Screening for alkaline keratinolytic activity in fungi isolated from soils of the biosphere reserve ‘‘Parque Costero del Sur’’ (Argentina)

A screening was carried out on 69 fungal strains isolated from alkaline-calcareous, neutral and alkaline-sodic soils, as well as from their associated plant material, to determine their ability to grow at alkaline pH. A total of 32 fungi were selected for their ability to produce alkaline keratinase activity in submerged shaken cultures supplemented with soybean meal (SM) and tryptone and on cow hair (CH) under solid state fermentation conditions. Although several fungal strains produced keratinolytic activity on both SM and CH, they differed in the levels detected. Among them, Aspergillus niger , Cladosporium cladosporioides , Metarrhizium anisopliae , Neurospora tetrasperma and Westerdikella dispersa were the best producers, with levels higher than 1.2 U ml - 1 . Different fungal species are here reported for the ﬁrst time for their ability to produce keratinolytic activity at alkaline pH.


Introduction
Keratins, which are structural proteins that represent the main constituents of the skin of vertebrates and appendages such as nails, hair, feathers, and wool, are water insoluble and extremely resistant to degradation (Gradisar et al. 2005).
Degradation of keratinous material has mostly been associated with the activity of dermatophytic fungi, yeast and bacteria.A lot of information is available on the abilities of these organisms to produce keratinase enzymes and on the catalytic properties of these enzymes (Friedrich et al. 1999;Mangiaterra et al. 2000).However, there are relatively few reports on the production and characterization of the keratinases produced by non-dermatophytic fungi, including those associated with extreme environments under stressful conditions (Gradisar et al. 2005;Elı ´ades 2009).An example of this kind of environment is the native xerophilic forest dominated by the tree species Celtis tala Gill ex Planch (Ulmaceae) and Scutia buxifolia Reiss (Rhamnaceae) and its associated Distichlis spicata (L.) Greene grassland, located in the eastern region of Buenos Aires province (Argentina, Arturi et al. 1996).This assemblage of ecosystems is part of the ''Parque Costero del Sur'', a biosphere reserve with biogeographic, landscape, cultural and historical features (MAB- UNESCO, Goya et al. 1992;Crous et al. 2005).This area is characterized by different soil types, including alkaline-calcareous (Rendolls), neutral (Argialbolls), and alkaline-sodic (Natracualfs) soils.These soils might be a source for the isolation of fungi able to produce enzymes tolerant to different pH values and/or highly active at extreme pH values.These enzymes can be used as biotechnological tools and have potential applications in the leather industry waste treatment (Galarza et al. 2007).
The aim of this study was to analyse the ability of 69 fungal strains, isolated on agar-medium at several pH values from various soils or from their plant material collected at the Biosphere Reserve ''Parque Costero del Sur'' (Argentina), to grow at alkaline pH, and on the ability of some strains to produce keratinase enzymes with activity at alkaline pH, when grownon medium with soybean meal and tryptone (SM) and on cow hair (CH) under various culture conditions, was also evaluated.

