Characterization of Aureobasidium pullulans isolated from airborne spores in Thailand

Abstract.

Isolates from air in several locations in Thailand were identified as Aureobasidium pullulans PR with dark pigmentation (Loei province), A. pullulans SU with an unusual conidial apparatus (Chiangmai province), and A. pullulans CU with burgundy-red pigmentation (from a shady area in Bangkok). The internal transcribed spacer sequences of the rDNA of A. pullulans SU and A. pullulans CU confirmed that they were A. pullulans. Both A. pullulans CU and A. pullulans PR preferred 30 °C and pH 7.5 for exopolysaccharide (EPS) production, while A. pullulans SU preferred 25 °C and pH 6.5. All three isolates preferred glucose over sucrose and (NH₄)₂SO4 over peptone for EPS production. Under optimal conditions, A. pullulans PR produced EPS yields of up to 0.225 g g⁻¹, followed by A. pullulans CU (0.185 g g⁻¹) and A. pullulans SU (0.158 g g⁻¹). Amylase activities were detected during the course of EPS production but gradually decreased as the EPS yields increased. IR spectra suggest that the EPS from these isolates was pullulan. EPS from the three isolates were partially sensitive to pullulanase.

Introduction

Aureobasidium pullulans is classified as a black yeast, in the Ascomycetes, order Dothideales. It is recognized as an important yeast in industry. The pullulan exopolysaccharide (EPS) produced by A. pullulans has applications in the food and plywood industries and in medicine [3]. A. pullulans is a saprophytic phylloplane fungus that occurs commonly in temperate zones. There are reports of the isolation of the fungus from tropical regions, such as Brazil, India, Malaysia, and Jamaica [3]. Tokumasu et al. [9] reported A. pullulans from a pine forest in Thailand. Microbiologists in Thailand occasionally find the yeast as a contaminant. However, attempts to isolate the culture intentionally are difficult, because secondary saprophytic fungi often predominate in a tropical climate. Therefore, this primary phylloplane colonist is not easily isolated from a plant specimen. In order to find this important yeast in Thailand, we attempted to collect airborne spores and isolate A. pullulans from several locations around the country. EPS production by these strains was also investigated.

Materials and methods

Sample collection

Samples were collected at several locations around Thailand, using corn meal agar (CMA; Difco, Detroit, Mich., USA) plates exposed for 5, 10, 15, 20, 25, or 30 min. The sampling locations included the Phurua mountain area in Loei province, the Doi Suthep pine forest (Chiangmai province), the Narm Now pine forest (Petchabun province), the Tung Salang Laung pine forest (Phitsanulok province), the Khao Yai forest (Nakornratchasima province), and a shady area of Chulalongkorn University (Bangkok).

Cultivation and identification

Exposed CMA plates were incubated at room temperature (30±2 °C) for 7 days. Experiments were performed in triplicate. Yeast-like colonies were picked and were transferred to potato dextrose agar (PDA; Scharlau Chemie, Barcelona, Spain) medium. Identification began with cultivation of the yeasts on malt extract agar (Scharlau Chemie) medium at 20 °C and at room temperature. Cells were collected for microscopic observation. A. pullulans ATCC 42023 and A. pullulans NRRL 6992 were used for comparison. In addition, identification of the yeast was made by using the characteristics described by Barnett and Barry [1], De Hoog and Guarro [2], and Hermanides-Nijhof [5]. Confirmation of the identification was done by sequencing the rDNA internal transcribed spacer (ITS) domains of each isolate, using primers ITS1 and ITS4. The ITS sequencing was performed at the Centraalbureau voor Schimmelcultures (CBS), Utretch, The Netherlands. The techniques described by Gerrits van den Esde and De Hoog [4] were used. Basic local alignment search tool (BLAST) searches (http://www.ncbi.nlm.nhi.gov.) were done in GenBank in order to look for similarity with the neotype strain of A. pullulans, CBS 584.75.

EPS production

EPS was produced from the three isolates of A. pullulans. The yeast was maintained on a PDA slant at 4 °C. EPS production started with the preparation of 5% (v/v) inoculum (10⁷ cells ml⁻¹) in potato dextrose broth (Scharlau Chemie). The optimal parameters for EPS production were determined, including temperature, carbon source, and nitrogen source. The pH optimization began with inoculation of A. pullulans into 95 ml of a production medium containing 5% glucose (Sigma Chemical, St. Louis, MO, USA) and 0.06% (NH₄)₂SO4 (Carlo Erba, Milan, Italy), with pH adjusted to 4.5, 5.5, 6.5, or 7.5 in individual 250-ml Erlenmeyer flasks. The flasks were incubated at 25 °C, 150 rpm for 5 days. The temperature optimization study was performed using the production medium at the optimal pH for each isolate and incubation temperatures of 25, 30, or 35 °C at 150 rpm for 5 days. Two carbon sources were tested, glucose and sucrose, and each sugar, at 5% (w/v), was used as the sole carbon source in the production medium at the optimal pH and temperature. EPS production was carried out at 150 rpm for 5 days. Two nitrogen sources, (NH₄)₂SO4 and bactopeptone (Difco), were tested at 0.06% (w/v) under the optimal conditions for pH, temperature, and carbon source. Again, EPS production was carried out at 150 rpm for 5 days.

