versão On-line ISSN 0717-3458
Electron. J. Biotechnol. v.9 n.1 Valparaíso jan. 2006
Physiological, morphological, and mannanase production studies on Aspergillus
Francisco J. Fernández
Mutant strains from Aspergillus
Filamentous fungi are important in industrial enzyme production, since they are able to synthesize and secrete large amounts of extra cellular proteins. These organisms grow in liquid and solid-state cultures by hyphal extension and branching. The importance of morphological and physiological studies on fungi in liquid cultures has been recently reviewed (Papagianni, 2004). Fungal macro- and micro-morphology affect the rheology of the fermentation medium, thereby having a significant impact on the mixing, mass transfer and aeration processes within the bioreactor. In addition, micro-morphology may influence metabolite productivity, which may lead to lower net specific growth rate (McIntyre et al. 2001), or to enhanced enzyme production by strains with altered morphology (McCarthy et al. 2005).
The potential of enzyme production by fungi using solid-state fermentation (SSF) techniques has been discussed (Viniegra-González, 1998). The difference of conditions between solid-state and submerged cultures can lead to altered expression of several genes, which in turn may affect various phenotypes, such as growth, development, mycotoxin and enzyme production (Iwashita, 2002). The advantages of fungal enzyme production in solid-state over liquid fermentation systems have also been pointed out (Viniegra-González et al. 2003). However, few criteria about fungal physiology and morphology in solid-state cultures have been established in the limited studies available. For instance, Trinci (1973) working with Neurospora crassa in solid culture defined the hyphal growth unit (total hyphal length/number of hyphal tips) as a measurement of the fungal invasive capacity. In contrast, Ferret et al. (1999), used the colony radial extension rate obtained by linear regression of colony diameter versus time. There are few studies relating morphology and physiology of fungi in solid-state cultures.
On the other hand, strain improvement has been achieved through mutation, selection, or genetic recombination. In many cases, mutations are harmful, but occasionally may lead to a better adapted organism to its environment with improved biocatalytic performance. The potential of a microorganism to mutate is an important property conferred by DNA, since it creates new variations in the gene pool. The challenge is to isolate those strains which are true mutants that carry beneficial mutations (Parekh et al.2000). UV rays are important inducers of strain mutations. The pyrimidines (thymine and cytosine) are especially sensitive to modifications by UV rays absorption. This may result in the production of thymine dimers that distort the DNA helix and block future replications (Sambrook et al. 2000).
The objective of this study was to characterize physiologically and morphologically Aspergillus
To carry out strain propagation for the mutagenesis experiments, a porcelain pearl was placed in an Erlenmeyer flask containing 30 mL of potato dextrose agar (PDA) and incubated at
Mutagenesis of Aspergillus
Selection of the hyper producing mutants.The parental and mutant strains were inoculated in Petri dishes containing ML1 medium, covered with cellophane 400, and incubated at
Mannanase activity determination was conducted using the Congo Red method (Downie et al. 1994), with modifications, to obtain a better definition of the activity zones. Since the mycelia interfered with the activity zone definition, a circle of sweet cellophane 400 was placed onto the ML1 medium, covering the Petri dish completely. The inoculum was placed over the cellophane, and was incubated for 48 hrs at
For enzyme (mannanase, xylanase and cellulase) activity determination, 25 µL of known amount of substrate (locust bean gum, carboxymetyl cellulose or xylan, respectively) and 25 µL enzymatic crude extract, were mixed and incubated during 10 min at
RYAM tubes with 20 mL of ML 1 medium were inoculated by biting an end of the tube, and incubating for 7 days at
Petri dishes containing the ML 1 medium and a cellophane paper covering the surface, were inoculated by bite (Loera-Corral and Viniegra-González, 1998) from a spore suspension adjusted to 1 x 107 spores/mL. After incubation during 4 days at
Erlenmeyer flasks containing 50 mL of ML 1 medium were inoculated with A.
SSF experiments were carried out using copra paste as substrate and support medium. The substrate was sterilized in autoclave during 15 min at
The fermented material from each column was weighed and acetate buffer
Sample preparation for electron microscopy studies.The parental strain (A.