Fungal isolation
The strains used for this work (Table 1) were isolated from alkaline-calcareous (Rendoll), neutral (Argialboll) and alkaline-sodic (Natracualf) soils, as well as from organic material such as decaying leaves of Distichlis spicata (Poaceae) collected at the Biosphere Reserve ''Parque Costero del Sur'' (MAB-UNESCO), located in eastern Buenos Aires province, Argentina (35°11 0 S, 57°17 0 W).The soil samples were taken from either the superficial horizon or that deeper than 20 cm.They were then processed as previously (Elı ´ades et al. 2006a) and incubated on agarmedium at several pH values (6, 8, or 11).Similarly, D. spicata debris was processed as previously (Elı ´ades et al. 2007).Plates inoculated with soil particles or debris were incubated at 25°C and observed microscopically after 5-7 days or at 1-week intervals for fungal development and isolated in axenic culture.Original taxonomic papers based on cultural and morphological features as well as the work by Domsch et al. (1993) were used to identify sporulating fungi (Cabello and Arambarri 2002).Stock cultures were kept at 4°C on 2% (w v -1 ) agar-malt extract slants and lyophilized according to Smith and Kolkowski (1996), being then deposited in the culture collection of the Instituto Spegazzini, UNLP, La Plata, Argentina (LPSC).
Screening for fungal growth at alkaline pH All fungi isolated were inoculated according to Saparrat and Guille ´n (2005) in a basal liquid medium as reported by Rojas et al. (2009), supplemented with ground soya meal (SM, 250-1,000 lm particles) at 1.5% (w v -1 ) and buffer-solutions according to Nagai et al. (1995) to adjust the pH to 9.0.Three replicates were incubated for 15 days at 28 ± 1.5°C under shaking (200 rpm).Mycelial development was estimated according to Hofrichter and Fritsche (1996).
Fungal culture for producing alkaline enzymatic activity and analytical methods Selected fungi from the screening were then grown at pH 9.0 both in submerged cultures supplemented with SM under shaking and on a solid-state fermentation SSF system using CH as growth substrate in order to analyse their ability to produce keratinase enzymes with activity at alkaline pH as well as to analyse their ability to modify the medium pH after 15 days of culture.Previous to inoculation, CH was defatted by soaking in dichloromethane, ground and fractionated into Petri plates (3 g), which were autoclaved (at 121°C for 25 min) and supplemented with buffer-solutions according to Nagai et al. (1995) for adjusting the water associated with the substrate to pH 9.0.Three replicates were carried out for each isolate in each culture system.Degradation of CH by each fungal strain was measured as percentage substrate mass reduction according to Pandey and Nagveni (2007).The pH of 15-day-old culture systems with each fungus, on the supernatants of the submerged cultures and from aqueous suspensions obtained from SSF cultures by mixing one part of substrate and five parts of water under shaking for 5 min, was also measured.

Preparation and determination of enzyme extracts
Enzyme extract from the supernatants of SM cultures was obtained by filtering the cultures with a glass OSMONICS micro-filter (size-pore 0.45 lm) and then centrifuging at 10,0009g for 10 min at 4°C.The clear supernatant was then passed through a PD 10 molecular exclusion column (Disposable PD-10 Desalting Columns IMPROVED) equilibrated with a 20 mM Tris-HCl buffer at pH 9.0.
Enzyme extract from CH-SSF cultures was obtained by mixing one part (wet mass) of substrate and three parts of 0.6 M NaCl solution and shaking the mixture at 50 rpm for 30 min at 25°C.The suspension obtained was filtered using a glass OSMONICS micro-filter (size-pore 0.45 lm).The supernatant was passed through a PD 10 molecular exclusion column (Disposable PD-10 Desalting Columns IMPROVED) equilibrated with a 20 mM Tris-HCl buffer at pH 9.0 and then concentrated 109 by lyophilization in an L-3 RIFICOR equipment.
Keratinase activity was determined using Keratin Azure (Sigma K-8500, 5 mg) as substrate, which was incubated with 0.4 ml borax buffer (0.1 mM pH 9.0) and 0.1 ml of   (2004).After 7 h, the absorbance at 595 nm of the supernatant of the mixture reaction, which had been previously stopped by adding 0.5 ml 10% trichloroacetic acid (TCA), was measured spectrophotometrically.A blank reaction without enzyme extract (replaced by buffer) was also carried out.The reactions were carried out in triplicate.Based on preliminary studies, optimal assay times were known to fall in the linear range of enzyme kinetics.One enzymatic activity unit (U) was defined as the amount of enzyme that produces 0.1 absorbance 595 nm unit per hour.Data were expressed as U ml -1 and analysed by a one-way ANOVA, and means were contrasted by Tukey's test (P \ 0.05, Statistix 8).

