinez, V. C. Furtado, B. B. Neto, M. F. Teixeira, J. L. Lima
Filho, A. L. F. Porto
DOI: 10.1007/s11274-004-3470-z
Diversity and dynamics of bacterial species in a biofilm at the end of
the Seoul water distribution system
(155 - 162)
Dong-Geun Lee, Jung-Hoon Lee, Sang-Jong Kim
DOI: 10.1007/s11274-004-2890-0





Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an
exopolysaccharide produced by Chryseomonas luteola TEM05
(163 - 167)
Guven Ozdemir, N. Ceyhan, E. Manav
DOI: 10.1007/s11274-004-1563-3
Production of extracellular alkaline proteases by Aspergillus clavatus (169 - 172)
Célia R. Tremacoldi and Eleonora Cano Carmona
DOI: 10.1007/s11274-004-2724-0
Barley-based medium for the cost-effective production of Bacillus
thuringiensis
(173 - 178)
P. S. Vimala Devi, T. Ravinder, C. Jaidev
DOI: 10.1007/s11274-004-2217-1
Induction, and production studies of a novel glucoamylase of
Aspergillus niger
(179 - 187)
M. Ibrahim Rajoka and Amber Yasmeen
DOI: 10.1007/s11274-004-1766-7
Characterization of Mucor miehei lipase immobilized on polysiloxane-
polyvinyl alcohol magnetic particles
(189 - 192)
L. M. Bruno, J. S. Coelho, E. H. M. Melo, J. L. Lima-Filho
DOI: 10.1007/s11274-004-3321-y
Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated
ionic liquid: significantly enhanced enantioselectivity and stability
(193 - 199)
Jia-Ying Xin, Yong-Jie Zhao, Yan-Guo Shi, Chun-Gu Xia, Shu-Ben Li
DOI: 10.1007/s11274-004-3108-1
Antimicrobial screening and active compound isolation from marine
bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve
(201 - 206)
Li Zheng, Haimin Chen, Xiaotian Han, Wei Lin, Xiaojun Yan
DOI: 10.1007/s11274-004-3318-6

Biotransformation of biphenyl by the filamentous fungus Talaromyces helicus
Maria C.Romero
1
,Elke Hammer
2,
*,Renate Hanschke
2
,Angelica M.Arambarri
1
and Frieder Schauer
2
1
Instituto Botanica Spegazzini,Facultad de Ciencias Naturales y Museo,Universidad Nacional de La Plata,La Plata,
Argentina
2
Institut fu
¨
r Mikrobiologie,Ernst-Moritz-Arndt-Universita
¨
t Greifswald,F.-L.-Jahn-Str.15,17487 Greifswald,
Germany
*Author for correspondence:Tel.:+49-3834-864211,Fax:+49-3834-864202,E-mail:hammer@uni-greifswald.de
Received 24 December 2003;accepted 15 June 2004
Keywords:Biphenyl degradation,filamentous fungi,oxidation products,ring fission,Talaromyces
Summary
The filamentous fungus Talaromyces helicus,isolated from oil-contaminated sludge,oxidizes biphenyl via 4-
hydroxybiphenyl to the dihydroxylated derivatives 4,4¢-dihydroxybiphenyl and 3,4-dihydroxybiphenyl,which,to a
certain extent,are converted to glycosyl conjugates.The sugar moiety of the conjugate formed from 4,4¢-
dihydroxybiphenyl was identified as glucose.Further metabolites:2-hydroxybiphenyl,2,5-dihydroxylated biphenyl,
and the ring cleavage product 4-phenyl-2-pyrone-6-carboxylic acid accumulated only in traces.From these results
the main pathway for biotransformation of biphenyl in T.helicus could be proposed to be the excretion of
dihydroxylated derivatives (>75%) and their glucosyl conjugates (<25%).
Introduction
Biphenyl and the monohydroxylated derivatives
2-hydroxy- and 4-hydroxybiphenyl are known to be
fungistatic substances.These compounds are widely
used for the conservation of citrus fruits,even though
biphenyl is known for its toxic effects on humans
(Hakkinen et al.1973).Furthermore,it has been found
that 4-hydroxybiphenyl shows estrogenic effects (Schultz
et al.1998).
In contrast to bacteria,complete mineralization of
biphenyl has never been found in fungi.However,the
transformation pathways for xenobiotics in these
organisms are of interest,due to their high similarity
to the metabolism of mammalian systems (Smith &
Rosazza 1974).The ability to hydroxylate such com-
pounds has been found in some yeasts and filamentous
fungi (Dodge et al.1979;Smith et al.1980;Cerniglia
1997) as well as in mammals (Meyer & Scheline 1976).
Beside monohydroxylated derivatives,di- and trihydr-
oxylated biphenyls were also found.Generally,the
transformation results in the formation of derivatives
with better water solubility,that sometimes even exceed
the parent compounds in toxicity and accumulate as end
products.
Recently,more and more data have accumulated
indicating that hydroxylated biphenyl derivatives can
also undergo ring cleavage in fungi.Products formed
during this biotransformation in yeasts (Lange et al.
1998;Sietmann et al.2001) and the filamentous fungus
Paecilomyces lilacinus (Gesell et al.2001) were identified
as phenylmuconic acid derivatives and the correspond-
ing lactones.
Presumably because of its fungistatic activity,few
data are available concerning biphenyl metabolism in
filamentous fungi.Biphenyl oxidation has only been
studied in the deuteromycetes Aspergillus parasiticus
(Cox & Golbeck 1985;Mobley et al.1993) and
Paecilomyces lilacinus (Gesell et al.2001) and in the
zygomycete Cunninghamella echinulata (Seigle-Murandi
et al.1991).
The aim of this work was to study the biotransfor-
mation of the fungicide biphenyl by an ascomycetous,
filamentous soil fungus,isolated from a hydrocarbon-
contaminated site,as well as to study the kinetics of the
product formation and compare the data obtained with
results for other filamentous fungi of the genus Talar-
omyces.
Materials and methods
Growth and culture conditions
Talaromyces helicus was isolated from contaminated
sludge of the East Channel,near the YPF-oil Refinery in
La Plata,Argentina.Cultures were maintained on malt
agar slants at 4 C.The fungus was identified by colony
morphology and morphology of hyphae,conidia as well
as ascospores,by scanning electron microscopy
World Journal of Microbiology & Biotechnology 2005 21:101–106
DOI:10.1007/s11274-004-2779-y

Springer 2005
(Figure 1),applying the method of Hanschke & Schauer
(1996).
The ability to grow on alkanes or aromatic com-
pounds was tested on a solid mineral salts medium.The
substrates were supplemented via the gas phase.
The strain was pre-cultivated in 40 ml Sabouraud (2%
glucose,1%peptone) medium,for 48 h at 180 rev min
)1
and 30 C.For degradation experiments 1 ml of this
culture was used to inoculate 100 ml of a mineral salts
medium (MM:Kaufman & Blake 1973) in 500 ml shake
flasks,supplemented with 10 g glucose l
)1
,pH5.4.After
incubation for 3 days at 30 C and 180 rev min
)1
on a
rotary shaker,the biomass was harvested by centrifu-
gation (5000 · g,5 min) and washed twice with sterile
MM.The fungal biomass (210 mg dry weight) was
resuspended in 100 ml MMand 1 g of biphenyl l
)1
were
added.Additional cultures (500 ml flasks with 100 ml of
MM and 0.32 g of mycelium) were incubated with 1 g
4-hydroxybiphenyl l
)1
,0.1 g 4,4¢-dihydroxybiphenyl l
-1
or 0.05 g 3,4-dihydroxybiphenyl l
-1
to enhance the yield
of intermediates.Assays with the fungus without sub-
strate in MM as well as biphenyl in MM without
biomass were used as controls.All assays were carried
out in duplicate.Standard deviation was no more than
10%.
Chemical analysis and identification of metabolites
At different sampling periods,2 ml of the culture liquid
were centrifuged (5000 · g,5 min).Formation of
metabolites was followed by analysing 100 ll of the
supernatant by reverse phase HPLC according to
Hammer & Schauer (1997).
For characterization of metabolites,culture superna-
tant was separated from the mycelium by centrifugation
after 129 h.The supernatant was then extracted twice
with ethyl acetate at pH 7,and once again after
acidification of the aqueous residue to pH 2.The
organic phases were dried over anhydrous sodium
sulphate,and the solvent was removed by evaporation.
The residues obtained were dissolved in methanol.
The mycelium obtained was washed twice with 5 ml
methanol;the extracts were dried over anhydrous
sodium sulphate and the solvent was reduced to 1 ml
by evaporation.
The three extracts were then analysed by HPLC.The
u.v–visible absorption spectra of the degradation prod-
ucts were determined in a diode array detector HP 1040
(Hewlett Packard,Bad Homburg,Germany).Extracts
were analysed by GC–MS on a gas chromatograph GC
8000 linked to a mass-selective detector MD800 (Fisons
Instruments,Mainz,Germany) operating at 70 eV,
fitted with a 30-m DB5-ms column (0.25 mm by
0.33 lm film;J & W Scientific,Folsom,Calif.,USA).