Covering with coal and gold. The fixed sample was evaporated under vacuum using a Baltec SCD050 equipment, followed by sample recovery with gold ions. Microscopic observations of samples were carried out in a Zellss DSM
From the raw data, a simple product moment correlation coefficient (Pearson's correlation) was calculated. The data were subject to analysis of variance and the sample means tested for significant differences using the multiple intervals test (
After UV radiation of a spore suspension containing 1 x 107 spores/mL, 50% lethality was reached after 3 min. This result agrees with that reported by Loera-Corral and Viniegra-González (1998), for Aspergillus
The cellophane paper used for the determination of fungal colony growth and enzymatic activity was chosen according to the growth characteristics and size of the colonies (Trinci, 1973). The relatively high tearing resistance and bursting strength of cellophane paper were also important considerations. For colony growth and size, the sweet cellophane types 300 and 400 allowed a better A.
The first selection of mannanase hyper producer mutants was conducted using a mutagenesis time of 3 min, with four replicates. From these experiments, 84 strains were obtained. The diameter of the hydrolysis zone was determined for each one in triplicate. The strains whose hydrolysis zone was larger than that of the parental strain, were selected for the next experiments. Five hyper producer strains were obtained after the first selection, which showed the biggest ratio colony diameter/hydrolysis zone, compared to the parental strain (Table 1).
The different standard enzyme concentrations used, made it possible to confirm the linearity of the relationship between enzymatic activity and area of the hydrolysis zone. The second selection was performed using this standard curve. The diameter of the hydrolyses zones produced by the enzymatic crude extracts from the SSF of the first selection mutants, were transformed to enzyme activity, using the above mentioned method. According to Table 2, the mutant strains showing more enzymatic activity were those labelled GS1-S059 and GS1-S067. These strains also showed the largest relationship between diameter of hydrolysis zone and colony diameter. These strains were used to perform our physiological and morphological studies.
Colony radial extension rate. It was not possible to appreciate differences among the results obtained for colony growth experiments of the tested strains. The colonies radial extension rates (Kr, mm d-1) were determined by plotting their length increment (mm) versus time (d). From the data of the wild type and the two mutants (GS1-S059 and GS1-S067), linear regression analysis was conducted to give the mean Kr values. Analysis of variance was used to test significant difference among treatments, while the
Spore production. In this experiment, samples were taken every 24 hrs. One notorious difference among the strains was an important delay in the sporulation time of strain GS1-S067, where spore production was observed until 144 hrs of incubation. Despite strain GS1-S067 showed a sporulation level 1.72 times smaller than strain UAM-GS1, there were small differences in the spore production levels among the tested strains (Table 4). Strain GS1-S059 showed the largest spore production. A delay in sporulation time could be beneficial, since longer fermentation times can result in increased enzyme production, leaving few spores in the fermented product, leading to its safe handling. However, this phenomenon might be due to either the mutation process, or the effect of
Enzymatic profile. The results of the enzymatic profile are shown in Table 5. Compared to the wild strain, mannanase activity increased 3.26 times for strain GS1-S059, and 2.85 times for strain GS1-S067. Cellulase production increased 3.70 times for strain GS1-S059, and 2.65 times for the mutant GS1-S067, as compared to the parental strain. The highest enzymatic activity increase of the mutant strains corresponded to xylanase, where strains GS1-S059 and GS1-S067 showed increases of 6.19 and 4.82 times that of the parental strain, respectively. Thus, mutant strains GS1-S059 and GS1-S067 significantly increased their levels of xylanase and cellulase production, improving in this way their potential industrial applications. These enzymes may be used in the bio-pulping processes (Ratto and Poutanen, 1988; Buchert et al. 1992), to increase digestibility of fodder and poultry feed (Wong and Saddler, 1993; Saki et al. 2005), fruit juice clarification and vegetable oil extraction (Sunna and Antranikian, 1997). Pearson's correlation analysis conducted on the enzymatic profile indicated a strong positive linear relationship between any two of the three enzyme activities (r≥0.939 with a 0.01 significance level, 2-tailed). According to these results, if the mannanase activity increased, enzyme activity levels for cellulase and xylanase also increased. We attributed this effect to possible changes in the promoter zones of the genes coding for these enzymes due to the ultraviolet exposure. This radiation might have deregulated the transcription of the mRNA corresponding to these enzymes, leading to an increased secretion production. Since ultraviolet radiation affects mainly the hydrogen bonds of pyrimidic bases (cytosine + thymine; C + T) the most vulnerable regulatory sequences must have been those containing the highest concentration of C + T. It can be hypothesized that mannanase, xylanase and cellulase production might be under the control of the same regulon. From an analysis of the promoters sequence of the genes coding for mannanase, cellulase and xylanase reported for several microorganisms (Table 6), it was observed that at least in 80% of those sequences the percentage of T-A links is predominant. This fact suggests that the promoter zone was strongly affected by the UV radiation and it might have affected the mechanism of hemicellulolytic and cellulolytic enzymes expression. The mutants produced here can therefore be used with advantage in processes were both hemicellulose and cellulose hydrolysis is required, such as in the degradation of municipal organic wastes.
