Note
Effect of Light on the Growth and Acid Protease Production of Aspergillus oryzae
2015 Volume 21 Issue 4 Pages 631-635
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2015 Volume 21 Issue 4 Pages 631-635
Aspergillus oryzae is known to have remarkable potential for the production of secreted enzymes. Submerged (Smf) and solid-state fermentation (SSF) are optimized with regard to temperature, pH, moisture, fermentation time, inoculum concentration, etc. Besides these parameters, the effect of light on growth and its role as a bioprocess variable in controlling enzyme production in A. oryzae is pivotal and hence the subject of study. A. oryzae was shown to be susceptible to light; growth was affected and the vegetative phase of the fungus was retarded. Biochemical alterations in biomass, growth diameter, chitin, and conidiation were also observed. The acid protease production by Smf and SSF showed improvement when incubated under darkness. Our study demonstrates for the first time that light must be considered as a bioprocess variable, as certain strains of A. oryzae are sensitive to light. Moreover, total darkness could be effectively employed in protease production on a pilot scale.
Among the filamentous fungi, Aspergillus oryzae is known to have high potential for the production of various secreted enzymes. In addition, developments in genetic engineering technology have enabled the application of A. oryzae to the production of industrial enzymes in modern biotechnology (Machida et al., 2008). One such application relates to fermentation, which is an inherently faster and more beneficial process compared to other chemical processes. The optimization of bioprocess variables is one of the most important fields of bioprocess engineering. This optimization focuses on the production of bio-industrial operations and is therefore essential for the fermentation industry. Fermentation optimization typically focuses on parameters such as moisture, temperature, fermentation time, inoculum concentration, carbon and nitrogen supplements, etc. (Murthy and Naidu, 2010; Murthy and Kusumoto, 2014). To date, most fermentation studies have paid little attention to the influence of light as a growth parameter and its effect on metabolite production. Fermentation processes were typically performed under darkness or according to the clock cycle (12h light:12h dark). For virtually all organisms, light, temperature, moisture etc., are crucial environmental signals in the regulation of developmental and physiological processes. Consequently, the capacity to sense and respond to light is extensively found, from archaea to fungi to humans (Babitha et al., 2008). Diverse filamentous fungi live in the soil, which is characterized by dark and stable conditions. When the fungi reach the soil surface they are exposed to UV radiation, desiccation or significant temperature changes. Therefore, most fungi use light as an environmental signal to adjust themselves to new habitats. In the course of adaptation to novel environmental conditions, fungal gene expression and metabolic pathways are drastically altered. The development of reproductive structures in a light-dependent manner has been reported in Aspergillus nidulans; sexual development was promoted under darkness while asexual sporulation was stimulated under illumination (Julian et al., 2013). Light signaling molecules are conserved in A. oryzae and can respond to light. In this paper, we studied the effect of light on growth, development and enzyme production in A. oryzae.
Materials Spray-dried potato pulp powder (PPP) produced during 2007—2008 was obtained from the Starch Factory of the Kamikawa North Agricultural Co-operative (Kamikawa Hokubu Noukyou Gourika Denpun Koujou, Hokkaido, Japan). Polypeptone (P) (Wako, Osaka, Japan), yeast extract (Y) (Difco BD Japan, Tokyo, Japan), potato dextrose agar (PDA) (Difco BD Japan)and Czapek Dox media (CD) (0.6% NaNO3, 0.1% KH2PO4, 0.05% KCl, 2 mM MgSO4, 1% glucose, and a 0.1% trace element solution consisting of 0.1% FeSO4·7H2O, 0.88% ZnSO4·7H2O, 0.04% CuSO4·5H2O, 0.01% Na2B4O7·10H2O, and 0.005% (NH4)6Mo7O24·4H2O) were obtained from Nacalai Tesque (Kyoto, Japan). All chemicals used were of analytical grade.
Effect of light on the growth of A. oryzae (F6) A. oryzae F6 was originally isolated from an improved protease-producing strain generated by UV mutagenesis of strain NFRI1599, and was maintained at the National Food Research Institute, NARO, Japan. A. oryzae was grown on PDA slants in an incubator for five days at 30°C and stored at 4°C. Spore suspensions were prepared using a sterile water solution containing 0.002% (v/v) Tween 80 and 0.5% (w/v) NaCl. A 1 mL aliquot of viable spores (106 spores) was used as the inoculum.