The temperature program was:5 min at 80–300 C at
10 min
)1
.Extracts were analysed again after derivati-
zation with diazomethane (formation of methyl deriv-
atives),as described by De Boer & Backer (1956).
Inhibition experiments
The same methodology mentioned above was employed
in the inhibition experiments,but in addition to the
substrates biphenyl or 4-hydroxybiphenyl,the cyto-
chrome P-450 inhibitor 1-aminobenzotriazole was
added in equimolar amounts.Samples were taken and
analysed by HPLC as described above.
Deconjugation experiments
Enzymatic deconjugation experiments were carried out
with b-glucuronidase and arylsulphatase according to
Cerniglia et al.(1982) and with xylosidase as described
by Sutherland et al.(1992).In all assays 50 mg
lyophilizate was used.Samples incubated without addi-
tion of enzymes served as controls.After enzyme
treatment 100-ll samples were analysed by HPLC and
another portion was subjected to characterize the sugar
moiety of the conjugate.The estimation of glucose was
carried out in an enzymatic assay based on glucose
oxidase and peroxidase (Bergmeyer et al.1974).This test
was shown to be specific for glucose and was negative
for xylose,rhamnose,mannose,and glucuronic acid.
Chemicals
Biphenyl,2- and 4-hydroxybiphenyl were obtained from
Merck (Darmstadt,Germany).3,4-Dihydroxybiphenyl
was purchased from Promochem (Wesel,Germany) and
1-aminobenzotriazole from Aldrich (Steinheim,Ger-
many).All other chemicals and solvents were of the
highest purity available.
Results
The ascomycete Talaromyces helicus was isolated among
a number of yeasts and filamentous fungi obtained from
oil-contaminated sludge.The strain was identified as
T.helicus by colonies showing typical yellow reverse,
Figure 1.Typical ascogonium with antheridium of the ascomycete
T.helicus.Scanning electron microscopic study at 5000-fold magni-
fication.
102 M.C.Romero et al.
ascospores,that are only delicately spinulose,and the
shaped ascogonia around which thin antheridia coil
tightly,soon growing out into a large terminal coil from
which the ascogonous hyphae originate (Figure 1).
The fungus was able to use hexadecane as well as
phenol supplemented via the gas phase for growth on
solid medium.In contrast,biphenyl was not used for
growth on solid or in liquid medium.However,incuba-
tion of T.helicus with biphenyl and analysis of the
culture supernatant by HPLC showed six products
accumulating,which were not found in control assays
(Figure 2).By comparing u.v.-spectral data as well as
retention times on the reverse phase HPLC column with
those of authentic standards,four of them were iden-
tified as 4-hydroxybiphenyl,2-hydroxybiphenyl,3,4-
dihydroxybiphenyl,and 4,4¢-dihydroxybiphenyl.In
contrast,four of six other Talaromyces strains studied
accumulated 4-hydroxybiphenyl as the only product
(Table 1).
For kinetic studies,the increase of the metabolites
mentioned and the formation of two unknown metab-
olites,I and II (retention time 4.8 and 5.8,respectively)
were determined by HPLC.4-Hydroxybiphenyl and
4,4¢-dihydroxybiphenyl were the major metabolites and
reached the maximum values at 48 h (Figure 3).The
relatively stable concentrations of all hydroxylated
products over the following 100 h analysed,indicated
that no further degradation of these products occurred
under the incubation conditions used.Metabolites I and
II showed u.v.spectra with high similarity to those of
3,4-dihydroxybiphenyl (I) and 4,4’-dihydroxybiphenyl
(II),but with lower retention times.Calculated on base
of the response factor of the biphenyl derivatives,these
unknown metabolites were produced in lower amounts
than the hydroxylated products.
For further characterization by GC–MS analysis,
metabolites were extracted from the culture fluid with
ethyl acetate at pH 7 and pH 2,and the methanol
mycelium extracts and ethyl acetate extracts of the
culture supernatant were analysed.The gas chromato-
graphic and mass spectral data of products adsorbed to
the mycelium and extracted from culture supernatant at
pH 7 confirmed the data obtained by HPLC-DAD
analyses.In the extracts 4-hydroxybiphenyl (170 [M
+
],
141 [M]-CHO,115 [M]-COC
2
H
3
),4,4’-dihydroxybiphe-
nyl (186 [M
+
],157 [M]-CHO,141 [M]-CHO-O,128
[M]-C
2
H
2
O
2
,115 [M]-COC
2
H
3
,77 C
6
H
5
),3,4-di-
hydroxybiphenyl (186 [M
+
],157 [M]-CHO,128 [M]-
C
2
H
2
O
2
),and traces of 2,5-dihydroxybiphenyl (186
[M
+
],157 [M]-CHO,128 [M]-C
2
H
2
O
2
) and 2-hydrox-
ybiphenyl (170 [M
+
],141 [M]-CHO,115 [M]-COC
2
H
3
)
were identified by comparison of chemical data with
those of standard compounds.
The main peak found in the acidic extract showed a
molecular ion peak at m/z 230 (C
13
H
10
O
4
) and main
fragment ions at m/z 202 (C
12
H
10
O
3
+
,
M
+
-CO),171
(C
11
H
7
O
2
+
,M
+
-COOCH
3
),115 (C
9
H
7
+
,M
+
-COOCH
3
-
2 · CO),which corresponds to the ring cleavage prod-
uct 4-phenyl-2-pyrone-6-carboxylic acid.
Incubation of the fungus with the hydroxylated
biphenyl derivatives 4-hydroxybiphenyl (1 g l
)1
),3,4-
dihydroxybiphenyl (0.05 g l
)1
),and 4,4¢-dihydroxybi-
phenyl (0.1 g l
)1
) showed that these substances were
also subject to further transformation.Thus,4-hydrox-
min
5
10
mAU
0
50
100
150
200
2.88
4.75
5.83
6.26
6.45
8.43
10.11
12.36
13.17
Biphenyl
SM
3,4diOHBP
4-OH-BP
I
II
4,4‘-diOH-BP
Figure 2.HPLC elution profile of an aqueous culture supernatant
after incubation of T.helicus with biphenyl (1 g l
)1
) after 48 h.Six
biphenyl transformation products were detectable after separation on
a reverse phase column.2-Hydroxybiphenyl could only be detected by
the typical u.v.spectrum next to the 4-hydroxybiphenyl peak.SM:
secondary metabolite,4-OH-BP:4-hydroxybiphenyl;3,4diOHBP:3,4-
dihydroxybiphenyl;4,4’diOHBP:4,4’-dihydroxybiphenyl;I;II:metab-
olites I and II.
Table 1.Biotransformation of biphenyl (0.01%) by fungal strains of the genus Talaromyces.Product formation after incubation for 14 days.
Strains Products
2-OH-BP
a
4-OH-BP 3,4diOHBP 4,4¢diOHBP Conjugates
T.flavus SBUG-M892
b
) + ) ) )
T.flavus SBUG-M941 ) + ) ) )
T.flavus SBUG-M1023 ) + ) + )
T.helicus SBUG-M + + + + +
T.rotundus SBUG-M1004 + ) ) ) )
T.stipitatus SBUG-M271 ) + ) ) )
T.wortmannii SBUG-M410 ) + ) ) +
a
2-OH-BP:2-hydroxybiphenyl;4-OH-BP:4-hydroxybiphenyl;3,4diOHBP:3,4-dihydroxybiphenyl;4,4¢diOHBP:4,4¢-dihydroxybiphenyl.
b
All Talaromyces strains except T.helicus were obtained fromthe Strain collection of the Institute for Microbiology of Greifswald University
and were isolated from natural habitats.
Biphenyl degradation by Talaromyces 103
ybiphenyl was transformed to 3,4-dihydroxybiphenyl
(7.5%) and 4,4¢-dihydroxybiphenyl (44.5%),within 22 h
incubation time.3,4-Dihydroxybiphenyl was trans-
formed to 4-phenyl-2-pyrone-6-carboxylic acid
(>90%) and compound I (traces);4,4¢-dihydroxybiphe-
nyl was slowly transformed to product II (10%).
Identification of products I and II
Both products (I and II) seemed to be more hydrophilic,
as indicated by their lower retention times on reverse
phase material compared to the parent compounds.
Neither of these two products was detectable after
extraction of the culture supernatant by ethyl acetate
followed by GC–MS analysis.In view of the high
similarity of u.v.spectra to those of hydroxylated
biphenyls and in comparison to the behaviour of
products found after biotransformation of dibenzofuran
by Penicillium canescens (Hammer et al.2001),these
compounds were assumed to be conjugates.This
assumption should be proven by deconjugation exper-
iments carried out with glucuronidase and arylsulpha-
tase.Culture supernatant containing products I and II
was concentrated by lyophilization,and individual 50-
mg-samples were treated with each of the enzymes.
