Journal of Non-Crystalline Solids 356 (2010) 15761580

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Journal of Non-Crystalline Solids

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n o n c r y s o l

Thermal and mass spectroscopic characterization of a sulphur-containing bacterial

melanin from Bacillus subtilis
Ana M. Gmez-Marn , Carlos I. Snchez
Escuela de Procesos y Energa, Universidad Nacional de Colombia, Sede Medelln, Cra 80 No. 65-223, Medelln, Antioquia, Colombia

a r t i c l e i n f o a b s t r a c t

Article history: Among technologies for melanin commercial-scale production, microbial synthesis is an attractive option. In
Received 10 September 2009 this study, a melanin producer, wild strain Bacillus subtilis, was isolated from the soil, and pigment from this
Received in revised form 28 April 2010 bacterium was puried and characterised using different techniques. The use of tandem pyrolysis/gas
Available online 22 June 2010
chromatography/mass spectrometry allowed us to classify the pigment as a sulphur-containing bacterial
melanin. Thermogravimetric and differential thermal analyses indicated that melanin might contain two
Keywords:
Biopolymer B125;
fractions of water, a strongly bound fraction and a weakly-bound fraction. Scanning electron microscopy
Differential scanning calorimetry D190; showed an amorphous structure without differential characteristics, and electronic conductivity measure-
Mass spectrometry M105; ments at normal atmospheric conditions and humidities suggest that melanin may be an insulator. However,
Thermal analysis T135 more work is underway to clarify this point.
2010 Elsevier B.V. All rights reserved.

1. Introduction Exploiting the microbial synthesis of these materials is an attractive

Organic conductors are interesting materials that are currently being
used in many different technological applications, including photo-
voltaics, sensors and other electronic devices. Melanins are disordered
bioorganic conductors with unique physical and chemical properties.
These properties give them a broad range of functions in nature,
including roles as photoprotectants, pigments, charge transport
mediators, free-radical scavengers and antioxidants [1]. They are an
attractive material with a wide range of possible industrial applications
in medicine, pharmacology, cosmetics and other elds and may prove to
be a novel class of semiconducting polymers, bio-compatibles and with
a readily available natural source as natural materials extracted from
pigmented cells.
However, despite signicant scientic effort over the past 30 years,
the basic functions of melanins are still a matter of controversy and
speculation. This uncertainty results from the few and poorly dened
structural and physicalchemical properties that are known in these
molecules that continue to perplex researchers even though their
adaptive importance has already been proven [2,3]. These materials
are particularly intractable from an analytical perspective because
they are chemically and photo-chemically very stable, and they are
virtually insoluble in most common solvents.
In microorganisms, melanins may be found in either extracellular
or intracellular environments, and they pose very interesting targets
for molecular structure determination and organic synthesis [3].
Corresponding author.
E-mail address: amgomezma@unal.edu.co (A.M. Gmez-Marn).
option to develop commercial-scale production. This method pro-
vides an alternative to other production methods that use puried
tyrosinase, expensive chemical synthesis based on the oxidation of
tyrosine and its derivatives or the cumbersome extraction of these
materials from animal or plant tissues. However, due to the high
diversity of biological sources for melanin production, there is no
common standard method for melanin isolation and purication [4].
A number of bacterial species have been reported to produce
melanins [5,6]. These materials can be produced in the Actinomiceto or
Streptomicetos groups, Bacillus and Azotobacter genuses [5,7,8]; howev-
er, very few of these pigment systems have been molecularly dened.
The best-understood melanisation pathway is the classic MasonRaper
pathway. In this pathway, tyrosinases yield the melanin intermediate
dihydroxyphenyl-alanine -DOPA-, dihydroxyindole -DHI- and dihy-
droxyindole carboxylic acid -DHICA- [1,9]. Other pathways that produce
melanins have also been reported [3,9]; for example, pyomelanin is
derived from the catabolism of tyrosine via -hydroxyphenylpyruvate
and homogentisic acid -HGA- [6].
Melanins constitute a general class of complex, polyphenolic
heteropolymers [10]. Traditionally, they are divided into brown
black melanins, described as eumelanins, that are primarily synthe-
sised by the classic MasonRaper biosynthetic pathway. Additionally,
brown, red or yellow melanins, described as pheomelanins, are
derived from sulphur-containing monomers like glutathione or
cysteinyldopa -CD-. Structural analogs of tyrosine or tyrosine
metabolites may also serve as precursors for eumelanin and
pheomelanin synthesis. Nitrogen-free melanins, described as allome-
lanins, are similar to eumelanins but instead form from catechol by a
mechanism that is not well understood [3,11]. Pyomelanins, also called