The major system responsible for carbon repression in Aspergillus is mediated by the carbon catabolite repressor protein CreA (Ruijter and Visser, 1997). CreA is a zinc finger protein which binds to specific sites in the promoters (SYGGRG) of a wide range of target genes, including xylanolytic (Shroff et al. 1996). Thus, more experiments should be conducted on the growth of the mutant strains obtained here, using various carbon and nitrogen sources to associate potential creA gene mutations. This could give more information about the possible deregulation observed for the three enzymes tested. However, there is also the possibility that the transcriptional activator XlnR may regulate xylanolytic, endoglucanase, arabinanase, and cellulolytic gene expression, as tested by van Peij et al. (1998). These authors analyzed the promoter region of these genes obtaining a binding site consensus sequence of GGCTAA. In addition, the presence of CCAAT boxes in the promoter regions and the involvement of HAP (heme activator protein)-like complexes in the regulation of xylanases and cellulases in Trichoderma reesei suggests that these systems might also be regulated by HAP-like complexes in Aspergillus (de Vries and Visser, 2001).
Sporangium and spore diameter. The sporangium or conidia heads are characteristic to distinguish the different groups of Aspergillus. These heads are formed by conidiophores, vesicle, and a series of primary sterigma, followed by a second series of secondary sterigma of which the conidia or spores sprout. Structures for the parental strain and mutants are shown in Figure 1. All pictures were taken to the same magnification and working distance. An average of 20 measurements made in different fields were obtained. The mutant strains GS1-S059 and GS1-S067 (Figure 1b and 1c, respectively) showed the same structures at the sporangium level as the parental strain (Figure 1a). However, the mutant strains showed variations in the sporangium diameter in relation to the original strain (Table 7). From the
Hyphal length. Filamentous fungi are commonly used in SSF processes because of their capacity to invade the substrate. Therefore, it is required that the microorganisms show a high invasive capacity to grow at the surface of the substrate. The possession of long and ramified hypha increases the number of interaction places with the substrate, being an interesting characteristic for a strain of high enzyme production. The mycelium structure of the parental and mutants strains are shown in Figure 3, where the hypha and the sporangium (analyzed previously) can be observed. Results on the measurement of 20 independent hyphae diameters and length for the three strains are shown in Table 7. The hypha length and diameter of the mutant strains significantly changed (P < 0.05) compared to the parental strain, i.e., the length of the hyphae increased twice for strain GS1-S059. Data analysis showed that the highest hyphal polarity index (hyphal length/hyphal width) was observed for strain GS1-S059 and the lowest one was for the strain GS1-S067. Harris et al. (1999), observed that Aspergillus nidulans mutants decreased their hyphal polarity index in relation to the wild strain. Microscopic observations (data not shown) of copra paste fermented with the GS1-S059 mutant corroborate that this strain showed a massive invasive capacity toward the substrate probably due to its high hyphal polarity index. Hyphal ramification data were not included due to sampling problems, since the hyphae form very compact networks. The samples were taken directly from an agar plate, where intersecting colonial fragments of the strains were clearly observed using the electron microscope.