Results and discussion
We have previously reported morphological and ecophysiological features of the most representative fungi growing on several soil types and on litter from forests of Celtis tala and Scutia buxifolia and Distichlis spicata grassland in Argentina, including the size of their reproductive structures and their response to pH (Elı ´ades et al. 2004(Elı ´ades et al. , 2006a(Elı ´ades et al. , b, 2007;;Saparrat et al. 2007Saparrat et al. , 2008)).In the present work, we isolated 69 fungal strains at pH 11, 8 and 6 (38, 27, 35%, respectively) from the three soil types tested and identified them taxonomically (Table 1).While 31 strains were isolated from Natracualf soils, 24 and 14 were isolated from Rendoll and Argialboll soil, respectively.Most of the fungi found were anamorphic states of Ascomycota, although teleomorphic ones as well as some representatives from Zygomycota such as Absidia spinosa, Cunninghamella elegans and Gongronella butleri, were also identified.
Fungi were screened for growth at alkaline pH in a basal liquid medium supplemented with SM at pH 9.0.Except for F. semitectum LPSC 935, all the strains revealed ability to grow in SM, showing formation of conspicuous hyphae and mycelial pellets in 30 and 15% of the strains tested, respectively.However, more than half of the strains presented poor growth (Table 1).
On selected fungi from the previous screening, they were grown at an initial pH of 9.0 both in submerged cultures supplemented with SM under shaking and on a SSF system using CH as growth substrate.Most of the fungi caused a decrease in the pH level of the system (Table 2), being A. niger LPSC 845 the isolate that caused the greatest decrease in the SM medium.This may be related to the typical fungal activity that generates acidification as a consequence of the use of C and N compounds (Moore-Landecker 1996).In contrast, Periconia minutissima LPSC 943 grown in SM medium increased the pH levels slightly to 9.5, being this increase probably due to  ? as a result of fungal degradation of organic N (Saparrat et al. 2007).
Table 2 shows the results of the screening of the 32 fungi selected, in both liquid (SM) and solid (CH) conditions.Although several of the fungal strains tested produced keratinolytic activity on both SM and CH, they differed in the levels detected.Aspergillus niger LPSC 845, Cladosporium cladosporioides LPSC 953, Metarrhizium anisopliae LPSC 996, Neurospora tetrasperma LPSC 837 and Westerdikella dispersa LPSC 830 were the highest producers in SM and CH systems.While Humicola grisea LPSC 933 and Pestalotiopsis guepinii LPSC 929 did not reveal extracellular keratinase activity in both culture conditions tested, A. luteo-albus LPSC 427 did not produce it only in SM.Doratomyces stemonitis LPSC 984, Phialocephala sp.LPSC 992 and Sporormia fimetaria LPSC 339 failed to show activity on CH.
All the strains that grew on CH revealed different levels of development on the solid substrate.About 50% of them showed a velvet-like mycelial surface, 12.5% of them mycelial mats, and 37% scarce growth.This fungal ability to develop more on CH than in SM could be related to the fact that the soil where fungi were isolated had animal waste produced by the livestock activity of the region (Sa ´nchez et al. 1976;Cabello and Arambarri 2002).
Reduction in CH mass by fungal activity was also measured, being observed for several fungal cultures (Table 3).Metarrhizium anisophliae LPSC 996 and Chrysosporium LPSC 951 sp.showed the highest reductions 26.86 and 24.98%, respectively.The scarce growth of Metarrhizium anisophliae on this substrate type and the high keratinase activity found are in agreement with previous findings about efficient performance of this fungal species as an entomopathogenic organism attaching organic N-rich substrates (Mohan and Pillai 1982).
In summary, in the present work we reported the ability of fungi isolated from three types of soils from the Biosphere Reserve ''Parque Costero del Sur'' (Buenos Aires, Argentina) to produce keratinolytic activity at pH 9.0.In this sense, these isolates growing on media with proteinrich substrates, such as CH and SM, differed in their enzyme production levels as well as in their contribution to CH mass loss.Further experiments are in progress to characterize the enzymes from strains revealing high enzyme potential.
at 28°C according to Galarza et al.

Table 1
Fungal strains used and their growth on SM cultures at pH 9

Table 2
Extracellular pH and keratinolytic activity from 15 day-old fungal cultures grown on SM and CH at pH 9 Letters indicate significant differences between fungi grown on each culture medium (Tukey's test, P \ 0.05).The following list includes the species (and LPSC number of strains) that showed levels lower than 0.5 U ml -1 : A. cerealis 928, A. ustus 981, F. oxysporum 961, S. chartarum 922, T. stipitatus 835 and T. trachyspermus 831 on both SM and CH cultures, and only on SM D. stemonitis 984 and Phialocephala sp.992 only on SM a