Studies on the production and dyeing properties of water soluble pigments from

filamentous fungi

Introduction

In recent years, pigments are envisaged to have extensive application in food,

dyeing, and pharmaceutical industries for coloring the products. Some of the plant
species such as Rubia tinctorum L (Angelini et al., 1997, Moresi et al., 2001), Isatis
tinctoria L, (Kokubun et al., 1998) and Reseda luteola L (Cerrato et al., 2002) were
found to be good sources of red (alizarin), indigo (indigotin) and yellow (luteolin) dyes
respectively. In fact, all three dyes were extensively exploited until the commercial
success of their synthetic analogues (Ball, 2002). Naturally derived colorants are also
extracted from fruit skins, seeds or roots and the manufacturers are usually dependent on
the availability of raw materials for the colour extraction. Further pigment profile in the
natural sources is prone to variation and it is influenced by the extraction procedures
employed. Thus, the chemical composition, including the presence of minor components,
and the colour stability of naturally derived colour additives vary significantly among
different suppliers and from batch to batch (Downham and Collins, 2000). The main
disadvantage of these natural dyes lies in the order of magnitude of their yield (a few
grams of pigment per kg of dried raw material). The red colorant carmine for instance, is
a product of the female cochineal insect and to extract 100 g carmine colorant,
approximately 14000 insects are required (Wissgott and Bortlik, 1996). This makes their
current market price high, thus limiting their application.

Though plant and animals are good sources for production of color pigment, the
inability of them to meet the world demand has led to increased interest in synthetic
colorant. Moreover, a great deal of concern has been raised by the effects of some
synthetic dyes on human health as sources of skin cancer, disorders and allergic reactions
(Francalanci et al.,2001). To overcome this limitation, it was suggested to exploit the
potentiality of other biological sources such as fungi, bacteria and algae with modern
biotechnological techniques to improve significantly the pigment production.

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 Micro algae are known to produce a wide range of water-soluble pigments, but
the low productivity of algal cultures is a significant bottleneck for their
commercialization (Hejazi and Wijffels, 2004). Pigments of basidiomycetous fungi have
been used in the past for dyeing wool and silk (Bessette and Besette, 2001). The
appropriate use of fermentation physiology together with metabolic engineering (Nielsen
et al., 2002) could allow the efficient mass production of colorants from fungi. The
present investigation is focused on fungi that produce industrially important pigments.

Review
Like plants, filamentous fungi synthesize natural products because they have an
ecological function and are of value to the producer (Firn and Jones, 2003). Depending
on the type of compound, they serve different functions—varying from protection against
lethal photo-oxidations (carotenoids) to environmental stress (melanins) and acting as
cofactors in enzyme catalysis (flavins). Filamentous fungi produce several characteristic
of carotenoid and non- carotenoid pigments (Baker and Tatum, 1998; Medenstev and
Akimenko, 1998; Adrio and Demain, 2003). Anthraquinone (octaketide) pigments like
catenarin, chrysophanol, cynodontin, helminthosporin, tritisporin and erythroglaucin are
produced by Eurotium spp., Fusarium spp., Curvularia lunata and Drechslera spp
(Duran et al., 2002). Yellow pigments-epurpurins A to C were obtained from Emericella
purpurea (Hideyuki et al., 1996) and azaphilone derivatives (hexaketides), falconensins
A–H and falconensones A1 and B2, were produced both by Emericella falconensis and
Emericella fructiculosa (Ogasawara et al., 1997). The red colorant is an extracellular
metabolite of the anthraquinone class and is claimed to be produced by a variety of
Penicillium oxalicum (Sardaryan et al., 2002; Sardaryan, 2004).

Monascus pigments, which are water-soluble, are of great biotechnological

interest. Monascus is a typical Ascomycete that produces a cleistothecium, a closed
fruiting body containing eight ascospores, but reproduces asexually by the formation of
conidiospores and a vegetative mycelium. The pigments may be produced both in the
mycelium and liberated to the fermentation broth (Margalith, 1992). However,

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Monascus-fermented rice has been found to contain the mycotoxin citrinin (Liu et al.,
2005). The production of citrinin together with pigments clearly limits the use of
Monascus as a producer of natural food colorants.

Optimization of growth medium is one of the essential steps to maintain a

balance between the medium components to minimize the amount of unutilized
components at the end of fermentation, and to have cost effective metabolic yield (Kumar
et al., 1999; Prakasham et al., 2005). Pigment production is often influenced by different
carbon and nitrogen sources. Lilly and Barnett (1962) reported that the biomass
production of Monascus purpureus was found to be increased in carbon source like
fructose, glucose and sucrose. McHan and Johnson (1970) showed that Monascus
purpureus grew better in glucose-peptone -yeast extract broth than in any other complex
medium. Zinc and a special combination of glycine, leucine and tryptophan yielded more
growth (McHan and Johnson, 1979). The overwhelming majority of extraction
procedures use the traditional liquid–liquid extraction (Bohm, 1999; Lacker, et al., 1999).
However, the successful applications of modern extraction techniques such as solid-phase
micro extraction (Emenhiser, et al., 1996) and microwave-assisted extraction
(Csiktusnadi et al., 2000) have also been reported.

The review clearly indicates that not much work has been undertaken on fungal
pigments, although variety of pigmented fungi are present in nature. Hence the present
work was planned with the aim of searching of novel water soluble pigments from
filamentous fungi. The work was designed as given below.

1. Isolation of pigment producing fungi from diverse ecosystems like agricultural

soils, forest soils and rhizoshpere of plants.

2. Selection of production medium and optimization of nutrient conditions for

pigment production.

3. Characterization of the pigments.

4. Genetic improvement of the selected fungi through mutagenesis for enhanced

pigment production

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 5. Optimization of the pigment production in solid substrate and cost effective
production of pigments.

6. Testing for mutagenesis, antioxidant, antimicrobial activities and toxicity

evaluation by seed germination

7. Determining the dyeing capacity on fabric and leather and to assess the
colorfastness.

Work undertaken:

Soil samples were collected from 51 different locations. Pigment producing

filamentous fungi were isolated. Potent and promising four isolates (Pacilomyces
farinosus-red, Emericella nidulans-yellow, Fusarium moniliforme-Brown and
Penicillium pupurogenum- thick red pigment) were selected on the basis of color and
type of pigment (intracellular or extracellular).

Culture conditions were optimized using different sources of carbon, nitrogen,

temperature, pH and light. Effects of various heavy metals on the production of pigment
were tested. Antimicrobial, MIC, antioxidant and mutagenic activity were determined.
Mutants were developed and tested for their pigment production.

Purification and characterization of pigment was done using column

chromatography, UV-VIS, IR, NMR and GC-MS spectrum. The compounds identified
were anthraquinone (Pacilomyces farinousus), di-o-acetyl-lanugon-j (Emericella
nidulans), Heptacosonic acid 25-methyl ester (Fusarium moniliforme) and
chrysanthemum hydrolyzed (Penicillium pupurogenum).

Dyeing property was tested on to cotton fabric and leather and the quality of the
dyed specimen was determined by employing various tests. The results are presented.

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 The thesis contains six chapters. The first chapter gives general introduction of
the study.

The chapter two covers literature review.

The third chapter describes various methodologies adopted to study the effect of
culture conditions on pigment production, purification, characterization, and the
application of the pigment on fabric and leather.

The chapter four deals with the results and chapter five focuses on the overall
discussion of the above study.

The sixth chapter highlights the results as summary, followed by the list of
references.

References:

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