Hydrolysis with glucuronidase led to the depletion of the
products I and II and produced increasing amounts of
3,4-dihydroxybiphenyl (from product I) and 4,4¢-di-
hydroxybiphenyl (from product II).In contrast,both
the control without enzyme and the sample treated with
arylsulphatase showed no hydrolysis and both conju-
gates remained intact.Because of the unspecific reaction
of glucuronidase towards several sugar conjugates,the
sugar moiety was analysed by a more specific enzymatic
assay for glucose with glucose oxidase.After glucuron-
idase treatment of separated product II,the assay
showed accumulation of glucose in the samples,whereas
the sugar was not detectable in the controls (medium,
buffer,arylsulphatase assay,assay without enzymes).
The amount of product I was too low for successful
separation and identification of the sugar.
Inhibition experiments and enzyme assays
1-Aminobenzotriazole affected the biphenyl transfor-
mation so that only about 50%of the 4-hydroxybiphe-
nyl was formed and no other metabolites could be
detected.Biotransformation of 4-hydroxybiphenyl was
nearly completely inhibited by 1-aminobenzotriazole.
Only traces of 3,4-dihydroxybiphenyl (2%) and 4,4¢-
dihydroxybiphenyl (8%) were observed.
Discussion
The fungus T.helicus cannot use biarylic compounds
like biphenyl,dibenzofuran,or naphthalene for growth.
However,the fungus is able to co-metabolize these
compounds.Cells grown in mineral salts medium with
glucose or grown on complex medium in addition,
transformed the hydrophobic parent compound to more
hydrophilic products:hydroxylated products in high
amounts and more hydrophilic sugar conjugates as well
as ring-cleavage products in lower amounts.
The fungus produced relatively large amounts of
hydroxylated intermediates,which included monohydr-
oxylated and dihydroxylated compounds,indicating the
involvement of monoxygenases in the biotransformation
pathway.From biphenyl,4-hydroxybiphenyl was the
major product,whereas production of 2-hydroxybiphe-
nyl was rather low.Hydroxylation of the C4- position
was also described as the main pathway for other
filamentous fungi,yeasts,and mammals (Meyer &
Scheline 1976;Smith et al.1980;Golbeck et al.1983;
Lange et al.1998;Sietmann et al.2000).A second
hydroxylation by T.helicus can occur in the same ring as
well as in the second ring.As a result,the dihydroxy-
lated derivatives 3,4-dihydroxybiphenyl and 4,4¢-di-
hydroxybiphenyl are produced in a ratio 1:3.Strong
accumulation of para,para¢-hydroxylated biphenyl was
also observed for Aspergillus strains by Mobley et al.
(1993).In contrast,hydroxylation at the unsubstituted
ring of 4-hydroxybiphenyl occurred only in traces or
could not be observed at all in yeast.In strains of the
genus Trichosporon (Sietmann et al.2002) or Debary-
omyces vanrijiae (Lange et al.1998) the main transfor-
mation pathway of biphenyl goes via 4-hydroxybiphenyl
and 3,4-dihydroxybiphenyl up to ring cleavage.Forma-
tion of considerable amounts of 2,5-dihydroxybiphenyl
as described for Trichosporon mucoides (Sietmann et al.
2000) was not observed in T.helicus.
The strong inhibition of hydroxylation reactions in the
presence of the cytochrome-P-450-inhibiting substance
1-aminobenzotriazole points to an involvement of such
enzymes in the transformation reactions.The differences
in the level of inhibition of the first and the second
hydroxylation may indicate the existence of different
0
1
2
3
4
50
100
150
Time (h)
( M)
µ
Figure 3.Time-dependent formation of products by glucose-
grown cells of T.helicus during incubation with biphenyl (1 g l
)1
).
(j) 4-Hydroxybiphenyl;(m) 3,4 hydroxybiphenyl;(r) 4,4¢-dihydroxy-
biphenyl.
104 M.C.Romero et al.
monooxygenases in the fungus.Moreover,the substrate
specificity of the cytochrome P-450 enzymes involved in
unspecific oxidation of hydrophobic compounds in
various fungi and yeast seems to be rather different.As
mentioned above,the hydroxylation pattern can differ
drastically in various eukaryotic organisms.
Although dihydroxylated biphenyl derivatives accu-
mulated in high amounts in the culture supernatant of
T.helicus,they are not dead-end products,but were
further transformed.As found in yeasts – mainly
described for Trichosporon strains (Sietmann et al.
2002) and Debaryomyces vanrijiae (Lange et al.1998)
but also for the imperfect filamentous fungus Paecilo-
myces lilacinus (Gesell et al.2001),3,4-dihydroxybiphe-
nyl can be oxidized up to ring cleavage.As a result
4-phenyl-2-pyrone-6-carboxylic acid was formed (Fig-
ure 4).In T.helicus this reaction only occured to a low
extent when biphenyl or 4-hydroxybiphenyl were used
as substrates.If 3,4-dihydroxybiphenyl was used as
substrate,nearly complete conversion to the pyrone was
observed.Perhaps the dihydroxylated biphenyl induces
a ring-cleaving enzyme,which does not occur at the low
level of 3,4-dihydroxylated biphenyl that accumulated
by transformation of biphenyl or 4-hydroxybiphenyl.
Furthermore,under these conditions 3,4-dihydroxybi-
phenyl and 4,4-dihydroxybiphenyl,were partially con-
verted to glucose conjugates.Therefore,detoxification
of harmful compounds in T.helicus seems to occur via
the so-called phase I (hydroxylation)/phase II transfor-
mation system (Zhang et al.1996),through which
excretion of toxic intermediates becomes possible.In
contrast,in yeast detoxification of such compounds is
achieved by ring cleavage of the dihydroxylated deriv-
atives produced (Lange et al.1998;Sietmann et al.2000,
2002).Surprisingly,conjugation of 4-hydroxybiphenyl
was not observed.Furthermore,it remains unclear why
hydroxylated biphenyl derivatives can be excreted par-
tially directly,whereas another part seems to be only
excreted after conversion to sugar conjugates.Here,it is
proven for the first time that a Talaromyces strain
isolated from oil- and PAH-contaminated water shows
higher potential to tolerate and to transform biphenyl
and its derivatives in comparison to Talaromyces
isolates from natural sites.
Acknowledgements
This research was supported by the National Research
Council,Argentina and the Bundesministerium fu
¨
r
Forschung und Technologie,Germany.We thank M.
Specht,Institute for Organic Chemistry,University of
Hamburg for providing 4-phenyl-2-pyrone-6-carboxylic
acid and H.Lehnherr for revising the manuscript.
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OH
OH
HO
HO
H
O
HO
OH
OH
OH
O
O
COOH
Metabolite I
A
B
C
D
E
F
G
H
Metabolite II
HO
O
OH
OH
OH
O
OH
OH
O
OH
OH
OH
O
OH
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106 M.C.Romero et al.
Solid-state fermentation production of tetracycline by Streptomyces strains using
some agricultural wastes as substrate
Agnes E.Asagbra
1,
*,Abiodun I.Sanni
2
and Olusola B.Oyewole
3
1
Biotechnology Division,Federal Institute of Industrial Research,Oshodi,Nigeria
2
Department of Botany and Microbiology,University of Ibadan,Nigeria
3
Department of Food Science and Technology,University of Agriculture Abeokuta,Nigeria
*Author for correspondence:Tel.:+234-1-080-23001173,E-mail:yemasagbra@yahoo.com
Received 23 December 2003;accepted 15 June 2004
Keywords:Optimisation,peanut shells,solid-state fermentation,Streptomyces,tetracycline
Summary
The ability of Streptomyces sp.OXCI,S.rimosus NRRL B2659,S.rimosus NRRL B2234,S.alboflavus NRRL
B1273 S.aureofaciens NRRL B2183 and S.vendagensis ATCC 25507 to produce tetracycline using some local
agricultural wastes as solid state media,were assessed.The wastes employed include peanut (groundnut) shells,
corncob,corn pomace and cassava peels.Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity.All
the strains produced tetracycline in a solid-state fermentation process containing peanut (groundnut) as the
carbohydrate source.Streptomyces sp.OXC1 had the highest ability for tetracycline production with peanut shells
as the substrate in solid fermentation (13.18 mg/g),followed by S.vendagensis ATCC 25507 (11.08 mg/g),
S.rimosus NRRL B1679 (8.46 mg/g),S.alboflavus NRRL B1273 (7.59 mg/g),S.rimosus NRRL B2234 (6.37 mg/
g),S.aureofaciens NRRL B2183 (4.27 mg/g).Peanut (groundnut) shells were the most effective substrate (4.36 mg/
g) followed by corncob (2.64 mg/g),cassava peels (2.16 mg/g) and corn pomace (1.99 mg/g).The composition of
the peanut (groundnut) shell mediumoptimal for tetracycline production were peanut shells 100 g,organic nitrogen
(peanut meal) 10 g,(NH
4
)
2
SO
4
1 g,KH
2
PO
4
0.5 g,CaCO
3
0.5 g,NaCl 0.5 g,MgSO
4
 7H
2
O 0.5 g,soluble starch
10 g,peanut oil 0.25 ml with initial moisture content of 65–68%,and initial pH 5.3–6.3.Substrate (1 g dry weight)
was inoculated with 1.0 · 10
8
conidia per ml and incubated at 28–31 Cfor 5–7 days,producing 13.18 mg/g of total
tetracycline.Tetracycline detection started on day 3 and attained its maximum level on day 5.