Relationship between physiological and morphological properties of A.
Müller et al. (2002) studied the Aspergillus oryzae morphology and α-amylase production during submerged cultivation in a wild-type strain (A1560) and in strains of in which chitin synthase B (chsB) and chitin synthesis myosin A (csmA) have been disrupted (ChsB/G and CM101). Despite hyphal tip extension rate of the mutant strains decreased and branching intensity did not show an expected pattern, α-amylase productivity was not significantly different in the three strains.
The relationship between physiology, morphology and enzyme production, if any, is poorly understood. Our results using A. niger mutants, obtained using UV rays, showed enhanced hemicellulolytic enzyme production and a good linear relationship between enzyme production (mannanase, cellulase and xylanase) and morphology (hyphal length and sporangium diameter).
Authors thank Dr. José D. Sepúlveda-Sánchez (Electron Microscopy Laboratory, Building "W" Environmental Science and Technology. UAM-I CENICA) for valuable help in the electron microscopy studies.
BUCHERT, J.; KANTELINEN, A.; RATTO, M.; SIIKA-AHO, M.; RANUA, M. and VIIKARI, L. Xylanases and mannanases in the treatment of pulp. In: KUWAHARA M. and SHIMADA M. eds. The 5th International Conference on Biotechnology in the Pulp and Paper Industry. Uni Publishers,
DE VRIES, R.P. and VISSER, J. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiology and Molecular Biology Reviews, December 2001, vol. 65, no. 4, p. 497-522. [ [ Links ]CrossRef]
DOWNIE, Bruce; HILHORST, Henk W.M. and BEWLEY, J. Derek. A new assay for quantifying endo-b-mannanase activity using Congo red dye. Phytochemistry, July 1994, vol. 36, no. 4, p. 829-835. [ [ Links ]CrossRef]
FERRET, E.; SIMÉON, J.H.; MOLIN, P.; JORQUERA, H.; ACUÑA, G. and GIRAL R. Macroscopic growth of filamentous fungi on solid substrate explained by a microscopic approach. Biotechnology and Bioengineering, 1999, vol.65, no. 5, p. 512-522. [ [ Links ]CrossRef]
FUJIWARA, M.; ICHINOMIYA, M.; MOTOYAMA, T.; HORIUCHI, H.; OHTA, A. and TAKAGI, M. Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development. Journal of Biochemistry, 2000, vol. 127, no. 3, p. 359-366. [ Links ]
HARRIS, Steven D.; HOFMANN, Amy F.; TEDFORD, Hugo W. and LEE, Maurice P. Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans. Genetics, March 1999, vol.151, no. 3, p. 1015-1025. [ Links ]
LOERA-CORRAL, Octavio and VINIEGRA-GONZÁLEZ, Gustavo. Identification of growth phenotypes in Aspergillus
MCCARTHY, Tracey C.; LALOR, Eoin; HANNIFFY, Orla; SAVAGE, Angela V. and TUOHY, Maria G. Comparison of wild-type and UV-mutant β-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications. Journal of Industrial Microbiology and Biotechnology, April 2005, vol. 32, no. 4, p. 125-134. [ [ Links ]CrossRef]
MONTIEL-GONZÁLEZ, Alba M.; VINIEGRA-GONZÁLEZ, Gustavo; FERNÁNDEZ, Francisco J. and LOERA, Octavio. Effect of water activity on invertase production in solid state fermentation by improved diploid strains of Aspergillus
MÜLLER, Christian; MCINTYRE, Mhairi; HANSEN, Kim and NIELSEN, Jens. Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis. Applied and Environmental Microbiology, April 2002, vol. 68, no. 4, p. 1827-1836. [ [ Links ]CrossRef]
NELSON, Norton Photometric adaptation of the Somogyi method for the determination of glucose. The Journal of Biological Chemistry, 1944, vol. 153, no. 2, p. 375-380. [ Links ]
