Introduction

Xylanases (Endo-1,4-Beta-xylanase or XYN, EC 3.2.1.8) are glycosidase which catalyzes the endohydrolysis of 1,4-β-D-xylosidic linkages in backbone of complex plant polysaccharides xylan. Filamentous fungi produce number industrial enzymes which have wide variety of applications (Kirk et.al 2002; Sharma et.al 2001) and hence studies on filamentous fungi have always been of prime importance. Among microorganisms filamentous microbes are one of the most prolific producers of extra cellular enzymes and they are also widely used for industrial enzyme production. Some other xylanase producing organisms are Humicola griesa, Trichoderma, Penicillium, Humicola insolens, Aspergillus sp., etc (Collins et al, 2005). Hemicellulytic microorganisms play a significant role in nature by recycling hemicelluloses. Thermomyces lanuginosus (formerly known as Humicola lanuginosa) is one such widely distributed filamentous thermophilic fungi. Mycelium is the characteristic growth form of filamentous fungi and it enables them to explore and exploit new environments and substrates. T. lanuginosus SS-8 is a filamentous fungus producing extracellular enzyme xylanases which has varied industrial application.

In the present study different growth forms of T. lanuginosus have been reported. During growth period the hyphal color of T. lanuginosus changes from white to dark brown as it matures.

It is a widely distributed thermophilic fungus commonly isolated from self heating masses of organic debris (Emerson, 1968). It is classified as a Deuteromycetes (imperfect fungus) that is unicellular or septate and reproduces asexually by forming aleurioconidia (Singh et al., 2003). This organism is non-cellullolytic and it has been reported that it probably grows commensally in composts with cellulolytic fungi by utilizing some of the sugars generated by the enzymes of these fungi and also by using their mycelial breakdown products (Deacon, 1997). T. lanuginosus are hyperproducers of extracellular xylanases. Along with this they also produce other hemicellulases. Apart from xylanase most glycosyl hydrolases are expressed in very low amount. T. lanuginosus also produces range of other secreted degradative enzymes such as alpha-amylase, glucoamylase, pectinase, phytase, protease and lipase (Singh et al., 2003).

Initially the colonies of T. lanuginosus appear white and felt like which turn grey or greenish grey, commencing from the centre of colony. Subsequently the colony turns purplish brown and the agar substratum stains a deep pink or wine colour. Mature colonies appear dull dark brown to black. Fungal hyphae grow by polarized (apical) growth (Cristina, 2005).

T. lanuginosus produces xylanases which are glycosidases which catalyze the endohydrolysis of 1, 4-β-D-xylosidic linkages in xylan; the major structural polysaccharide in plant cell and is the second most abundant polysaccharide in nature. Xylanase has varied industrial applications such as fruit and vegetable processing, brewing, wine production, baking, animal feed, paper and pulp, starch, textiles, bioremediation/bioconversion. Xylanases produced from T. lanuginosus are. thermostable and good activity at varied pH range have made this enzyme applicable in various industries. The present study was conducted to understand the various morphological forms of T. lanuginosus SS-8 isolated from garden soil sample with organic debris and correlative study of change in hyphal morphology to xylanase production.

Despite considerable progress in understanding the different stages in hyphal developments during enzyme production by various morphological forms of filamentous fungi the present hyperxylanolytic isolate T. lanuginosus SS-8 is yet to be explored by various workers. Consequently in the present study we investigated the various morphological forms of T. lanuginosus SS-8 and its correlation with xylanase production.

Materials and methods

Microorganism and growth conditions

Microscopy & Image analysis

Enzyme Activity Assay

Results

The indigenous T. lanuginosus SS8 colony was white in color which with maturity changed to yellowish followed by brown and then blackish brown colony [Fig a]. Light microscopic study of white colony suggested that these were only filamentous. The filaments were of 1µm and the conidia are of about 0.75µm [Fig b]. As the colony matured it turned to yellowish. At this stage young aleurioconidia of about 1µm emerged laterally from the mycelial hyphae and matures to about 2.5µm. [Fig c] and [Fig d]. The hyphal width at this stage is about 2µm. This feature is novel to the organism as it does not turn greenish or purplish like other reported organisms of same species. On further maturation the colonies turn brown. At this stage the aleurioconidia matures and they are of about 6µm to 9µm in diameter [Fig d], [Fig e] and [Fig f]. The hyphal diameter at this stage is about 2.5µm.

When the colonies completely mature, they appear blackish brown with dark brown globbose aleurioconidia of diameter size 10µm to 11µm. The hyphal diameter at this stage is about 3µm [Fig g]. These aleurioconidia have wrinkled surface. Finally the conidia detaches from hyphae [Fig h] and [Fig i].

The changes in hyphal width with respect to different colony color have been plotted in Graph 1. Changes in aleurioconidia diameter with change in colony color have been plotted in Graph 2. Activity of crude extracellular enzyme collected after 48, 72, 96, 120, 144 and 168 hr incubation was calculated [Table 1] in IUml ^-1 min ^-1 and change in enzyme activity with respect to change in fungal morphology have been plotted in Graph 3.

Figure 1

Figure 1: (a) SS-8 in YpSs Medium. (b) Aleuroconidia (0.75 µm Hyphae: 1 µm. at 40X (c) Aleuroconidia (1 -2.5 µm), hyphae: 2 µm. at 40X (d) Hyphae and Aleuroconidia at 100X oil emersion. (e) Hyphae and mature aleuroconidia at 100X, oil emersion, Aleurioconidia: 6-9 µm and hyphae: 2.5 µm. (f) Mature aleuroconidia on the verge of detaching from hyphae (100X oil immersion). (g) Hyphal elements and aleuroconidia from mature colony at 40X. (h) Aleuroconidia: 11 µm; Hyphae 3 µm. (i) Mature aleuroconidia detached from hyphae.

Figure 2

Table 1: Change in colony color, hyphal width, aleurioconidia diameter and enzyme production with respect to incubation time

Figure 3

Graph 1

Figure 4

Graph 2

Figure 5

Graph 3

Discussion

From the study it was concluded that as fungal cells mature the enzyme production increases. There was no enzyme present in production medium upon 24hr incubation. Thus suggesting that white colony with budding conidia could not produce any enzyme. Subsequent incubation for 48, 72, 96, 120, 144 and 168 hr showed gradual increase in enzyme activity. It was seen that as conidia matures its capacity of enzyme production increases and amount of enzyme produced shows a measurable increase with the change in colony color from yellow to brown.

The increase in hyphal diameter from white to yellowish colony is by a factor of 2. The complete increase in hyphal diameter from white to blackish brown colony is by a factor of 3. The yellow color colony of the organism is unique to this novel form as no earlier literature reports this feature. Previous literatures suggest the purplish or green colony which is absent in this strain.

Acknowledgement