Introduction
Streptomyces is the largest antibiotic-producing genus in
the microbial world so far (Watve et al.2001).However
it is becoming increasingly apparent that 99% of the
diverse bacterial species with antibiotic potentials are
still unexplored (Ward et al.1990;Watve et al.2000).
The need to continue to explore for more antibiotic-
producing microbial strains of microorganisms has been
stressed because of the resistance of disease-causing
microorganisms to known antibiotics and the varying
diversities in the properties of antibiotics produced by
different organisms (Pelczar et al.1993).
Antibiotic imports,including tetracycline and ampi-
cillin constitute a major percentage of medical imports
in Nigeria.Unfortunately,while the raw materials for
antibiotic production are readily available in the coun-
try,there is no single facility for commercial antibiotic
production (Ifudu 1986a,b).
Tetracyclines are broad-spectrum antibiotics with an
octahydronaphthacene structure with four annelated six-
membered rings.They are useful in a variety of infections
caused by bacteria,rickettsias,trachoma,coccidia,
amoebae,balantida and mycoplasma (Yang & Swei
1996).They are used for the control of plant diseases,
stimulation of acid fermentation and inhibition of
material biodeterioration (Somerson & Phillips 1961;
Huang 1972).Submerged cultures (Bhatnagar et al.
1988) and culture media are usually used to produce
antibiotics;culture conditions affect the kind and quan-
tity of antibiotic production (Komatsu et al.1975;Yang
and Ling 1989).Solid-state fermentation gives a product
which is more stable than that of submerged culture and
also requires less energy input (Wang 1989).
Cellulolytic materials are abundantly available glob-
ally and can be used by a number of microorganisms
(Yang & Swei 1996).Peanut (groundnut) and its
residues are abundantly available in Nigeria,as at
1996 the annual peanut (groundnut) production was
7608 tons (Rake 1998).This study was embarked upon
to compare the antibiotic-producing potentials of some
reference standard Streptomyces spp.with a locally
isolated strain,using different agricultural wastes in
Nigeria as substrates in tetracycline production.
World Journal of Microbiology & Biotechnology 2005 21:107–114
DOI:10.1007/s11274-004-2778-z

Springer 2005
Materials and methods
Agricultural wastes
The agricultural wastes used for the work include
peanut shells,corn cob,corn pomace and cassava peels.
They were obtained from the pilot plant of the Federal
Institute of Industrial Research Oshodi,Lagos,Nigeria.
The wastes were dried separately and were screened with
4–16 mesh sizes to remove the dust and large aggregates.
Determination of moisture contents of substrates
Samples were dried at 60 C under vacuum for 8–12 h
to a constant weight.The weight difference after drying
was considered as the moisture content.
pH
Initial pH of substrates was determined directly by
immersing the electrode into the substrate,but the final
value was determined after mixing a sample with 4
volume distilled water (Yang & Swei 1996).
Bulk density
The dry weight or wet weight of samples per unit volume
(1 ml) was the bulk density in dry weight or wet weight,
respectively (Baver 1956).
Water activity (A
w
)
Samples with different moisture contents were placed in
a sealed container at 25 C and water activity was
determined by a hygrometer (Yang 1977).
Microorganisms
Streptomyces sp.OXC1 was obtained from topsoil
around the pilot plant building of the Federal Institute
of Industrial Research Oshodi and was characterized by
16SrRNA gene sequencing.S.rimosus NRRL B2659,
S.rimosus NRRL B2234,S.alboflavus NRRL B1273
S.aureofaciens NRRL B2183 and S.vendagensis ATCC
25507 were obtained from the Northern Regional
Agricultural Research Center in USA.All the Strepto-
myces isolates produced antibiotics.Bacillus subtilis
ATCC 6633 was used to assay antimicrobial activity.
Culture media and culture conditions
The Streptomyces strains were initially grown at 26 C
in solid medium containing (g 1
1
):soluble starch 10;
(NH
4
)
2
SO
4
2;CaCO
3
2;NaCl 1;K
2
HPO
4
1;MgSO
4

7H
2
O 1,trace elements 1 ml (contained FeSO
4
 7H
2
O
1.0 g,CuSO
4
 5H
2
O 0.5 g) ZnSO
4
 7H
2
O 1.0 g and
MnSO
4
 2H
2
O 1 g) and agar 20 g.Spores were washed
with 5 ml 0.05% Tween-80 in sterile water and har-
vested by centrifugation at 3000 · g for 10 min.Each ml
of the suspension contained about 10
8
spores.The solid
state medium contained (g):peanut (groundnut) shells
100,(NH
4
)
2
SO
4
1.5,CaCO
3
1.0,NaCl 0.5,KH
2
PO
4
0.5
and MgSO
4
 7H
2
O.The medium was mixed thoroughly
with seed culture and the initial moisture content was
then adjusted to 65% with distilled water.Incubation
was at 28 C for 5–7 days.
Extraction of antibiotics
After fermentation,the culture mass was extracted with
four times volume of distilled water with shaking at
room temperature for 5 min (Yang & Ling 1989;Yang
& Swei 1996).
Determination of antibiotic activity
Antimicrobial activity of the extract was measured by
the paper disc method in antibiotic medium 1 (DIFCO
Laboratory,USA) at 30 C as described by Yang &
Ling (1989).Total tetracycline equivalent potency was
calculated from the clear zones for tetracycline in the
range of 1 lg/g to 15 mg/g.The regression equation for
this curve was y ¼ 0.349485x + 0.6 where y was the log
concentration of tetracycline (mg/g and x was the
diameter of the zone of inhibition (mm) (r
2
¼ 0.950).
Effect of substrate
The peanut shells in the solid state medium were
replaced by other agricultural substrates using the
Streptomyces strains.The waste producing the highest
tetracycline level in the solid state fermentation process
was chosen.The substrates investigated included corn
cob,peanut shells,corn pomace and cassava peels.The
composition of the medium is as stated earlier.Peanut
shells were chosen because they produced the highest
tetracycline concentration.
Process optimization
Following earlier investigations to choose the best
producing strain and the best substrate,the optimum
conditions for tetracycline production were determined
by employing the methods of Yang & Ling (1989) and
Yang & Swei (1996).pH and antibiotic bioassay were
monitored in all cases.
Effect of initial moisture content
The effects of different initial moisture contents on the
production of tetracycline by Streptomyces sp.OXC1
were investigated.The modified solid-state fermentation
medium described by Yang & Swei (1996) was used:
peanut (groundnut) shell 100 g,CaCO
3
,1.0 g;KH
2
PO
4
,
0.5 g;MgSO
4
 7H
2
O,2 g;(NH
4
)
2
SO
4
,2 g was used for
all the tests.The moisture contents tested were between
50 and 80%.The medium was sterilized at 121 C for
20 min.The sterile medium was inoculated and the
108 A.E.Asagbra et al.
appropriate volume of sterile distilled water was added
to make up the desired moisture content.These were
then incubated statically in flasks (the thickness of
medium was about 2 cm).Incubation was at 31 C for
5–7 days with stirring once a day.
Effect of pH
pH values of 4.3,5.3,6.3,7.3 and 8.3 were used.The
medium was as stated above.The sterile medium was
inoculated and the moisture content adjusted to 68%
moisture.Duplicate sets of experiments were carried
out.Incubation was at 31 C for 5–7 days.The statically
incubated flasks were stirred once daily.
Effect of temperature
The effect of different temperature ranges on the
production of tetracycline was investigated.The mod-
ified medium as described by Yang & Ling (1989) was
used.The different temperatures tested were 28,31,34,
37 and 40 C.The medium were sterilized at 121 C for
20 min.The sterile media were inoculated with 1 · 10
8
conidia and the moisture content made up with sterile
distilled water to 68%.Duplicate flasks were incubated
at all the above stated temperatures.
Effect of inoculum size
Different inoculumsizes of Streptomyces sp.OXC1 were
used to inoculate sterile medium,to produce a high yield
of tetracycline.The inoculum sizes used were 1.0 · 10
5
,
1.0 · 10
6
,1.0 · 10
7
,1.0 · 10
8
,and 1.0 · 10
9
.After the
addition of the inoculum in duplicates,the moisture
content was made up to 68%.Incubation was at 31 C
for 5–7 days with stirring once daily.The samples were
analysed for pH change and bioassay.
