Introduction

Filamentous fungi of the genus Aspergillus are widely used for the production of fermented foods, organic acids and enzymes. A. niger has a long history of industrial usage, which means many strains already have a GRAS (“generally regarded as safe”) status (Wongwicharn et al. 1999). The submerged cultivation of the fungi is based on the unsteady-state operation of reactors with changes of several parameters among which temperature, pressure, feed concentration, flow rate, etc. are maintained through externally forced methods to enhance the productivity. The high fermentation temperature is critical for the production of many biological-active compounds (Bai et al. 2003; Li et al. 2009). Long-term exposure to temperatures above the optimum can however exert stress effects, which are identical to oxidative stress events (Aggarwal et al. 2012).

Heat stress responses have been well documented in a wide range of organisms. Published data demonstrates denaturation of proteins resulting in weakening of polar bonds, unfolding, and exposure of hydrophobic groups. One major secondary consequence of heat stress is the production of the reactive oxygen species (ROS) and the induction of antioxidant defense. The protection against the oxidative stress damage in aerobic cells is performed by the enzymatic and non-enzymatic constituents of the antioxidant defense system that detoxify ROS and repair the damage they cause (Fridovich 1995). Our current understanding of the relationship between oxidative stress and temperature treatment in filamentous fungi is relatively limited and the data about industrially used fungi are scarce. The most significant contribution to clarifying this problem is by the group of Prof. McNeil (Kreiner et al. 2000; Bai et al. 2003; Li et al. 2009).

Our previous investigations showed that A. niger 26 is a good producer of superoxide dismutase (SOD). Short-term high temperature treatment of the spores and mycelia in mild-exponential growth phase markedly enhanced SOD activity (Abrashev et al. 2005, 2008).

Most industrial processes however involve longer-term exposures to high temperatures. This motivated the aim of the present study: to examine the effects of longer-term exposure to elevated temperature on cell structure, protein denaturation, and SOD and CAT activities in A. niger 26.

Materials and methods

Strain, media and cultivation conditions

The fungal strain A. niger 26 from the Mycological Collection at the Institute of Microbiology, Sofia, was used throughout and maintained at 4 °C on beer agar, pH 6.3. The composition of the culture medium AN-3, the preparation of the inoculum, and the cultivation in shake flasks and laboratory bioreactors were described earlier (Abrashev et al. 2008). The cultures were grown at 30, 35, 40, or 45 °C with a stirrer speed of 600 rpm air flow, 1 v.v.m. for up to 120 h.

For electron microscopy, the bioreactor cultures were grown 18 h at temperature 30 °C with a stirrer speed of 600 rpm air flow, 1.0 v.v.m. After that, the temperature was raised to 40 °C. This up-shift was reached approx. for 40 min and incubation continued for 24 h under heat stress conditions. The control variants were grown at optimal temperature during the whole period. Samples were taken at the beginning of the stress as well as at 6 and 24 h.

Morphological measurements

The morphological characteristics of A. niger 26 cells produced in cultures were examined by phase-contrast microscopy (Docuval, Carl Zeiss, Germany) and registered using a digital camera (Docuval, Carl Zeiss, Germany). Samples taken from late exponential growth phase were prepared for measurements as described by Papagianni et al. (1998). A magnification of 100× was applied for measurements on mycelial particles. The main hyphal length is the tip-to tip length of the main (longest) hyphal length in an ‘organism’ (Kreiner et al. 2003). The hyphal growth unit was calculated by dividing the total hyphal length for each organism by the number of tips, as defined by Thomas (1992). For each sample we calculated the mean results of hyphal parameters.