0022-3093/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jnoncrysol.2010.05.054
 A.M. Gmez-Marn, C.I. Snchez / Journal of Non-Crystalline Solids 356 (2010) 15761580
alkaptons, are produced from tyrosine through HGA [6]. While most
melanins fall into these categories, we should note that other types of
melanins do exist as well.
Melanins are usually classied by essential differences in their
chemical composition and their monomer subunit structure [11].
Nevertheless, this classication remains ambiguous and diagrams that
classify melanin structures are an oversimplication [10]. Melanin
formation is an oxidative, radical process that happens to phenolic
compounds and is inuenced by light, radiation, temperature and the
presence of metals. Unlike the synthesis of most other biopolymers,
this process does not follow a discrete pattern. A given sample of
melanin contains molecules with various structures and, due to the
reactivity of melanin intermediates, different components, including
proteins and lipids [10,12].
The structural study of melanins has encountered great difculties
because these pigments are difcult to separate from other cellular
components of the organisms in which they occur. Physio-chemical
tests that are usually employed to characterise these types of materials
do not differentiate between eumelanins and allomelanins [13]. In
addition, analyses such as infrared transmission spectroscopy IR,
ultravioletvisible UVVIS transmission spectroscopy, magnetic
nuclear resonance NMR, elemental composition and colour
identication are also not sufcient to differentiate these types of
molecules [14].
New information about melanin structure has been obtained by two
approaches, both involving the direct analysis of these pigments and the
study of their degradation products using pyrolysis/gas chromatogra-
phy/mass spectrometry PyGCMS [15]. The usefulness of this
method is well documented [1618], especially in its ability to
differentiate between eumelanins and pheomelanins [19]. Additionally,
some reports suggest that melanin precipitation by HCl acidication
does not have any inuence on the melanin pyrolytic pattern [15,18].
The Bacillus subtilis strain that produces an orange pigment is
classied as B. subtilis var. niger (Bacillus globigii DSM 2277), which
was recently re-classied as Bacillus atrophaeus [20]. In the past, this
pigment has been characterised as a melanin [21]; however, neither
the type of melanin nor the thermal and electrical properties of this
pigment are known.
In the present study, we describe a procedure for the isolation and
purication of melanin from the culture medium of a coloured strain
of B. subtilis. The extracted pigment was characterised by conductivity
measurements under atmospheric conditions using energy dispersive
spectroscopy EDS, PyGCMS, differential thermal analysis DTA
and thermogravimetric analysis TGA analyses.
2. Experimental part
2.1. Cell culture
A B. subtilis native strain that was isolated from soil and is
considered to be a strong melanin producer was used throughout the
study. This strain was preserved in a spore solution that was a ten on
the Mac-Farland scale [21]. LuriaBertani medium was used for
inoculum preparation and pigment production. Approximately 0.5 mL
of spore solution was added to 50 mL of LuriaBertani broth in 250-mL
asks. This solution was then incubated at 37 C until the stationary
phase was reached. Media were sterilised by autoclaving. Erlenmeyer
asks containing 18 mL of medium were inoculated with 2 mL of the
inoculum suspension. These solutions were then incubated for 24 h at
28.0 C and 95 rpm in a rotary shaker [21].
2.2. Extraction and purication of bacterial melanin
After the incubation time, the medium was centrifuged at 3000 rpm
for 30 min to separate the broth and the cells. The solid pellet was then
separated and resuspended in distilled water. Again, the cells were
1577