PAREKH, S.; VINCI, V.A. and STROBEL, R.J. Improvement of microbial strains and fermentation processes. Applied Microbiology and Biotechnology, September 2000, vol.54, no. 3, p. 287-301. [ Links ]
PITT, John I. and HOCKING, Ailsa D. Fungi and Food Spoilage. 2nd ed. Blackie Academic and Professional,
RAIMBAULT, M. and ALAZARD, D. Culture method to study fungal growth in solid state fermentation. European Journal of Applied Microbiology and Biotechnology, 1980, vol. 9, no. 3, p. 199-209. [ [ Links ]CrossRef]
RATTO, M. and POUTANEN, K. Production of mannan-degrading enzymes. Biotechnology Letters, 1988, vol. 10, no. 9, p. 661-664. [ Links ]
REGALADO, Carlos; GARCIA-ALMENDÁREZ, Blanca E.; VENEGAS-BARRERA, Luz M.; TÉLLEZ-JURADO, Alejandro; RODRIGUEZ-SERRANO, Gabriela; HUERTA-OCHOA, Sergio and WITAKER, John R. Production, partial purification and properties of endo-b-mannanases obtained by solid substrate fermentation of spend soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger. Journal of the Science of Food and Agriculture, June 2000, vol. 80, no. 9, p. 1343-1350. [ [ Links ]CrossRef]
SAKI, A.A.; MAZUGI, M.T. and KAMYAB, A. Effect of mannanase on broiler performance, lleal and in vitro protein digestibility, uric acid and litter moisture in broiler feeding. International Journal of Poultry Science, 2005, vol. 4, no. 1, p. 21-26. [ Links ]
SAMBROOK, J.; FRITSCH, E.F. and MANIATIS, T. Molecular cloning, a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory,
SHROFF, R.A.; LOCKINGTON, R.A. and KELLY, J.M. Analysis of mutations in the creA gene involved in carbon catabolite depression in Aspergillus nidulans. Canadian Journal of Microbiology, September 1996, vol. 42, no. 9, p. 950-959. [ Links ]
SUNNA, A. and ANTRANIKIAN, G. Xylanolytic enzymes from fungi and bacteria. Critical Review in Biotechnology, 1997, vol. 17, no. 1, p. 39-67. [ Links ]
SUZUKI, Takahito; MIYAMAE, Yuri and ISHIDA, Ikuko. Variation of colony morphology and chromosomal rearrangement in Candida tropicalis pk 233. Journal of General Microbiology, January 1991, vol. 137, no. 1, p. 161-167. [ Links ]
TRINCI, A.P.J. Influence of the peripheral growth zone on the radial growth rate of the fungal colonies. Journal of General Microbiology, 1971, vol.67, p. 325-344. [ Links ]
VAN PEIJ, N.N.M.E.; GIELKENS, M.M.C.; DE VRIES, R.P.; VISSER, J. and DE GRAAFF, L.H. The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus
VINIEGRA-GONZÁLEZ, Gustavo. Solid state fermentation: Definition, characteristics, limitations and monitoring. In: ROUSSOS, S; LOSANE, B.K; RAIMBAULT, M. and VINIEGRA-GONZÁLEZ, G. eds. Advances in
VINIEGRA-GONZÁLEZ, Gustavo. Strategies for the selection of mold strains geared to produce enzymes on solid substrates. In: GALINDO, E. and RAMÍREZ O.T. eds. Advances in Bioprocess Engineering II. Kluwer Academic Publishers, 1998, p. 123-136. ISBN 0792349237. [ Links ]
VINIEGRA-GONZÁLEZ, Gustavo; FAVELA-TORRES, Ernesto; AGUILAR, Cristóbal N.; ROMERO-GÓMEZ, Sergio J.; DÍAZ-GODÍNEZ, Gerardo and AGUR, Christopher. Advantages of fungal enzyme production in solid-state over liquid fermentation systems. Biochemical Engineering Journal, 2003, vol.13, no. 2-3, p. 157-167. [ [ Links ]CrossRef]
WONG, K.K.Y. and SADDLER, J.N. Application of hemicellulases in the food, feed and pulp and paper industries. In: COUGHLAN M.P. and HAZLEWOOD, G.P. eds. Hemicellulose and Hemicellulases. Portland Press,
Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.