Effect of inorganic nitrogen sources
The effects of the nitrogen sources on pH and tetracy-
cline production were investigated.Inorganic nitrogen
sources such as ammonium chloride (NH
4
Cl),ammo-
nium nitrate (NH
4
NO
3
) and ammonium sulphate
(NH
4
)
2
SO
4
were used in the following concentrations
(0.25 0.5 0.75,1%).A modification of the solid state
medium as described by Yang & Swei (1996) was used.
100 g of the medium containing each concentration of
the nitrogen source was distributed into flasks in
duplicates.The medium was sterilized at 121 C for
20 min and moisture level adjusted to 68% prior to
inoculation with Streptomyces sp.OXC1 in different
flasks.The inoculated flasks were incubated statically at
31 C with stirring once a day.
Effect of organic nitrogen sources
Rice bran,soybean meal and peanut meals were used as
organic nitrogen sources.The effects of the nitrogen
sources on pH and antimicrobial compound production
were investigated.A modification of the solid state
medium as described by Yang & Swei (1996) was used.
The organic nitrogen sources were used to replace
inorganic sources in the following concentrations (10,
20,30,40 and 50%) and urea was used at the following
concentrations (0.25,0.5,0.75,1%).The medium was
adjusted with the required organic nitrogen and steril-
ized at 121 C for 20 min.The sterile medium was
inoculated and the moisture content adjusted to 68%
with incubation carried out statically at 28 C for 5–
7 days with stirring once a day.
Effect of combined nitrogen sources
Combined inorganic and organic nitrogen sources were
studied.A modification of the solid state medium as
described by Yang &Ling (1989) was used.The medium
comprised 100 g of peanut shells,CaCO
3
1 g,NaCl 0.2 g
and 0.5% (NH
4
)
2
SO
4
.The organic nitrogen sources
(peanut meal,soybean meal and rice bran) were used in
addition to the inorganic nitrogen source [1%
(NH
4
)
2
SO
4
].The concentrations of the organic nitrogen
used were 10%.The mediumwas treated as stated above.
Effect of additional carbon source
Effect of additional carbon sources on the antimicrobial
compound production was investigated.A modification
of the solid state medium described by Yang & Ling
(1989) was used:peanut shells 100 g,(NH
4
)
2
SO
4
2.4 g,
CaCO
3
1 g,NaCl 0.2 g,and 1 · 10
8
conidia at 65%
moisture content was the inoculum load.The carbon
sources tested were glucose,sucrose,maltose and starch
at 10% concentration.The sugars were filter sterilized
while the medium was sterilized at 121 C for 20 min.
Effect of inorganic salts
The effects of different concentrations of inorganic salts
on the production of antimicrobial compound by
Streptomyces sp.OXC1 were investigated.The modified
medium described by Yang & Swei (1996) was used as
earlier stated.The salts tested were KH
2
PO
4
CaCO
3
,
MgSO
4
 7H
2
O and NaCl at 0.0,0.5,1.0,1.5 and 2.0%
concentrations.The sterile medium was thoroughly
mixed with 1 · 10
8
conidia and the moisture content
adjusted to 68% with sterile distilled water.The
experiments were carried out in duplicate.
Effect of oils on production of antimicrobial compound
Vegetable oils such as palm oil,peanut (groundnut) oil,
melon oil,soybeans oil and palm kernel oil (PKO) was
used in the experiment.Ifudu (1986a,b) described the
method used.To the medium were added different
concentrations of the oils (0.25,0.50,0.75 and 1.0%).
The medium was sterilized at 121 C for 20 min.The
sterile medium was inoculated and the appropriate
Tetracycline production on wastes 109
volume of sterile distilled water was added to raise the
moisture content to 68%.These were incubated statically
in flasks (the thickness of medium being about 2 cm).
Incubation was at 31 Cfor 5–7 days with stirring once a
day.The experiments were carried out in duplicates.
Results
Table 1 shows the tetracycline production of the differ-
ent strains when cultivated on the basal medium.
Streptomyces sp.OXC1 demonstrated the potential for
tetracycline production using peanut (groundnut) shells
as the substrate in solid fermentation (13.18 mg/g),
followed by S.vendagensis ATCC 25507 (11.08 mg/g),
S.rimosus NRRL B1679 (8.46 mg/g),S.alboflavus
NRRL B1273) (7.59 mg/g),S.rimosus NRRL B2234
(6.37 mg/g),S.aureofaciens NRRL B2183 (4.27 mg/g).
Therefore Streptomyces sp.OXC1 was chosen as the
best strain in this study.
Agricultural wastes
Results of proximate analyses of the substrates used
revealed that peanut shells had total carbohydrate
16.99%,crude protein 6.77%,crude fibre 70.38% and
ash 2.65%,corn cob (total carbohydrate 49.64%,crude
protein 5.30%,crude fibre 38.18%and ash 2.53%),corn
pomace (total carbohydrate 75.46%,crude protein
5.65%,crude fibre 12.45%and ash 2.93%) and cassava
peels (total carbohydrate 76.47%,crude protein 4.11%,
crude fibre 10.35% and ash 3.79%).
Effect of substrate on isolates for tetracycline production
Experimental results indicated that peanut shells was the
most effective substrate (4.36 mg/g) followed by corn
cob (2.64 mg/g),followed by cassava peels (2.16 mg/g)
and corn pomace (1.99 mg/g).Therefore peanut shells
were chosen as the substrate for the model system of
antibiotic production.
Process optimization
Initial moisture content
The effect of initial moisture content of substrate on
tetracycline production is shown in Table 2.The initial
moisture content of the substrate increased between
1.53% and 1.66% from an initial of 65% whereas it
increased between 4.49%and 5.10%for initial moisture
content of 75%.During fermentation,tetracycline was
first detected on day 3,reached a maximumyield on day
5 and gradually decreased.
Tetracycline production increased with initial mois-
ture content of 50–70%,having its maximum at 68%
(4.1 mg/g).When the initial moisture content was less
than 50%,tetracycline production was low as the
substrate was too dry for cell growth and antibiotic
production.At initial moisture of 68%,A
w
of the
substrate was 0.995,and bulk densities on dry and wet
weight bases were 0.20 and 0.39 g/m
3
,respectively.At
moisture contents higher than 68%tetracycline produc-
tion gradually decreased while at initial moisture con-
tent of 80%,total tetracycline equivalent was only
0.01 mg/g dry substrate,since the togetherness of the
substrate prevented gas exchange.
Initial pH
Tetracycline production at different initial substrate pH
is shown in Figure 1.The optimal pH for tetracycline
production was the same as the original pH of peanut
shells (5.35–5.60).When the pH was lower than 5.3,
tetracycline production was low and each gram of the
dry substrate produced about (0.15 mg/g) total tetracy-
cline equivalent,when the initial pHwas higher than 6.3,
tetracycline production,a slight decrease was observed.
At initial pHof 8.3 each gramof dry substrate produced
1.70 mg/g of total tetracycline equivalent during fer-
mentation.The pH change of substrate was not signif-
icant when the initial pH was lower than 6.3 but the
reverse was observed at higher pH.
As the original pHof the peanut shells ranged between
5.35 and 5.55,it was not necessary to adjust the pH for
tetracycline production in solid state fermentation.
Incubation temperature
Tetracycline production was optimal at 31 C,and
decreased sharply when incubation temperature was
higher than 40 C or less than 28 C.Each gram of dry
substrate produced 3.60 and 2.52 mg/g of total tetra-
cycline equivalent at 31 C and 34 C respectively.
Table 1.Tetracycline production by the different strains when
cultivated on peanut shells at 68% moisture content at 31 C for
5 days.
Streptomyces spp.Initial
pH
Final
pH
Total tetracycline
equivalent (mg/g)
S.vendagensis ATCC 25507 5.40 6.60 11.08
S.rimosus NRRL B1679 6.29 7.05 8.46
S.alboflavus NRRL B1273 5.80 6.30 7.59
S.rimosus NRRL B2234 5.15 7.40 6.37
S.aureofaciens NRRL B2183 5.30 5.50 4.27
Streptomyces sp.OXC1 5.60 5.90 13.18
Table 2.Tetracycline determination with bioassay method at different
initial moisture content.
Initial moisture
content
%increase in
moisture content
Final pH Bioassay (mg/g)
50 7.58 5.35 0.82
55 6.16 5.35 1.65
60 5.05 5.31 2.70
65 1.53 5.70 3.40
67 1,66 6.00 3.75
68 1.58 6.05 4.10
69 1.49 6.20 3.57
70 5.10 6.01 3.40
75 4.49 6.33 0.69
80 3.73 6.36 0.01
110 A.E.Asagbra et al.
Inorganic salts
Addition of CaCO
3
regulated the substrate pH value
and stimulated tetracycline production.Addition of
0.5% CaCO
3
resulted in the optimal production of
3.40 mg/g tetracycline equivalent.Supplementation with
1.0%MgSO
4
 7H
2
O produced 2.70 mg/g of total tetra-
cycline equivalent.NaCl and MgSO
4
 7H
2
O stimulated
antimicrobial production.However NaCl stimulated it
by 140%while KH
2
PO
4
stimulated tetracycline produc-
tion by 40% (Table 3).