Enzyme assay

Total amylase activity was assayed in the production medium. The reaction mixture (0.5 ml) contained 1% (w/v) boiled soluble starch (Carlo Erba) solution, 50 mM acetate buffer, pH 5.0, and the enzyme solution. The incubation period was 30 min at 50 °C. The dinitrosalicylic acid method was used to measure the liberated reducing sugar [7]. One unit of amylase activity was the amount of enzyme that produced 1 µmol glucose min⁻¹ in the reaction mixture.

Analysis of EPS

EPS was precipitated from spent medium using ethanol. Dry precipitate was ground prior to the addition of 95% KBr. An infrared (IR) spectrophotometer (Perkin-Elmer, Norwalk, Conn., USA) was used for the IR analysis. Pullulanase sensitivity was determined by the method of Leathers et al. [6]. Dry EPS was suspended at 0.1% (w/v) in 50 mM sodium acetate, pH 5.0. Pullulanase from Klebsiella pneumoniae (Sigma) was added at 0.1 unit ml⁻¹ and incubated for 21 h at 25 °C. Glucose-reducing sugar equivalents were determined by the dinitrosalicylic acid method [7]. Levels of pigmentation were judged by visual observation.

Data on EPS production were averaged from quadruplicate samples. All experiments had a completely randomized design. Statistical analyses included Duncan's multiple range test.

Results and discussion

Sampling sites which showed the presence of A. pullulans are indicated in Fig. 1. There was no obvious correlation between recovery of Aureobasidium spp and environmental factors in Thailand. The organism was found at both low (2 m) and high (1,000 m) altitudes, at ranges from 25.4 °C to 28.0 °C.

Fig. 1.

Collection sites in Thailand where Aureobasidium pullulans was (+) or was not (−) found. The elevation and mean temperature at collection sites were: A 900 m, 26.1 °C in March 1999 and 25.4 °C in August 1999, B 900 m, 25.8 °C in August 1999, C 800 m, 26.7 °C in August 1999, D 1,000 m, 26.7 °C in August 1999, E 900 m, 28 °C in May 1998, and F 2 m, 27.7 °C in November 1999

Although sample collections were not done throughout the year, A. pullulans was detected in both the rainy season (August) and the dry seasons (March, November).

The microbial flora that emerged from exposure of CMA plates included yeast, bacteria, fungi, and actinomycetes. When the exposure exceeded 15 min, only filamentous fungi were detected. Rapid proliferation of filamentous fungi probably hindered yeast growth. These fungi, common to the tropics, were identified as Aspergillus sp., Cladosporium sp., Curvularia sp., Neurospora sp., Nigrospora sp., Penicillium sp., Trichoderma sp., and Xylaria sp.

Identification of Aureobasidium pullulans was accomplished initially through microscopic examination of morphological characteristics. A. pullulans is polymorphic, consisting of blastospores, swollen cells, chlamydospores, hyphae, and pseudohyphae. The conidia are hyaline, smooth, and ellipsoidal. Endoconidia are present. Melanin pigmentation is common. Using the classic guidelines for identification, A. pullulans PR, isolated from Phurua, resembled A. pullulans var. melanigenum, as indicated by the culture rapidly becoming black or dark olivaceous-green [5]. The culture also contained dark arthroconidia (Fig. 2). A. pullulans SU, isolated from the Doi Suthep pine forest, also resembled A. pullulans var. melanigenum but the conidial apparatus was elongated, as in the variety pullulans. Therefore, this A. pullulans isolate appeared to be a hybrid between var. pullulans and var. melanigenum (Fig. 3). A. pullulans CU, isolated from Bangkok, also resembled A. pullulans var. melanigenum (Fig. 4). However, its burgundy-red pigment is not typical of A. pullulans var. melanigenum.

Fig. 2A–F.

A. pullulans PR from Loei province. A Colony, B conidial apparatus, C dark hypha with a chlamydospore at the hyphal tip, D dark hyphae with chlamydospores, E dark arthroconidia, and F conidia

Fig. 3A–L.

A. pullulans SU from Doi Suthep pine forest. A Colony, B–E conidial apparatus, F, G dark hypha, H–K dark chlamydospores, and L conidia

Fig. 4A–F.

