Identification of Strain F119
The strain F119 can grow at 4 ℃-30 ℃, the optimal culture temperature is 28 ℃, and resistant to 15% NaCl. the initial colonies were all yeast-like, off-white on Czapek Dox Agar at 28 ℃. In the later period, the fungal mycelium was embedded in the culture medium. After 3 days of culture, the colonies turned dark or blackish-brown, and raised (Fig. 1). After 7 days of cultivation, the colony size was 0.2 cm-1.0 cm; the optimal initial growth pH was 6.0. Microscopic observation showed that the vegetative mycelium was transparent, smooth, thin-walled, and had a septum, which was converted into black mycelium in the middle and late stages of culture (Fig. 2). The conidia were transparent to dark brown and they were single cells, smooth, oval, 4.5–10 × 3.0–10 µm.
Comparisons with LSU rDNA D1/D2 domain sequences from GenBank database revealed that strain F119 (557 bp, GenBank accession number JN854147) shared 95.1-99.5% similarity with those of the species of the genus Aureobasidium, and the most closely related strains were A. melanogenum CBS 105.22T and A. melanogenum CBS 621.80T. Based on the analysis of phylogenetic trees, strain F119 merits recognition as a distinct genomic species of the genus Aureobasidium.
Metabolic profiling of A. melanogenum F119 in the Biolog FF Microplate
The metabolic abilities of this isolate were tested by using the Biolog FF Microplate which included 95 different carbon sources. The absorbance values at different times (0-144 h) were obtained to study the abundance index of 95 carbon sources metabolized by the strain. It can be seen from Fig. 4, the strain used the most types of carbon source at 72 h. The strain F119 was able to efficiently metabolize 46 carbon sources for growth of the fungus. It included 24 types of carbohydrates: D-arabitol, arbutin, D-cellobiose, D-arabinose, L-arabinose, i-erythritol, D-fructose, gentiobiose, α-D-glucose, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, β-methyl-D-glucose glycosides, palatinose, D-psicose, D-raffinose, L-rhamnose, D-ribose, stachyose, sucrose, D-trehalose, D-xylose; 6 types of amino acids: L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid; L-proline, L-pyroglutamic acid; 8 types of carboxylic acids: D-galacturonic acid, D-glucuronic acid, fumaric acid, y-hydroxybutyric acid, α-ketoglutaric acid, L-malic acid, quinic acid, succinic acid; 1 type of amines: succinamic acid; 3 types of polymers: Tween 80, dextrin, glycogen; 4 types of miscellaneous: salicin, glycerol, bromosuccinic acid, succinic acid mono-methyl ester (Fig. 5).
Metabolic profiling of A. melanogenum F119 with exposure to radiation
In order to explore the utilization capacity of carbon sources from the strain F119, AWCD value was used to measure it [14]. The collected data was analyzed and the average absorbance value was calculated. The results showed: in the control strain, the AWCD value increased with the increase of the cultivation time, tended to be stable at 96–168 h, and eventually reached the maximum at 144 h. The AWCD values of the strain showed the same trend under different radiation doses. Under 60Co γ-ray 2,500 Gy irradiation, the cell metabolic activity was close to the control group, which were respectively 0.2357 and 0.1628; when the radiation dose was greater than 5,000 Gy, the cell metabolic activity dropped rapidly to 0.0782, lower than 50% of the control group; when the radiation dose reached 10,000 Gy, the cell metabolic activity was 0.0162, only about 6% of the control group. It showed that under low dose radiation, the growth and metabolism of the strain were less affected. However, when the radiation dose was greater than 5,000 Gy, the cell growth rate and metabolic activity decreased significantly. When the radiation dose reached 10,000 Gy, the cell growth was almost stagnant and the metabolic activity was extremely low (Fig. 6).