removed by centrifugation, and melanin was extracted from the
supernatant in the following manner: acidication at a pH b 4.0 using
6 M HCl; a 24-h incubation at room temperature in a rotary shaker
(95 rpm); and centrifugation at 3000 rpm for 30 min to obtain the crude
melanin. The pigment was puried according to literature precedent
[21]. The extracted melanin was carefully rinsed with ultrapure water
and then was centrifuged three times at 4 C and 10,000 rpm for 10 min.
The residue was puried by treatment with organic solvent (glacial
acetic acid, hexane, and ethanol) and then repeated centrifugation at
3000 rpm for 30 min. Finally, acetone was added, and the resulting
solution was evaporated to dryness at room temperature. The obtained
dry solid was kept from light.
2.3. Physicochemical characterization of bacterial melanin
Powders of the puried bacterial melanin were compressed into
pellets, and the conductivity was measured using a two-point method
under a direct current regime with a device that was fabricated in house.
The xed area was 7.9 10 3 cm2, and the applied pressure was 4 MPa.
These measurements were done at atmospheric conditions. Some
samples of the melanin compressed-powders were coated with a thin
lm of gold and observed by scanning electron microscopy SEM
with an accelerating voltage of 5 kV. The supercial chemical compo-
sition was analysed by EDS.
DTA and TGA data were recorded using Thermal Analyst equip-
ment under an atmosphere of air and a heating rate of 5 C min 1.
Puried melanin was pyrolysed and the decomposition gas was
analysed using gas chromatography/mass spectrometry. Pyrolytic
products were separated on a capillary column using helium as a
carrier gas at a ow rate of 1.5 mL min 1. The separations were carried
out at programmed temperatures ranging from 60 C to 290 C, with
an increase rate of 15 C min 1. Mass spectra of the obtained pyrolytic
products were compared to standard spectra in the Instituto de
Capacitacin e Investigacin del Plstico y Caucho ICIPC Library
database.
3. Results
The puried bacterial melanin showed qualities that are typical of
melanins in general. The bacterial melanin was insoluble in organic
solvents, such as acetone, hexane, chloroform, methanol, and ethanol,
precipitated in acid aqueous solutions (pH b 4.00) and was slightly
soluble in alkalis (pH N 10.00). The melanin was bleached with NaClO
and H2O2 and yielded a brown precipitate after treatment with FeCl3.
The UVVIS spectrum (data not shown) showed an exponential decay
without distinguishing characteristics, including small shoulders and
no peaks in the region between 200 and 500 nm. The Fourier
Transform IR analysis of the puried pigment also had bands that
were characteristic of these compounds (data not shown) [21].
Fig. 1 shows SEM images of pigment obtained with acid extraction
and solvent purication methods. The material obtained is best
characterised as an amorphous deposit without differentiable struc-
tures, similar to other investigations using melanin puried with
similar procedures [19,22]. Although SEM/EDS analysis is not a reliable
method to quantify low molecular weight elements, including C, O,
and/or N, these analyses reveal the nature of the polymer constitutive
elements (Table 1). Additionally, these analyses give approximate O/C
and C/N relations, in this case equals to 0.17 and 5.71, respectively.
Electronic conductivity for melanin compressed-powder at room
humidity was 2.39 10 9 S cm 1, suggesting that melanin may be an
insulator at environmental conditions.
Melanin extracted and puried from bacterial broth was subjected to
thermal degradation. Table 2 shows the characteristic pyrolysis
products, retention times and percentage of participation of identied
substances. The most abundant product of bacterial melanin pyrolysis
was toluene (H), which comprised 28% of the sample. In addition,
1578 A.M. Gmez-Marn, C.I. Snchez / Journal of Non-Crystalline Solids 356 (2010) 15761580