Conidial production was examined using point inoculation or spread plate methods under both light and dark conditions. White fluorescent light was used for light illumination experiments. The photon flux density at the surface of the culture was approximately 25 µmol m−2 s−1. With respect to point inoculation, 1 µL of 106 conidia/ mL was deposited in the middle of the plate. The spread plating method was conducted by inoculating 105 spores on CD plates and incubating under light and dark conditions at 30°C for 5 days. The linear growth of the fungal mycelium was measured daily from the area of inoculation along four diameters, and the mean of these values was recorded. A mycelial disk 5 mm in diameter was taken from the center of the plate and homogenized with 1 mL of water containing Tween 80 and NaCl. The conidia were counted using a hemocytometer and a light microscope.
Submerged fermentation (Smf) and Solid-State Fermentation (SSF) The effect of light on A. oryzae F6 growth and fermentation for the production of acid protease was determined. CD media was used for Smf, and PPPYP media (1 g PPP, 1 g Y and 1 g P) was used for SSF (3). The cooled sterilized CD (100 mL) and PPPYP solid substrate (100 g) of 60% moisture content were individually placed in 250-mL conical flasks, inoculated with 10% inoculum containing 2.6 × 106 conidia/mL, mixed uniformly and incubated at 30°C under darkness or continuous white light for 120 h without shaking. Light illumination experiments employed a photon flux density at the surface of the culture of approximately 25 µmol m−2 s−1 (Murthy and Kusumoto, 2014). In the case of Smf, the mycelial mat was filtered using Whatman filter paper No 1. Biomass determinations were made by heating at 105°C to a constant weight and the supernatants were assayed for acid protease. The protease in SSF was extracted by suspending the fermented substrate in distilled water (1:5) using an orbital shaker at 30°C and 150 rpm for 60 min. The extracts were filtered through Whatman No. 1 filter paper, centrifuged at 5,000 × g at room temperature for 10 min, and the obtained supernatant was then subjected to the acid protease assay.
Acid protease assay Acid protease activity was determined as described by Rowan and Buttle (1994) with modifications. The reaction mixture containing 0.4 mL of 0.2% azocasein (dissolved in 20 mM NaPO4 buffer, pH 5.7) and 0.5 mL of the extracted extracellular enzyme was heated at 55°C for 60 min. The mixture was cooled on ice for 5 min, and then 0.5 mL of 10% trichloroacetic acid (TCA) was added, followed by incubation on ice for 20 min and centrifugation at 10,000 × g for 15 min. The control sample contained the same reaction mixture, but the enzyme was added after the TCA. The supernatants were transferred to fresh tubes, and absorbance at 440 nm was measured using a spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan). One unit of protease activity was defined as the amount of enzyme needed to produce an increase of one absorbance unit per 30 min at 440 nm under defined reaction conditions.
Chitin measurement The amount of chitin in the cell wall was determined by measuring the amount of N-acetyl glucosamine (GlcNAc) liberated from the cell wall after digestion with a cell wall degrading enzyme. GlcNAc determinations were conducted as previously described by Suzuki et al. (2010). Briefly, 2 g of mycelia from Smf was suspended in 10 mL of 50 mM phosphate buffer (pH 7.0). The mycelia were washed three times with 10 mL of 50 mM phosphate buffer (pH 7.0) and then re-suspended in 10 mL of the same buffer. To liberate GlcNAc from the fungal cell wall, 10 mg of yatalase (Takara Bio, Otsu, Japan) was added to the sample suspension and incubated at 37°C for 1 h. The amount of GlcNAc in 500 µL of sample supernatant was determined according to the procedure of Reissig et al. (1955).
Statistical analysis All analyses were conducted in triplicate. Results are presented as the mean ± standard deviation.
Rhythmic growth and sporulation are principally heterogeneous in fungal populations. Exposure of growing cultures to white light and darkness caused the subsequent formation of a conidia-bearing mycelial ring in A. oryzae F6 (Fig. 1). This indicated that A. oryzae is light sensitive and could also indicate growth and metabolism. A. oryzae showed enhanced growth when incubated under darkness compared to under light. The growth diameter when incubated under darkness was 32 mm compared to 28 mm with illumination. There was 12.5% increase in growth diameter due to the effect of dark incubation (Table 1). Secondly, A. oryzae incubated under darkness had 19.36 × 109 conidial counts per dish while that for light incubation had 14.50 × 109 conidia per dish. Our findings are supported by the report of Hatakeyama et al. (2007), which showed that light exerted repressive effects on conidial production in A. oryzae. In many fungi, conidiation requires a light pulse to be initiated. The processes relevant for conidiation and its regulation are diverse. Several stages lead to the initiation of conidiation and the mechanisms triggering enzyme production (Tisch and Schmoll, 2010). Biomass production on the minimal media showed a similar trend; a biomass production of 22.5 mg was observed when grown under darkness compared to 20 mg when grown under light, an 11% increase in growth. Light frequently induces or represses sporulation in filamentous fungi, and light can have diverse effects on growth, metabolism, and the balance between asexual and sexual development (Atoui et al., 2010).