Utilization of carbon sources of A. melanogenum F119 under different radiation doses
According to the change of AWCD value and the utilization of carbon sources, this study selected 72 h results to analyze the utilization percentage of six major carbon sources for each sample. It can be seen from Fig. 7 that obvious differences in the utilization percentage of the six major carbon source of strain. With the increase of radiation dose, the utilization percentage of carbohydrates showed an upward trend, while the utilization percentage of carboxylic acids and amino acids showed a downward trend. The utilization percentage of other types of carbon sources was basically the same. But under the radiation of 10,000 Gy, except for carbohydrates, no other carbon sources are used.
doses
Top 10 types of carbon sources that were utilized by the strain under different radiation doses were analyzed. The results were shown in Table 1. A total of 22 carbon sources were involved. Among them, there were 11 kinds of carbohydrates, which accounted for 50% of the total; 2 kinds of amino acids, for 9%; 4 kinds of carboxylic acids, for 18%, only 1 kind of polymer, for 5%, and other 4 kinds of carbon sources were miscellaneous, accounting for 18%.
Table 1
Utilization of top 10 carbon sources by strain under different radiation doses
Radiation Dose(Gy) |
0 Gy | 2500 Gy | 5000 Gy | 10000 Gy |
Carbon source | Absorbance (Ci –R) | Carbon source | Absorbance (Ci –R) | Carbon source | Absorbance (Ci –R) | Carbon source | Absorbance (Ci –R) |
L-Malic Acid | 0.716 | Arbutin | 0.608 | Arbutin | 0.562 | D-Ribose | 0.188 |
Fumaric Acid | 0.656 | Fumaric Acid | 0.555 | D-Xylose | 0.391 | Sucrose | 0.138 |
Succinic Acid | 0.609 | L-Proline | 0.537 | D-Raffinose | 0.242 | D-Arabinose | 0.126 |
L-Glutamic Acidine | 0.602 | L-Malic Acid | 0.497 | Palatinose | 0.239 | D-Trehalose | 0.126 |
L-Proline | 0.597 | Succinic Acid | 0.444 | D-Melezitose | 0.232 | Arbutin | 0.106 |
Arbutin | 0.576 | Salicin | 0.402 | Dextrin | 0.217 | D-Xylose | 0.105 |
Palatinose | 0.531 | D-Xylose | 0.389 | Bromosuccinic Acid | 0.215 | L-Arabinose | 0.101 |
Salicin | 0.481 | Dextrin | 0.37 | D-Fructose | 0.197 | | |
D-Glucuronic Acid | 0.476 | L-Glutamic Acid | 0.367 | D-Trehalose | 0.195 | | |
D-Xylose | 0.468 | D-Trehalose | 0.336 | Succinic Acid Mono-Methyl Ester | 0.192 | | |
Change of carbon source utilization by A. melanogenum F119 under different radiation doses
The differences among the 10 carbon sources most used by A. melanogenum under different radiation doses were compared. It was found that some carbon sources were used under all radiation doses, such as arbutin, D-xylose, D-trehalose. These carbon sources were critical for cellular growth and metabolism. With the increase of radiation intensity, some carbon sources were no longer used by strain in large quantities, such as fumaric acid, L-proline, salicin, L-malic acid, succinic acid, L-glutamic acid. When the radiation dose reached 5,000 Gy and above, the utilization of these carbon source dropped significantly. It was worth noting that D-ribose, sucrose, D-arabinose, and L-arabinose were not used in large quantities at low radiation doses. However, the utilization increased significantly under 10,000 Gy irradiation. This suggested that under high radiation, strain adapted to extreme conditions and began to use these carbon sources to maintain survival.
In addition, 95 carbon sources of 3 treatment groups and control group were counted. the utilization of 7 carbon sources increased significantly with radiation intensity increased, all of which were carbohydrates: D-arabinose, L-arabinose, D-ribose, sucrose, D-trehalose, D-xylose, arbutin. The utilization of these carbon sources increased significantly when the radiation intensity increased, which may indicate that the cells use these substances to participate in the maintenance of growth metabolism and stress response under extreme conditions.