Fig. 1. SEM images of bacterial melanin powders that were obtained using acid extraction and solvent purication methods.

signicant amounts of pyridine (C), benzene derivatives (A) and
sulphur-containing compounds (S) were found. These ndings are
distinctive characteristics and they allow us to classify this polymer as a
pheomelanin.
Based on chemical composition, the major products of the
pyrolysis were divided into following groups: derivatives A benzene
(A, A1, A2 and A3), C pyridine (C and C1), S sulphur-containing
compounds (S, S, S1 and S2), H toluene (H), E benzenenitrile (E)
and O oxygen (O) containing compounds (Fig. 2).
One interesting characteristic of melanin is its high thermal stability
compared to other natural polymers, as seen in Fig. 3. Thermograms
show that, apart from H2O desorption up to about 140 C, the polymer
structure is relatively stable. However, when approaching 500 C, the
mass loss is signicant and only about 33% of the initial mass remains.
This result is not surprising considering that although the polymer
growth process has proceeded at room temperature, melanins have
well-developed graphitic-like structures, capable of supporting such
high temperatures [23]. TGA data corroborate the existence of this
structure in the bacterial melanin.
The DTA curve shows an endothermic peak between 34 and 140 C
and two exothermic peaks between 170 and 700 C, the second wider
than the rst. The nature of the exothermic peaks can be better
analysed with a plot of the weight percent of each derivative with
respect to temperature Fig. 4. This gure clearly shows three peaks
that correspond to at least three different mass losing processes.
our acid extraction and solvent purication procedures removed all
bound-proteins and the polymer nitrogen content belongs to the
constitutive units. On the other hand, the very low aluminium content
found by EDS analysis indicates that our purication process removes
almost all metallic ions, similar to other studies [19].
McGuinness and Proctor reported that melanin conductivity was
similar to that seen in semiconductor materials [26]; nevertheless,
other authors have reported that melanin is an insulator [27,28]. This
is a controversial matter because the origin of conduction in these
species (ionic or electronic) is not known [2629]. In our case,
melanin conductivity is one thousand times greater than previously
reported [2729], but the conductivity is nonetheless comparable to
insulating compounds. Because the conductivity properties of the
powder form are largely determined by grain boundaries, more work
is underway to clarify this point. Conjugate polymers, however, can be
either insulators or conductors, according to their doping degree [30].
Other tests that we have performed have revealed the possibility of
adjusting melanin conductivity with a change in the molecule's
oxidation state, similar to what had been seen in conjugate polymers
[30].
Pyrolytic patterns of synthetic melanins that were obtained from
cysteinyldopamine CysDA and their copolymers with dopamine
DA are characterised by large quantities of benzothiazine-,
benzothiazole- and thiazoloisoquinoline-derivatives, in addition to
benzothiopyrano-imidazol. Some authors have postulated that these