Sporulation and growth of A. oryzae F6 incubated under (a) 12 h light : 12 h dark culture producing a conidia-bearing ring-like structure, (b) point inoculation culture under light (L) and dark (D) conditions, and (c) spread-plating culture under (L) and (D) conditions.
| Incubation | ||
|---|---|---|
| Parameters | Light | Dark |
| Biomass (mg) | 20.2 ± 0.41 | 22.0 ± 0.11 |
| Conidia (109/dish) | 14.50 ± 0.21 | 19.36 ± 0.32 |
| Growth diameter (mm) | 28.0 ± 0.20 | 32.0 ± 0.15 |
| Chitin (mg/mL) | 0.027 ± 0.05 | 0.034 ± 0.05 |
The various patterns of growth recorded for A. oryzae indicates that it is sensitive to light and growth is retarded. With respect to physiological effects, the transition from the vegetative state (favored inside the substrate, i.e., under darkness) to the reproductive state (which mostly occurs on the surface of the substrate, i.e., under illumination) is likely to require drastic alterations to the metabolism (Tisch and Schmoll, 2010). Upon illumination of the fungal mycelia, the cell wall is the first site of impact of the photons. The results showed that the amount of chitin in fungi cultured under darkness was 0.034 mg/mL compared to 0.027 mg/mL under illumination. Similar findings from Farkas et al. in Trichoderma reesei indicate a decrease in endogenous reserves, as indicated by the lower glycogen content with light cultivation (Farkas et al., 1990). Olmedo (2001) established the presence of mad genes in Phycomyces blakesleeanus, which show an altered response to light, and growth regulation of the cell wall in the growing zone, due to the interplay of chitinases and new chitin synthesis.
Studies of the environmental factors that affect the growth and metabolism of filamentous fungi are necessary because they contribute to the control of cellular metabolism and the optimization of certain biosynthetic products (Velmurugan et al., 2010). The investigation of A. oryzae resulted in interesting observations in both Smf and SSF with the effect of light illumination. The Smf and SSF carried out under darkness produced more acid protease compared to under light. Light repressed sporulation and enzyme production (Table 2). This indicates that reproduction in A. oryzae is greatly influenced by light illumination, resulting in decreased enzyme production. Incubation under darkness produced a 2-fold greater enzyme level compared to under light. This could be explained by the existence of fungal photoreceptors responsive to dark and light conditions. This observation suggested that incubation under total darkness is the most effective in inducing biomass production and extracellular enzyme production. Elucidation of the precise molecular mechanisms regulating enzyme production by light response, including the differences between Smf and SSF, is currently in progress.
| Incubation | ||
|---|---|---|
| Light | Dark | |
| Smf (U/mL) | 77 ± 0.75 | 172 ± 0.57 |
| SSF (U/g) | 302 ± 5.01 | 365 ± 4.28 |
The capacity to secrete large amounts of proteins (and other metabolites such as organic acids) in combination with established fermentation technology and molecular biology make Aspergilli such as A. niger, A. oryzae, A. awamori, A. sojae, and A. terreus attractive industrial microorganisms for the production of homologous and heterologous proteins (Meyer et al., 2011). The optimization of fermentation has typically focused on parameters such as moisture, temperature, fermentation time, inoculum concentration, carbon and nitrogen supplements, etc. (Murthy and Naidu, 2010; Murthy and Kusumoto, 2014). A. oryzae is generally regarded as safe GRAS) status organism and its application to enzyme production must take into consideration its response to light, since growth is typically directly related to enzyme production. As bioprocesses are optimized for various parameters, light as an environmental parameter must also be taken into consideration. Our results clearly show the interesting observations that fungi respond to light and initiate considerable adaptations in their metabolic pathways during growth. Alterations in response to light have been noted with respect to carotenoid metabolism, polysaccharide and carbohydrate metabolism, fatty acid metabolism, nucleotide and nucleoside metabolism, and in the regulation of secondary metabolite production (Tisch and Schmoll, 2010). Notably, considering the differences in favorable conditions for sexual and asexual development in various fungi, regulation of these changes may differ accordingly.
This study is the first attempt to evaluate light as a bioprocess variable, and illustrates the physiological consequences of light on extracellular enzyme and biomass production in A. oryzae F6. Furthermore, the effects of light illumination on filamentous fungi and fermentation could be used to efficiently manipulate enzyme production. Hence, functional identification and investigation of fungi insensitive to light is anticipated to have positive biotechnological implications.
Acknowledgements This work was partly supported by M/s Kirin Holdings Co., Ltd., Tokyo, during the UNU-Kirin Fellowship program at the National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Japan.