Nitrogen sources
The effect of inorganic nitrogen source on tetracycline
production and substrate pH showed that 1.0% of
(NH
4
)
2
SO
4
was the best inorganic nitrogen source and it
produced 4.62 mg/g of total tetracycline equivalent per
gram of dry substrate.NH
4
NO
3
was the next best at
2.0% concentration giving 4.27 mg/g total tetracycline
equivalent per gram of dry substrate.Agricultural
wastes were tested as alternative organic nitrogen
sources.Of the wastes tested 10.0% soybean meal and
10.0%peanut meal enhanced production of tetracycline
producing 4.27 and 4.62 mg/g tetracycline equivalent,
respectively.To improve the utilization of the agricul-
tural wastes the combined organic nitrogen source and
inorganic source was found to have a complementing
effect on tetracycline production.When 1.0% of
(NH
4
)
2
SO
4
was added to 10.0% of peanut meal there
was an increase in the tetracycline produced.Each
gram substrate produced 11.78 mg/g of tetracycline
(Table 4).
Figure 1.Effect of pHon tetracycline production in solid fermentation
of peanut shells at 31 C for 5 days by Streptomyces sp.OXCI.
Table 3.Effect of inorganic salts on tetracycline production in solid-
state fermentation of peanut shells.
Inorganic salts Final pH Total tetracycline
equivalent (mg/g)
KH
2
PO
4
0.0 5.50 2.35
0.5 5.50 3.05
1.0 5.90 2.90
1.5 5.90 2.85
2.0 6.05 2.70
CaCO
3
0.0 4.90 0
0.5 5.90 3.40
1.0 6.05 2.75
1.5 5.15 2.00
2.0 6.35 1.70
MgSO
4
Æ 7H
2
O
0.0 5.50 0
0.5 6.30 2.08
1.0 6.40 2.70
1.5 6.10 2.53
2.0 6.10 2.47
NaCl
0.0 5.80 1.70
0.5 5.90 4.80
1.0 5.90 2.70
1.5 5.90 2.70
2.0 5.90 2.35
Table 4.Effect of different nitrogen sources on tetracycline production in solid-state fermentation.
a
Nitrogen source (%) Initial pH Final pH Bioassay method (total
tetracycline equivalent) (mg/g)
None 5.60 6.90 1.47
(NH
4
)SO
4
(1.0%) 5.35 5.80 4.02
NH
4
Cl (1.0%) 5.20 5.35 2.00
Urea (0.5%) 6.10 8.20 3.49
NH
4
NO
3
(0.5%) 5.30 5.65 4.27
Rice bran (10%) 5.30 5.40 6.28
Soybean meal (10%) 5.30 5.60 6.28
Peanut meal (10%) 5.30 6.00 10.21
Peanut meal (10%) +(NH
4
)
2
SO
4
(1.0%) 5.30 5.80 11.78
Rice bran (10%) +(NH
4
)
2
SO
4
(1.0%) 5.50 5.80 8.64
Soybean meal (10%) +(NH
4
)
2
SO
4
(1.0%) 5.30 5.60 8.99
*a
Peanut shells 100 g,CaCO
3
0.5 g,NaCl 0.5 g and 1 · 10
8
were mixed thoroughly,and the initial moisture content was adjusted with distilled
water to 68%.The substrate was incubated at 31 C for 5 days.
Tetracycline production on wastes 111
Additional carbon source
Effect of additional carbon source on tetracycline
production is shown in Table 5.Maltose and soluble
starch stimulated tetracycline production by 1.29–1.95
times.Glucose and sucrose inhibited the production of
tetracycline by 10.0 and 40.0% respectively.
Additional vegetable oil
The experiments showed that peanut oil was the best oil
for tetracycline production.0.25%peanut oil produced
4.02 mg/g of tetracycline equivalent.At 0.25%,palm
kernel oil and palm oil did not synthesize any antimi-
crobial compound but rather inhibited the production.
As the concentration of the oil increased the rate of
tetracycline production reduced (Figure 2).
Inoculum size
Each gram of dry substrate inoculated with 1.0 · 10
8
conidia could produce 3.22 mg total tetracycline equiv-
alent when the inoculum size was 1.0 · 10
10
(2 mg of
total tetracycline equivalent) or less than 1.0 · 10
6
(0.86 mg of total tetracycline equivalent) conidia.
Tetracycline production decreased after 5 days incuba-
tion.
The above results reveal the following.The optimum
conditions for tetracycline production using soybean
meal as the organic nitrogen source were peanut shells at
an initial moisture content of 65–70%,initial pH 5.6–
5.8,(NH
4
)
2
SO
4
0.5%,CaCO
3
0.5%,NaCl 0.5%,
KH
2
PO
4
0.55%,MgSO
4
 7H
2
O 1.0%,incubation at
31 C for 5 days.Each gram of the dry substrate
produced 11.78 mg/g of total tetracycline equivalent.
In the case of peanut meal as the organic nitrogen
source:the optimum conditions for tetracycline produc-
tion by Streptomyces sp.OXC1 were peanut shells at
initial moisture content of 65–70%,initial pH 5.6–5.8,
supplemented with (NH
4
)
2
SO
4
0.5%,CaCO
3
0.5%,
NaCl 0.5%,KH
2
PO
4
0.55%,MgSO
4
 7H
2
O 1.0%,
soluble starch 10%,peanut oil 0.25% and incubated
at 31 C for 5 days.Each gram of the dry substrate
produced 13.18 mg/g of tetracycline (Table 6).
Discussion
Tetracycline was detected on the third day of incubation
and had maximal activity at 5 days and decreased
gradually within a 1 month period.Tetracycline in
Streptomyces sp.OXC1 was a secondary metabolite
synthesized and secreted in the late lag phase or in the
stationary phase (Okami &Oomura 1979).In submerged
fermentation,antibiotic activity sharply decreased after
prolonged incubation due to cell autolysis (Yang &Ling
1989).During this study it is observed that the moisture
content of the substrate increased,possibly due to the
production of metabolic water by the Streptomyces.This
result compares with that obtained by Yang & Ling
(1989),Yang & Cheng (1991) in the solid-state fermen-
tation of sweet potatoes and when sweet potatoes were
enriched with amylolytic fungi.It also compares with
corncob fermentation by Trichoderma and oxytetracy-
cline by S.rimosus in solid-state fermentation of corncob
(Yang 1993;Yang & Swei 1996) and during protease
production from sweet potato residue by amylolytic
fungi (Yang & Huang 1994).The maximal initial
moisture content for tetracycline production was at 65–
68%,and the final moisture content was 67–70%.
The substrate pH was regular and did not become
acidic;this could have been due to the initial pH of the
substrate,and the high level of nitrogen source.Yang
(1988) observed that the presence of other nitrogen
sources apart from inorganic nitrogen source helped to
maintain substrate pH.Calciumcarbonate also played a
role in counteracting acidity and enhancing tetracycline
production (Yang & Ling 1989).
The optimal temperature for tetracycline production
was dependent on the test organism(Yang 1993).In this
study Streptomyces sp.OXC1 had an optimal temper-
ature of 31 C for tetracycline production in solid state
Table 5.Effect of additional carbon source on tetracycline production
by Streptomyces sp.OXC1.
Carbon source
(10%)
Initial pH Final pH Bioassay (mg/g)
None 5.40 4.00 2.35
Glucose 5.50 5.50 2.17
Maltose 5.30 5.20 2.78
Sucrose 5.35 5.90 1.70
Soluble starch 5.30 4.40 4.01
Figure 2.Biosynthesis of tetracycline produced by Streptomyces sp.
OXCI in groundnut shells supplimented with various vegetable oils.
112 A.E.Asagbra et al.
fermentation.In 1989 Yang & Ling found that S.virid-
ifaciens had an optimal temperature of 26 C for
tetracycline production using sweet potato residue.Also
Yang & Swei (1996) recorded an optimal temperature of
25–30 C for oxytetracycline production using corn cob.
This study also shows that (NH
4
)
2
SO
4
was the best
inorganic nitrogen source.This result was consistent
with Yang & Ling’s (1984) result during tetracycline
production with sweet potato residue.This study
revealed that supplementation with high concentrations
of (NH
4
)
2
SO
4
(1.5%) inhibited tetracycline production.
This result is consistent with Yang & Swei’s (1996)
finding with oxytetracycline production using corncob.
High concentration of nitrogen sources also inhibited
b-lactam production by Cephalosporium acremonium
(Shen et al.1984) and spiramycin production by
S.ambofaciens (Ahmed et al.1987).This study showed
that combination of inorganic nitrogen [(NH
4
)
2
SO
4
] and
organic nitrogen source (peanut meal and soybean meal)
enhanced tetracycline production.Yang & Ling (1989),
Yang & Yuan (1990) using sweet potato residue
observed similar results,while Yang & Swei (1996)
observed the same with corn cob.