4. Discussion
Table 2
In this work, the O/C ratio, which was 0.4 for natural melanin and Compounds obtained after pyrolysis/GC/MS of bacterial melanin.
up to 0.62 for synthetic melanin, is smaller than the O/C ratios found Compound Abbrev.a Retention time (min) % Total area
in other investigations [24]. This difference can be due to intrinsic
3 compounds N.I n.i 1.1911.333 6.634
characteristics of the bacterial melanin, or alternatively it may reect Benzene A 1.350 4.428
the decarboxylation of polymer constituent units due to the extensive Furan, 2, 5dimethyl O 1.444 0.834
extraction process that was employed [19,22]. N.I n.i 1.525 4.443
Although some investigations have reported signicant quantities Thiazole S 1.571 2.594
Pyridine C 1.621 9.285
of proteins that are bound to polymer structure for melanins obtained
Toluene H 1.703 27.993
by acid extraction [19], our results showed a C/N ratios lower than Tiophen, 3 methyl S 1.764 1.308
those seen in other works, oscillating from 8 to 33 [24,25]. Possibly, N.I n.i 1.837 1.807
Thiazole, 2 methyl S1 1.908 2.270
Pyridine, 2 Methyl C1 1.982 1.545
Table 1 2 compounds N.I n.i 2.0182.084 4.430
EDS elemental composition of puried bacterial melanin (% molar). Benzene ethyl- A1 2.24 2.137
Benzene, 1,4 dimethyl A2 2.293 2.108
1 2 3 4 Averagea
N.I n.i 2.349 0.997
C 71.30 64.74 69.49 75.75 70 5 Isothiazola, 3,4 dimethyl S2 2.404 0.965
N 15.81 12.54 14.35 6.53 12 3 Styrene A3 2.447 5.203
O 9.54 16.31 10.81 12.19 12 2 6 compounds N.I n.i 2.5823.172 3.476
Al 0.31 0.86 0.32 0.5 0.3 Benzenenitrile E 3.208 1.267
S 2.86 5.27 3.88 4.30 4.1 0.6 20 compounds N.I n.i 3.28611.244 16.276
Cl 0.48 0.83 0.62 0.72 0.7 0.9 a
Abbreviation of selected group of compounds; n.i, non-identied compound; A,
a
Al and Cl quantitative determinations are very imprecise because of the low benzene type; C, pyridine type; S, sulphur-containing compounds; E, benzenenitrile
accelerating voltage employed and their low molar composition. type; H, toluene type; and O, oxygen containing compounds.
 A.M. Gmez-Marn, C.I. Snchez / Journal of Non-Crystalline Solids 356 (2010) 15761580
Fig. 2. Pyrolytic products divided into chemical groups obtained after Py/GC/MS
(legend see Table 2). Derivatives are as follows: A Benzene (A, A1, A2 and A3), C
pyridine (C, C1) S sulphur-containing compounds (S, S, S1, and S2), E
benzenenitrile (E), H Toluene (H) and O oxygen containing compounds (O).
compounds are specic markers of CysDA-derived units in pheomela-
nins [17]. Thermal degradation of these compounds during pyrolysis
may generate thiazole and their alkyl derivatives. Also, isoquinoline, a
thermal degradation product of pheomelanin, may generate pyridine
and its alkyl derivatives.
Bacterial melanin pyrogram contains little benzeneacetonitrile
and high quantities of pyridine and benzene derivatives, similar to
those reported for natural melanins [15], although in major
percentages, ascending to 42% between benzene, toluene, ethyl
benzene, styrene and 4-dimethylbenzene. This indicates that these
compounds arise not only from noncyclic, amino acid-type units, but
also as a result of the degradation of other polymeric subunits, such as
hydroxyindole, indolequinone, benzothiazole, benzothiazine, thiazo-
loisoquinoline and benzothiopyrano-imidazol [1518].
Additional compounds arise during the thermal degradation of
DOPA melanin that is synthesised in the presence of metal ions. These
compounds include pyrazines and pyridines and their alkylated
derivatives. Furthermore, the amount of benzene is enhanced and the
levels of pyrrole, phenol, benzenenitrile and indole-derivatives are
considerably reduced when compared with their copper- and zinc-free
melanin counterparts [22]. In our case, the high concentration of
pyridine among pyrolytic products can reect the presence of metal ions
during the pigment synthesis, which is normal in biological environ-
ments [12,16]. These metal ions were removed during the extraction
and purication processes.
Regarding the previous paragraph, some non-identied compounds
in the pyrolytic pattern could be, according to its retention time on
GC/MS, pyrazine, which is seen between 2, 5-dimethyl furane and
thiazol, and phenol, which is seen between styrene and benzene-nitrile.
Fig. 3. TGA and DTA data for bacterial melanin.
1579
Fig. 4. Derivative of the thermogram from the TGA analysis of bacterial melanin. The y-
axis is the derivative of weight percent of the sample with respect to temperature,
where the initial weight of the sample is 100%.
In the same way, although they have been reported for natural melanin,
neither 1, 2-benzenediol nor indoles were detected in the bacterial
melanin pyrolysates. These molecules would be visible between the
retention times of 2-methyl pyridine and styrene. Indolequinone,
benzothiazole, benzothiazine, thiazoloisoquinoline and benzothiopyr-
ano-imidazol derivatives could be the non-identied compounds that
have a retention time greater than benzenenitrile.