Electron microscopy and lectin-gold cytochemistry

Part of the samples were fixed in 2 % aqueous KMnO4 for 15 min and washed extensively in water. There followed a 2 h fixation in 1.5 % uranyl acetate, dehydration in graded ethanol series and propylene oxide, and embedding in Durcupan. For cytochemical labeling, the samples were fixed for 2 h in 2 % glutaraldehyde in 0.05 M Na cacodylate, pH 7.2, dehydrated and embedded in LR White resin (Sigma) according to the producer’s instructions. The ultrathin sections were prepared on Reichert-Jung ultramicrotome. To localize chitin in the cell wall we used the N-acetyl glucosamine-binding lectin, wheat germ agglutinin (WGA). A single-step post-embedding labeling protocol with WGA-10 nm gold conjugates (Sigma) was applied (Stoitsova et al. 2006). The sections from permanganate-fixed samples were counterstained with lead citrate. The sections from LR-White embedded materials were counterstained in 2 % uranyl acetate and lead citrate. The observations were made on Opton 10C electron microscope.

Cell-free extract preparation and biochemical assays

The cell-free extract was prepared as described earlier (Abrashev et al. 2005). SOD activity was measured by the nitro blue tetrazolium reduction method of Beauchamp and Fridovich (1971). CAT was assayed by the method of Beers and Sizer (1952). Protein was estimated by the procedure of Lowry et al. (1951) using crystalline bovine albumin as standard. Protein oxidative damage was measured spectrophotometrically as protein carbonyl content using DNPH binding assay as described earlier (Abrashev et al. 2008).

PAGE electrophoresis

The SOD isoenzyme profile was performed on polyacrylamide gels. Total protein (40 mg) was applied to 10 % nondenaturing PAGE and was stained for superoxide dismutase activity, as described by Beauchamp and Fridovich (1971).

Statistical evaluation of the results

The results obtained from at least three repeated experiments were evaluated using three or five parallel runs. The statistical significance of the differences between the different treatments of conidiospores was determined by the Student’s t test for MIE (mean interval estimation) and by one-away analysis of variance (ANOVA) followed by F test, with a significance level of 95 % (α = 0.05).

Results

Our unpublished results showed that the A. niger 26 is a mesophilic strain with maximum biomass accumulation at 30 °C. Temperatures above 30 °C slowed down growth and gradually reduced biomass production. At the same time the growth mode did not change. Four basic phases of growth were clearly distinguished at all temperatures tested: lag, exponential, stationary and decline phases. The duration of these phases depended on the growth temperature.

Elevated temperature effects on A. niger hyphal morphology

The mean main hyphal length, the branch length, and the values for the hyphal tips and hyphal growth units are shown in Fig. 1. The results confirmed that the most suitable temperature for growth is 28–30 °C at which the main hyphae and branches were the longest, and the highest number of growth units was observed. Experiments under the condition of increased temperature (35, 40 and 45 °C) revealed much shorter hyphal elements with a large number of branches (Fig. 1a–c). A t test between the values of the mean main hyphal length and mean branch length, obtained at temperature 35, 40 and 45 °C and in control cultures, showed that the hyphae were significantly shorter (P > 0.05) at temperature above optimum. The values for the hyphal growth unit decreased in temperature stressed cultures, with the lowest values at 45 °C (Fig. 1d).

Fig. 1
figure 1

Comparison of morphological indices between the control and temperature treated batch cultures of A. niger 26

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Ultrastructural changes of A. niger cells in response to heat exposure

To study the temperature-induced ultrastructural changes, mycelia from bioreactor cultures were submitted to a heat stress (40 °C) for 6 or 24 h and compared to the control cultures (30 °C). When grown at the optimum temperature, the samples were characterized by a predominance of thin hyphae (c.a. 9–20 μm in diameter) with moderate electron density (Fig. 2a). The nuclei were euchromatic, with single prominent nucleoli (Fig. 2b). Mitochondria had a predominantly condensed conformation, and the vacuoles varied in size dependent on the differentiation stage of the hypha. After growth at 40 °C, a variety of morphologies occurred. Together with hyphae that were like these observed at 30 °C, there were cells at various stages of degeneration. Some nuclei contained increased amounts of heterochromatin (Fig. 2c). Some otherwise unaltered hyphae contained mitochondria with abnormally dilated cristae (Fig. 2d). A frequent occurrence of autolytic hyphae with detachment of the degenerated cytoplasm from the cell wall was also registered (Fig. 2e).