Phosphate had a stimulatory effect when its concen-
trations was not greater than 0.5% but became inhib-
itory in higher concentrations.Similar results were
obtained in nikkomycin production by S.tendae
(Treskatis et al.1992) and cephamycin production by
S.clavuligerus (Ahmed et al.1987).NaCl stimulated
tetracycline production in this study.This could be
attributed to the absorption of Cl

ions,which are
essential for antibiotic production (Yang & Ling 1989).
Monosaccharides were good for cell growth,but
inhibited the production of tetracycline in this study.
Disaccharides and polysaccharide stimulated its pro-
duction.Yang & Ling (1989) obtained similar results
when they observed that a small amount of soluble
starch or other fermentable polysaccharide was good for
secondary metabolites production.
Ifudu (1986a,b) indicated that the use of 0.5%
vegetable oils was important in biosynthesis of antibi-
otics since they functioned as antifoaming agents and as
a source of carbon and energy for the microorganism.In
this study it was found that 0.25% of peanut oil
enhanced tetracycline production.
Solid-state fermentation gave a product which was
more stable than that from submerged culture,and also
required less energy input (Wang 1989).Each gram of
substrate produced 13.18 mg of total tetracycline equiv-
alent while the submerged produced 3.05 mg of total
tetracycline equivalent.The solid-state product also had
an advantage that it could be stored temporarily without
significant loss of activity.
It is concluded that solid-state fermentation maybe an
economic alternative in the production of value added
pharmaceuticals and agriculture chemicals in the Nige-
rian economy.An added advantage of this study was
that the local strain isolated was even more effective
than the standard strains.
Table6.EffectofcombinedingredientsontetracyclineproductionbyStreptomycessp.OXC1bysolid-statefermentationofpeanutshellswithinitialmoisturecontentof68%at31Cfor5days.
MediumitemDifferentcombinationsofmediumitems
ABCDEFGHIJKL
Peanutshells100100100100100100100100100100100100
Inorganicnitrogen
source(NH
4)2SO4
1.01.01.01.01.01.01.01.01.01.01.01.0
CaCO3
0.50.50.50.50.50.50.50.50.50.50.50.5
NaCl0.20.50.50.50.50.50.50.50.50.50.50.5
KH2PO4
0.00.50.50.50.50.50.50.50.50.50.50.5
MgSO
4
0.00.50.50.50.50.50.50.50.50.00.010.0
Solublestarch0.00.00.00.00.010.00.010.010.00.00.00.0
Peanutmeal0.00.010.00.010.010.00.00.010.010.00.020.0
Soybeanmeal0.00.00.020.00.00.020.00.00.00.020.00.0
Melonoil0.00.01.00.00.00.01.00.00.00.00.00.0
Soybeanoil0.00.00.00.250.250.00.00.00.250.250.00.25
Peanutoil0.00.00.00.00.00.250.00.00.00.00.00.0
InitialpH5.605.605.505.605.505.605.505.805.405.505.905.60
FinalpH6.106.006.006.005.906.005.906.005.806.307.105.90
Bioassaymethod(Total
tetracyclineequivalent)(mg/g)
1.473.057.5911.086.1913.186.803.7311.7810.378.8911.78
Tetracycline production on wastes 113
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114 A.E.Asagbra et al.
Influence of glucose and oxygen on the production of ethyl acetate and isoamyl
acetate by a Saccharomyces cerevisiae strain during alcoholic fermentation
C.Plata,J.C.Mauricio*,C.Milla
´
n and J.M.Ortega
Department of Microbiology,Faculty of Sciences,University of Cordoba,Campus Universitario de Rabanales,Edificio
Severo Ochoa,14014 Co
´
rdoba,Spain
*Author for correspondence:Tel.:+34-957-218640,Fax:+34-957-218650,E-mail:mi1gamaj@uco.es
Received 29 January 2004;accepted 15 June 2004
Keywords:Alcohol acetyltransferase,esterases,ethyl acetate,isoamyl acetate,Saccharomyces cerevisiae
Summary
The effect of glucose and dissolved oxygen in a synthetic mediumsimulating the standard composition of grape juice
on the production of ethyl acetate and isoamyl acetate by a Saccharomyces cerevisiae strain during alcoholic
fermentation was studied.The specific in vitro activity of alcohol acetyltransferase (AATase,EC 2.3.1.84) and
esterases (ESase,EC 3.1.1.1;hydrolysis and synthesis of esters) in cell-free extracts was also examined.The specific
activity of AATase for ethyl acetate was found to peak at the beginning of the exponential growth phase and that
for isoamyl acetate at its end.While the glucose concentration only affected the maximum specific activity of
AATase,and only slightly,oxygen inhibited such activity,to a greater extent for isoamyl acetate than for ethyl
acetate.On the other hand,esterases were found to catalyse the synthesis of ethyl acetate only at a low or medium
glucose concentration (50 or 100 g l
)1
,respectively),and to reach their maximum hydrolytic activity on isoamyl
acetate during the stationary growth phase.The highest ethyl acetate and isoamyl acetate concentrations in the
medium were obtained with a glucose concentration of 250 g l
)1
and semianaerobic conditions.
Introduction
Esters are byproducts of the alcoholic fermentation of
sugars by wine yeasts.The factors most strongly affecting
the ester content in wine are the particular yeast species
and strain,the must composition and the fermentation
conditions (Soles et al.1982;Mauricio et al.1993;Rojas
et al.2001;Plata et al.2003).After higher alcohols,esters
constitute the family of major compounds accounting to
the greatest extent for wine aroma.The ethyl esters of
succinic and lactic acids are the most abundant among
them,followed by acetates and the ethyl esters of fatty
acids (Schreier 1979,1984;Soles et al.1982).
Among acetates in wine,ethyl acetate is the most
abundant and isoamyl acetate that most markedly
contributing to wine aroma (Van Der Merwe & Van
Wyk 1981).With a given yeast species,their production
is governed by the must composition and fermentation
conditions (Soles et al.1982).
Saccharomyces cerevisiae,the well known principal
wine yeast species,produces esters via an intracellular
process that is catalysed by an alcohol acetyltransferase
(EC 2.3.1.) using energy provided by the acyl-coenzyme
A compounds.The synthesis of acetate esters during
fermentation of wine has been widely studied and
ascribed to the activity of at least three acetyltransfe-
rases (AATase,EC 2.3.1.84),namely:alcohol acetyl-
transferase,ethanol acetyltransferase and isoamyl
alcohol acetyltransferase (Lilly et al.2000).Two distinct
AATase activities for isoamyl alcohol and other alco-
hols have been studied in S.cerevisiae (encoded by
ATF1 and ATF2 genes) that exhibit different mecha-
nisms of regulation and,probably,also different phys-
iological roles (Fujii et al.1996;Lilly et al.2000;Mason
& Dufour 2000).The overexpression of the ATF1 gene
in wine yeasts was reported to significantly increase the
concentrations of ethyl acetate,ethyl caproate,hexyl
acetate,isoamyl acetate and 2-phenylethyl acetate in
wine produced by these transgenic microorganisms
(Lilly et al.2000).Recently,analysis of the fermentation
products confirmed that the expression levels of ATF1
and ATF2 greatly affect the production of ethyl acetate
and isoamyl acetate (Verstrepen et al.2003).The atf1D
atf2D double deletion strain did not form any isoamyl
acetate,showing that together,Atf1p and Atf2p are
responsible for the total cellular isoamyl alcohol acetyl-
transferase activity.However,the double deletion strain
still produced considerable amounts of certain other
esters,such as ethyl acetate (50% of the total concen-
tration produced by the wild-type strain),suggesting the
presence of as yet-unknown ester synthases in the yeast
proteome (Verstrepen et al.2003).It has also been
shown that S.cerevisiae AATase is strongly repressed
under highly aerobic conditions and by the addition of
World Journal of Microbiology & Biotechnology 2005 21:115–121
DOI:10.1007/s11274-004-2780-5

Springer 2005
unsaturated fatty acids to the culture medium(Malcorps
et al.1991;Fujii et al.1997).Esterases (ESase,EC
3.1.1.1.) function mainly by hydrolysing esters;in S.
cerevisiae,however,esters can also be synthesized via the
reverse reaction in the absence of coenzyme A.In
contrast,the relevance attributed to the ester synthetase
as an ester-synthesizing activity is rather limited:two
esters (ethyl caprylate and ethyl acetate) have been
reported as being produced by breadmaking and beer
yeast strains,respectively,of S.cerevisiae from ethanol
and the respective acids (Rojas et al.2002).Campbell
et al.(1972) succeeded in electrophoretically separating
two esterase activities in S.cerevisiae,probably corre-
sponding to the enzymes encoded by the EST1 and
EST2 genes identified by Schermers et al.(1976).The
latter gene was cloned and sequenced by Fukuda et al.
(1996) and its product is a carboxyesterase that hydro-
lyses mainly isoamyl acetate.Thus,the production of
esters is widely believed to rely on the balance of ester
synthesis by AATase and ester hydrolysis by ESase
(Inoue et al.1997;Fukuda et al.1998).