Finally, the presence of nitrogen and sulphur compounds indicates
that the studied melanin is different than the catechol-melanins, as
phenolic-type melanins only have nitrogen traces, or from 1, 8 dihy-
droxynaphthalene-melanins [24].
Our TGA of bacterial melanin is quite similar to that previously
reported [23]. In our studies, the mass that was lost is greater than the
mass of the lost L-Dopa melanin that was obtained by electro-oxidation
during 9 days and auto-oxidation during 21 days. In those cases, 40%
and 55% of the total mass are retained at temperatures up to 500 C,
respectively [23,31]. However, other works with different synthetic and
natural melanins report a permanence of 1226% of the initial weight
[32]. Some authors have proposed a relationship between melanin
resistance to thermal degradation and the melanin origin [25,27,32].
Our results suggest that microbial melanins are thermally more stable
than melanins produced by superior organisms. However, one study has
found that 17.5% of the initial mass of the L-Dopa melanin, obtained by
auto-oxidation over 48 h, remains stable up to 500 C [32], which
suggests a close relationship between thermal stability and the degree of
polymerisation of the melanin.
It is clear from Fig. 4 that the polymer suffers at least three different
mass losses. The rst and the last losses correspond to the release of
weakly bonded water and polymer decomposition, respectively. The
middle negative peak (Fig. 4) is consistent with the release of
intramolecular, strongly-bonded water, as this process extends only
until approximately 280 C and there are two minima in the region
between 150 and 400 C with polymer decomposition at low
temperatures of approximately 280400 C [25].
Considering the TGA curve (Fig. 3), if all mass lost up to 280 C
caused by water loss, approximately 32% of the initial mass of the
sample is adsorbed water. Furthermore, if all mass loss below 95 C,
the boiling point of water, is due to the loss of weakly-bound water, its
initial mass is then approximately 7.4%. This would mean that 27% of
melanin dry weight is strongly bound water, 6% less than the reported
value for L-Dopa melanin [31]. Between 280 and 400 C, the weight
loss is 21%, a high percentage that only can be explained by considering
polymer degradation because the extraction process has removed all
bound protein.
Our DTA curve (Fig. 3) is similar to those previously reported from
melanin pigments, both synthetic and natural. A strong exothermic peak
was attributed to the beginning of rapid polymer decomposition and an
endothermic peak was specied as weakly bonded water [25,27,32].
1580 A.M. Gmez-Marn, C.I. Snchez / Journal of Non-Crystalline Solids 356 (2010) 15761580
However, our curve also has an extra exothermic peak that L-Dopa and
natural melanins, eumelanins mainly, do not have [23,32]. Pheomela-
nins have sulphur units in their structure, and the decomposition of
these molecules could be responsible for the appearance of this new
peak. Previous ideas suppose that the natural melanins analysed by
Simonovic et al. are eu or allomelanins [32]. Unfortunately, the TGA and
DTA curves of pheomelanin have not been reported before. These data
could be used to validate or reject this hypothesis.
5. Conclusions
In this work, an advanced characterisation of bacterial melanin is
presented using different techniques. SEM images revealed that acid
extractions caused modications of melanin structure that resulted in
an amorphous material. EDS and Py/GC/MS analyses suggested that
our studied melanin is a pheomelanin; however, sulphur and nitrogen
content would indicate that the melanin precursor is different from
catecholic (only nitrogen traces) or -DHN- precursors. Thermogravi-
metric and differential calorimetric data suggest that bacterial
melanin is thermally more stable than other natural melanins,
showing stability to approximately 400 C. The water content appears
to consist of a weakly-bound fraction, which is lost up to 140 C, and a
strongly bound fraction, which can remain in the structure at higher
temperatures and may make up approximately 27% of the melanin
dry weight. Finally, direct current melanin conductivity, at room
temperature, would suggest that this compound is characterised as an
insulator.
Acknowledgements
We thank the help of Professors Olga Ins Montoya Campuzano,
Daro de Jess Gallego Surez and Alejandro Toro. The Instituto de
Capacitacin e Investigacin del Plstico y Caucho ICIPC and the
Laboratorio de Procesos Fisicoqumicos Aplicados from the Universidad
de Antioquia for the PyGCMS experiments and Differential thermal
analysis and thermogravimetric analysis, respectively, as well as Juan
Felipe Santa M., Leonardo Fabio Velasquez Vallejo and all the members
of the Grupo de Ingeniera Electroqumica GRIEQUI from the
Universidad Nacional de Colombia, Sede Medelln are gratefully
acknowledged.
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