A. pullulans CU from Bangkok. A Colony, B conidial apparatus, C dark hyphae, D, E intercalary and discrete chlamydospores, and F conidia

Sequencing of the rDNA ITS domains suggested that A. pullulans SU (GenBank accession number AY 139393, CBS 110376) and A. pullulans CU (GenBank AY 139391, CBS 110377) were similar to the neotype strain of A. pullulans. BLAST searches in GenBank also revealed the highest similarity with A. pullulans. The ITS sequencing of A. pullulans PR was not conclusive (data not shown). However, this isolate was also likely to be A. pullulans (G.S. De Hoog, personal communication).

In modern yeast taxonomy, the classification of A. pullulans into A. pullulans var. melanigenum based on morphology and melanin pigmentation should be considered inadequate (G.S. De Hoog, personal communication).

Recently Yurlova and De Hoog [12] reported a new variety of A. pullulans, var. aubasidani, characterized by its EPS structure. By determining the EPS structure, nutritional requirement and molecular features, A. pullulans was classified into A. pullulans var. pullulans and A. pullulans var. aubasidani. To identify the true variety of these isolates, further investigations will be required.

A pH of 7.5, 25 °C yielded the greatest dry weight of isolates A. pullulans CU (0.167 g g⁻¹ of carbon source) and A. pullulans PR (0.052 g g⁻¹) after 5 days, while isolate A. pullulans SU yielded the most EPS at pH 6.5 (0.238 g g⁻¹) after 5 days.

At pH 7.5, 30 °C yielded the maximum EPS dry weight of isolates A. pullulans CU (0.186 g g⁻¹) and A. pullulans PR (0.225 g g⁻¹) after 5 days. A. pullulans SU produced the most EPS (0.158 g g⁻¹) at 25 °C and pH 6.5 after 4 days, while 30 °C yielded significantly less EPS (0.076 g g⁻¹).

As carbon source, Glucose was preferred over sucrose for EPS production by all three isolates. Glucose yielded the greatest dry weight of A. pullulans PR (0.225 g g⁻¹) [followed by A. pullulans CU (0.185 g g⁻¹)] after 5 days and the greatest dry weight of A. pullulans SU (0.158 g g⁻¹) after 4 days, while sucrose yielded only 0.020 g g⁻¹(A. pullulans PR), 0.122 g g⁻¹ (A. pullulans CU), and 0.007 g g⁻¹ (A. pullulans SU) during the same period.

(NH₄)₂SO4 was more suitable than peptone for EPS production by all three isolates. (NH₄)₂SO4 yielded the greatest dry weight of A. pullulans PR (0.225 g g⁻¹) [followed by A. pullulans CU (0.185 g g⁻¹)] after 5 days and the greatest dry weight of A. pullulans SU (0.158 g g⁻¹) after 4 days, while peptone yielded only 0.168 g g⁻¹ (A. pullulans PR), 0.131 g g⁻¹ (A. pullulans CU), and 0.030 g g⁻¹ (A. pullulans SU) during the same period.

During the course of EPS production, amylase activity was detected in the extracellular medium of all three isolates. The highest amylase activity (0.875 units ml⁻¹) was from A. pullulans CU on day 2, followed by 0.432 units ml⁻¹ from A. pullulans PR and 0.435 units ml⁻¹ from A. pullulans SU (Fig. 5). After reaching maxima on day 2, amylase activities gradually decreased, while EPS yields increased. Since endogenous glucoamylases may attack alternan, it is possible that accumulation of pullulan in late cultures is related to this decrease in amylase activities [8, 11].

Fig. 5A–C.

Exopolysaccharide (EPS) yields and amylase activities (U/ml units of enzyme activity per milliliter of medium) during the cultivation of A. pullulans SU in production medium. A SU isolate at pH 6.5 and 25 °C, B PR isolate at pH 7.5 and 30 °C, and C CU isolate at pH 7.5 and 30 °C

Analysis of the precipitated EPS by IR suggested that the precipitated polymer was pullulan (Fig. 6). These EPS were partially sensitive to pullulanase, ranging from 46.8% sensitivity for the EPS from A. pullulans SU to 20.0% (A. pullulans CU) and 2.6% for the EPS from A. pullulans PR. While the degree of pullulanase sensitivity could be related to the pullulan content and purity, it was observed that highly pigmented EPS (A. pullulans PR) was less sensitive to pullulanase. Leathers et al. [6] noted that the presence of melanin could be inhibitory to pullulanase. West and Reed-Hamer [10] suggested the same possibility. However, it is equally possible that these new isolates produce novel polysaccharides analogous to the EPS produced by the recently described variety aubasidani. Further studies are planned to resolve this question.

Fig. 6A–D.

Infrared (IR) spectra. A Pullulan standard (Sigma), B EPS from A. pullulans CU, C EPS from A. pullulans PR, D EPS from A. pullulans SU