In winemaking yeasts,isoamyl acetate is only synthe-
sized in the presence of acetyl-CoAby acetyltransferases;
by contrast,ethyl acetate is produced both in the
presence of acetyl-CoA (by acetyltransferases) and in
the presence of ethanol and acetic acid,by a reverse
reaction of esterases (Plata et al.1998).Both can be
hydrolysed in the wine,whether spontaneously or under
the action of esterases.
The production of esters during alcoholic fermenta-
tion depends on the extent of yeast growth and increases
during the second half of the growth phase as the
synthesis of lipids and sterols stops and the availability
of acetyl-CoA increases.After the fermentation ends,
the esterase activity increases and the production of
esters in the wine decreases during the cell lysis phase
(Mauricio et al.1993).
In previous work we studied the potential of various
wine yeast species for producing ethyl acetate and
isoamyl acetate,which contribute substantially to the
aroma of wine (Plata et al.2003),in a model grape must.
In the present work,we examined the influence of the
glucose and dissolved oxygen in the must on the activity
of alcohol acetyltransferases and esterases in S.cerevi-
siae,as well as on the production of ethyl acetate and
isoamyl acetate during alcoholic fermentation.
Materials and methods
Yeast strain
The yeast strain used was Saccharomyces cerevisiae E-1
(ATCC Number MYA 425),isolated from spontane-
ously fermenting must in the Montilla–Moriles designa-
tion of origin (southern Spain).The strain was stored in
Agar YM(0.3%yeast extract,0.3%malt extract,0.5%
peptone,1% glucose,and 2.5% agar;pH 6.5) at 4 C
prior to use.
The total number of cells was determined by counting
under a light microscope in a Thoma cell-counting
chamber.
Culture medium,fermentation conditions and sampling
The synthetic fermentation medium used to simulate a
standard natural grape juice was that reported by Singh
& Kunkee (1976),which was modified by adding 250,
100 or 50 g glucose l
)1
.Medium containing (g l
)1
):
malic acid,3;potassium bitartrate,5;citric acid,2;
casamino acids,3;K
2
HPO
4
,1;MgSO
4
Æ7H
2
O,0.25;
CaCl
2
Æ2H
2
O,0.50;as well as vitamins and trace ele-
ments.The medium was adjusted to pH 3.5 with 50%
KOH,sterilized by passage through a filter of 0.45 lm
pore size.Inoculum was prepared in a flask with cotton
plug containing 75 ml culture grown in the same
synthetic fermentation medium and incubated at 28 C
for 48 h without shaking.
Fermentation tests were conducted in 2-l flat-bot-
tomed flasks that were filled with 1800 ml of the above-
described synthetic medium,inoculated with
1 · 10
6
cells ml
)1
and incubated at 28 C under differ-
ent oxygen availability conditions,namely:semiaerobic,
semianaerobic and strict anaerobic.Semiaerobic condi-
tions were obtained by continuous shaking at 150 rev
min
)1
on an orbital shaker from New Brunswick
Scientific (Edison,NJ,USA).Semianaerobic conditions
were established by allowing the flasks to stand with
their mouths stopped by cotton plugs,these conditions
are similar to the usually carried out for winemaking.
Finally,strict anaerobic conditions were accomplished
by using a three-mouthed flask,one mouth being fitted
with a special air lock filled with mercury and the other
two with two taps connected to vacuum and to a
nitrogen gas supply,respectively.The fermentation
vessel was previously degassed and then refilled with
nitrogen by bubbling for 15–20 min.
Samples were collected for analysis at 0,6,12,24,48,
72 and 240 h of fermentation,and immediately supplied
with 20 lg cycloheximide ml
)1
and 20 lg chloramphe-
nicol ml
)1
to prevent the potential synthesis of proteins
upon contact of the cells with oxygen during the later
procedure.Each sample was adjusted by cellular recount
in a microscope to obtain 1 · 10
8
to 1 · 10
9
cells that
were collected by centrifugation at 3.500 · g at 4 C for
5 min.
Analytical procedures
The supernatant of the fermentation medium was used
to determine the amount of ethanol produced,using the
method of Crowell & Ough (1979).Reducing sugars
were determined enzymatically,using specific kits from
Boehringer–Mannheim GmbH (Mannheim,Germany)
as recommended by the manufacturer.
Isoamyl alcohol,ethyl acetate andisoamyl acetate were
extracted with Freon-11 in a continuous extractor for
24 h,and concentrated to 200 ll in a microconcentrator.
116 C.Plata et al.
2-Octanol at a 481 lg l
)1
concentration was added as an
internal standard in all samples.These compounds were
quantified by gas chromatography (GC).A volume of
2 ll was injected in the injector,with a split ratio of 60:1,
from a Hewlett-Packard 5980 gas chromatograph
equipped with a Supelco SP-1000,60 m · 0.32 mm i.d.
fused silica capillary column.The oven temperature
programmer was as follows:9 min at 50 C,followed by
a 6 C min
)1
ramp to 185 C,which was held for 1 min.
The injector and detector temperatures were kept at 275
and 300 C,respectively,and nitrogen at a flow-rate of
26 ml min
)1
was used as carrier gas.
Determination of enzyme activities
The enzyme activities studied were determined as
described in a previous paper by the authors (Plata
et al.2003).
Results and discussion
Yeast growth,glucose consumption and ethanol
production
Figure 1 shows the variation of yeast growth over a
period of 10 days under the different fermentation
conditions used.As can be seen,the glucose concentra-
tion had no substantial effect on yeast growth.On the
other hand,as expected,growth was influenced by the
available oxygen in the medium;thus,it was maximal
under semianaerobic conditions,followed by semiaero-
bic and,finally,anaerobic conditions,where growth
ceases after 12 h presumably due to a lack of essential
lipids (Mauricio et al.1998).
Table 1 shows the glucose consumption and ethanol
production after 10 days under the operating conditions
assayed.In the semianaerobic tests with 50 and 100 g l
)1
glucose,the sugar was completely fermented to ethanol
by the S.cerevisiae strain used by the end of the
experiment.In the same period,with 250 g l
)1
glucose,
however,the sugar was incompletely fermented to
ethanol:to a greater extent under semianaerobic condi-
tions than under semiaerobic and anaerobic conditions.
Ester production
Figure 2A and B shows plots of the data of AATase
specific activities corresponding to the assayed fermenta-
tions.In all cases,the specific activity for the synthesis of
ethyl acetate by AATase (Figure 2A) was high at the
beginning of fermentation,after which it decreased
exponentially (the curve was a hyperbola running along
the x-axis and y-axis).The highest value in activity was
observed at hour 6 under semiaerobic conditions,but
later on diminished more drastically than the rest of the
other cases assayed.The anaerobic fermentationtests and
those conducted under semianaerobic conditions with 50
or 100 g glucose l
)1
showed intermediate values of
activity,the lowest values being those obtained with
250 g l
)1
under semianaerobic conditions.The specific
activity for the synthesis of isoamyl acetate by AATase
(Figure 2B) peaked during the exponential growth phase
under semianaerobic conditions (12–24 h),and 24–48 h
(stationary phase) under anaerobic conditions.This
differential behaviour of the kinetics of AATase activity
for the synthesis of the two acetates may be consistent
with the presence of various AATases (Lilly et al.2000).
Althougha number of AATases have beenreportedfor S.
cerevisiae,their activity cannot be unequivocally ascribed
to a specific acetate ester as the different AATases are
somewhat non-specific for the different alcohols.
Oxygen was found to inhibit the synthetic activity of
AATase for the twoacetates,particularly that for isoamyl
acetate (see Figure 2).According to Fujiwara et al.
(1998),this inhibitory effect is a result of the increased
Figure 1.Growth kinetic of Saccharomyces cerevisiae strain in the
fermentation conditions used in this study:(d) semianaerobic and
250 g of glucose l
)1
;(￿) semianaerobic and 100 g of glucose l
)1
;(.)
semianaerobic and 50 g of glucose l
)1
;(,) semiaerobic and 250 of
glucose g l
)1
;(j) anaerobic and 250 g of glucose l
)1
.
Table 1.Glucose consumed,ethanol produced and ethanol yield coefficient (g ethanol produced per g glucose consumed) after 10 days of
fermentation.
Fermentation Conditions Initial glucose (g l
)1
) Glucose consumed (g l
)1
) Ethanol produced (%v/v) Ethanol yield coefficient
Semianaerobic 50 50.0 3.30 0.52
Semianaerobic 100 99.9 6.82 0.53
Semianaerobic 250 182.2 11.10 0.48
Semiaerobic 250 86.0 5.25 0.48
Anaerobic 250 46.0 3.24 0.55
Glucose and oxygen effects on ester production 117
synthesis of unsaturated fatty acids and their conse-
quently increasedconcentrationinthe plasma membrane,
which,according to Fujii et al.(1997) and Yoshimoto
et al.(1998),inhibits the transcription of the ATF1 gene.
Also,according toFujiwara et al.(1998),the effect is more
marked in synthetic than in naturally rich culture media.