Fig. 2
figure 2

Effects of temperature stress on A. niger 26 mycelium ultrastructure. Samples from control cultivated at 30 °C (a, b, f, g) or subjected to temperature stress, cultivation for 24 h at 40 °C (c, d, e, h, i). a Longitudinal section through two adjacent cells of a hypha. N, nucleus, m, mitochondria, V, vacuole. Bar 6 μm. b Transverse section at the level of the nucleus. The nucleus (N) is euchromatic with a prominent single nucleolus; m, mitochondria. Bar 3 μm. c A cell with heterochromatic nucleus (N) and a cytoplasm with uneven electron density. Bar 3 μm. d A hypha with otherwise unaltered ultrastructure containing mitochondria (m) with dilated cristae (asterisks). Bar 2 μm e Autolytic hypha with cytoplasm detached from the cell wall (cw) leaving an empty cavity (asterisk). Bar 5 μm. (f–i) Hyphal septation. f Ultrathin section through the centre of a normal septum (s) and the cell wall (cw). Black arrow points to the septal pore and white arrows—to Woronin bodies. Bar 2 μm. g The localisation of chitin as demonstrated by WGA-gold, sample from 30 °C. The septum (s) and the adjacent areas of the cell wall (cw) are intensively labelled with gold grains. Bar 2 μm. h Wavy septum (s), sample cultivated at 40 °C, sparce WGA-gold labelling. Bar 3 μm. i Intrahyphal hyphae with uneven septation; cw, cell walls. Bar 5 μm

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One important abnormality caused by the elevated temperature involved cell wall septation. In the 30 °C control specimens, the longitudinal fission between hyphal cells was accompanied by the formation of cell wall septa which proceeded from out to inwards (Fig. 2f). The young septa were intensively labelled with WGA-gold particles indicating the abundance of chitin (Fig. 2g). Abnormal wavy septation occurred in some hyphae grown at 40 °C. Such aberrant septa bound only insignificant amount of the chitin-labeling lectin, WGA (Fig. 2h) and may hypothetically be considered as a step to intrahyphal hyphae development. Intrahyphal hyphae were often observed. Figure 2i shows an example of such alteration in combination with an advanced state of cytoplasmic autolysis.

Total and oxidative damaged protein content

To determine whether the long-term exposure to temperature stress affects fungal development, we measured the total protein content in bioreactor cultures (Fig. 3a). When A. niger 26 cells were cultivated at optimal temperature (30 °C) the intracellular protein content increased up to 60 h of cultivation followed by a significant decrease that coincided with the late stationary phase. Long-term exposure to higher growth temperatures caused sharp reduction in total protein content (Fig. 3a).

Fig. 3
figure 3

Effect of long-term temperature treatment on content of intracellular protein (a) and damaged protein (b) in A. niger mycelia: (filled circle) 30 °C; (open circle) 35 °C; (filled triangle) 40 °C; (open triangle) 45 °C

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In addition to changes in protein synthesis, stress exposure also elicited distinct changes in protein structure. As a biomarker of stress effect, the level of carbonylated proteins at different temperatures was measured (Fig. 3b). Both the control culture (30 °C) and the variant grown at 35 °C had similar content of carbonyl groups. In contrast, the exposure to enhanced temperature (40 and 45 °C) resulted in significantly higher, sixfold resp. tenfold carbonylated protein level.

Levels of antioxidant enzyme defense in temperature tolerance of A. niger 26

The time courses of SOD activities are shown in Fig. 4a. Registered SOD activity consists of two different parts—true enzymatic (protein) SOD and non-protein SOD-like with unknown nature. In the control, the activities of all SOD forms (total, enzyme and SOD-like) remained stable, about 30–38 U/mg ml, during the whole cultivation period, as the proportion of the enzyme component is 65–68 %. A temperature increase of 5 °C (growth temperature 35 °C) increased SOD activity by about 30 % in parallel with the production of total protein content (Fig. 3a). This increase was mainly due to the higher level of the enzyme component. However, the activity of the enzyme was significantly induced by exposure to 40 °C, and was further enhanced at 45 °C. The SOD activity in the mycelium was more pronounced during the 18 to 60 h, which coincided with the exponential and the early stationary phase. The strain showed a maximal total activity two- or three-times higher at 40 and 45 °C, respectively, than that at optimum temperature. Higher total activity is on account of both enzyme and non-enzyme component, but with a predominance of the former.

Fig. 4
figure 4

Antioxidant enzyme activity of A. niger 26 cells cultivated under condition of temperature stress. a SOD activity (total, enzyme and SOD-like); b changes in Cu/Zn–SOD level from cell-free extracts of A. niger 26 cultivated at 30 (lane 1) and 40 °C (lane 2); significant increase in Cu/Zn–SOD is apparent after exposure to 40 °C; c CAT activity

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The isoenzyme profile of SOD was demonstrated by the native gel electrophoresis technique (Fig. 4b). The non-denaturing PAGE of the cell-free extract of 48-h culture grown at optimal temperature (30 °C) showed only one band with SOD activity, which was identified as Cu/Zn–SOD (inhibited by both KCN and H2O2, data not shown) (Fig. 4b, lane 1). The staining intensity of Cu/Zn–SOD isoenzyme increased significantly at higher growth temperature (Fig. 4b, lane 2).

The activity of CAT was also significantly affected by the enhanced temperature treatment. The enzyme activity observed for the cell-free extracts from cultures grown at temperatures above the optimum (35, 40, and 45 °C) were statistically significantly higher than that in the control variant, having mean maximal values of 12.1 ± 1.1, 18.0 ± 1.5, and 21.4 ± 1.09, respectively. Notable increase in CAT activity was established between 18 and 72 h of cultivation that coincided with exponential and early stationary growth phase.

Discussion

The present study demonstrated significant changes in the morphological characteristics of A. niger 26 when grown at temperatures above optimal. The microorganism decreased the size of its hyphal elements and increased the proportion of active cytoplasm in each active compartment (“active length”) by switching from mostly inactive long filaments with a few branches, to numerous active-cytoplasm branched forms (Wongwicharn et al. 1999). The observed alterations are in agreement with earlier-observed stress responses to a variety of factors including O2 enriched air and H2O2 by A. niger cultures and other filamentous fungi (Wongwicharn et al. 1999; Kreiner et al. 2000). However, the intensification of cytokinesis could often result in improper septation as illustrated presently by electron microscopy. At 40 °C we registered alterations that are usually ascribed to cell wall stress, i.e., wavy septation and the formation of intrahyphal hyphae. Notably, when gold conjugates of the chitin label, WGA, was applied, the wavy septa were insignificantly marked. This indicated suppressed chitin incorporation in such loci.

The proper cell division in fungi is a complicated process requiring a harmonized interaction between cell polarity related to the cytoskeleton (Harris et al. 2005) and the incorporation of chitin (Fukuda et al. 2009; Walker et al. 2013) and mannans or glucans (Takeshita et al. 2005). The importance of chitin incorporation is demonstrated by the presence of several classes of chitin synthases. Both the disruption of actin cytoskeleton (Boyce et al. 2003) and the mutations in chitin synthase genes (Martín-Urdíroz et al. 2008; Fukuda et al. 2009; Walker et al. 2013) caused variable degree of cell wall stress revealed as wavy septation and the formation of intrahyphal hyphae. Due to the variety of chitin synthase genes, some enzymes can possibly compensate for the absence of other enzymes, however this is a complicated process related to post-transcription mechanisms as well (Rogg et al. 2011). Some of the mutations in cell wall biosynthetic genes were demonstrated as altered morphology only at elevated temperatures (Takeshita et al. 2005).

A possible switch for the changes in hyphal morphology could be temperature-induced oxidative stress as suggested earlier (Bai et al. 2003). Exposure to elevated growth temperatures is known to accelerate the formation of ROS. Our previous investigations also showed the temperature upshift from 30 to 40, 50 and 60 °C caused a significant increase in the ROS level in the fungal strain A. niger 26 (Abrashev et al. 2008). Some recent reports show that under stress conditions ROS can influence apical tip growth, hyphal branching and cellular differentiation and development (Li et al. 2009). On the other hand, the accumulation of ROS may cause acceleration of autolysis (Emri et al. 2004). High temperature-generated ROS could thus explain the increase of autolytic hyphae shown presently.

Comparatively unaltered hyphae were also observed in the 40 °C-grown mycelium, but they were characterised by dilated mitochondria. This indicates that the mitochondria are one of the initial targets of the high-temperature stress, as has been found for the copper stress (Krumova et al. 2012) and the action of H2O2 (Qin et al. 2011).

The present results clearly show that the increase in growth temperature coincided with a markedly decreased intracellular protein content (Fig. 3a). Proteins are the main targets of the temperature-induced stress (Abrashev et al. 2008). Such a decrease in the intracellular protein content during oxidative stress appeared in parallel with a decrease in the adenosine-5′-triphosphate (ATP) pool, hence the available energy can limit important cellular activities such as de novo protein synthesis (O’Donnell et al. 2011). Our results show that the temperature-induced oxidative stress affects the level of carbonyl proteins (Fig. 3b). Oxidative modifications of proteins result in physical changes in protein structure, which causes inactivation and chemical fragmentation (see Pena et al. 2012). Carbonylated proteins are irreversibly damaged proteins. The majority of them escape degradation by forming aggregates. The accumulation of nondegradable carbonylated proteins in aggregated state probably contributes to the increase in the carbonyl content observed during temperature-induced oxidative stress.

Enhanced temperature also modulated the antioxidant enzyme response in the A. niger cells. The exposure at temperatures above the optimum growth temperature induced an increase in SOD and CAT activity, i.e., the enzymes of importance for temperature stress tolerance in mycetes (Kumar et al. 2011). The present results demonstrated that the increase in superoxide scavenging activity in the model strain A. niger 26 is on account of enzyme SOD component that is notably the major antioxidant in the stationary fungal cultures grown at 35, 40 and 45 °C. We have shown that heat shock induces increased activity of Cu/Zn-SOD. Similar results were reported for A. niger B1-D cultivated in the presence of either 25 % oxygen-enriched air or heat shock (Kreiner et al. 2000; Bai et al. 2003).

On the other hand, the enhanced rate of ·O2 – scavenging by SOD contributed to increase in H2O2 content and hence in CAT activity. Such thermal stress response has been reported about in different mycetes (Morsy et al. 2010; Kumar et al. 2011).

Conclusion

  1. 1.

    Two patterns can be distinguished in the cellular response of the model strain. The first one involves the temperature range of 30–35 °C, when the level of oxidative stress biomarkers does not change significantly. The activity of antioxidant defense is probably an adequate response to the level of generated ROS. The second pattern (40–45 °C) is characterised by a significant increase in the level of oxidative stress biomarkers and induction of SOD and CAT biosynthesis. The enhanced activity of the antioxidant enzyme defense was unable to cope with the enhanced ROS level. Thus, A. niger stress response includes various changes of the hyphal morphology: from increased proportion of active cytoplasm in each active compartment to formation of intrahyphal hyphae, altered mitochondria, and accelerated hyphal autolysis.

  2. 2.

    The model strain A. niger 26 cultivated at 30 or 35 °C can be used as a producer of Cu/Zn–SOD. Although an increase in SOD biosynthesis was identified at 40 or 45 °C, the decrease in biomass and intracellular protein content caused a significant reduction in enzyme yield.