Abstract
The influence of inoculum preparation on Aspergillus awamori morphology, broth rheology and glucoamylase synthesis in submerged cultures was investigated. A series of runs were performed in fermenters, using initial total reducing sugar concentrations of 20 g/L and 80 g/L. The inocula were prepared in a rotary shaker, at 35oC and 200 rev/min, using a spore concentration of 9.2 x 105 spores/mL and varying both cultivation time and medium pH during the spore germination step. Three types of inocula were used: inoculum cultivated for 24 hours at an initial pH of 5.0, and inocula cultivated for 7 hours at both a pH of 2.5 and a pH of 5.5. Regarding glucoamylase production, the inoculum which provided the best results was shaker cultivated for 7 hours at a pH of 2.5. This inoculum produced glucoamylase of about 1,221 U/L in the fermenter, which was between 20% and 30% higher than those obtained using other inocula.
Aspergillus; glucoamylase; inoculum
RELATIONSHIP BETWEEN MORPHOLOGY, RHEOLOGY AND GLUCOAMYLASE PRODUCTION BY Aspergillus awamori IN SUBMERGED CULTURES
C.R.D. Pamboukian, M.C.R. Facciotti and W. Schmidell
Escola Politécnica da Universidade de São Paulo - Departamento de Engenharia Química
Caixa Postal 61548 CEP 05424-970 - São Paulo - SP - Fax (011) 211-3020
(Received: March 5, 1998; Accepted: May 18, 1998)
Abstract - The influence of inoculum preparation on Aspergillus awamori morphology, broth rheology and glucoamylase synthesis in submerged cultures was investigated.
A series of runs were performed in fermenters, using initial total reducing sugar concentrations of 20 g/L and 80 g/L. The inocula were prepared in a rotary shaker, at 35oC and 200 rev/min, using a spore concentration of 9.2 x 105 spores/mL and varying both cultivation time and medium pH during the spore germination step. Three types of inocula were used: inoculum cultivated for 24 hours at an initial pH of 5.0, and inocula cultivated for 7 hours at both a pH of 2.5 and a pH of 5.5. Regarding glucoamylase production, the inoculum which provided the best results was shaker cultivated for 7 hours at a pH of 2.5. This inoculum produced glucoamylase of about 1,221 U/L in the fermenter, which was between 20% and 30% higher than those obtained using other inocula.
Keywords: Aspergillus, glucoamylase, inoculum.
INTRODUCTION
Filamentous fungi in submerged cultures may grow either as free mycelium dispersed throughout the culture medium (filamentous form) or as pellets, which are spherical agglomerates of hyphaes. In the filamentous form, hyphal entanglement can cause the suspension to be highly viscous and pseudoplastic. Growth in the form of pellets causes lower broth viscosity, which enhances mixing and mass transfer properties of the suspension, but creates other problems such as nutrient limitations in the pellet interior (Pedersen et al., 1993). Factors which affect growth morphology are strain characteristics, inoculum size, medium composition, pH and shear forces (Whitaker and Long, 1973).
Fungal fermentations generally require different growth morphologies for optimum product yield. As reported in recent papers, the filamentous form is recommended for glucoamylase production (Ruohang and Webb, 1995; Pamboukian, 1997).
In Aspergillus awamori submerged cultures, pellets are formed by spore aggregation in the early stages of germination (Carlsen et al., 1996). As reported by Wainwright et al. (1993), culture medium pH plays an important role in the process of spore aggregation. When pH was maintained between 5.0 and 7.0 during germination, spore agglomerates were formed and growth occurred mainly in pellet form. Utilization of a lower pH (between 2.0 and 3.0) prevented spore aggregation and pellet formation, leading to dispersed filamentous growth.
Aspergillus awamori cultivations for the purpose of producing glucoamylase are usually carried out in aerated fermenters, using a mycelium suspension, obtained by precultivation in a rotary shaker, as the inoculum. Depending on conditions of the culture, this shaker cultivation can lead to the formation of a pellet suspension, with almost no dispersed filamentous growth in the broth (Pamboukian, 1997).
This paper focuses on the relationship between inoculum preparation, Aspergillus morphology, broth rheology and glucoamylase production in batch fermentations.
MATERIALS AND METHODS
Strain
Spores of Aspergillus awamori NRRL 3112 stored in test tubes containing Czapek agar medium (Raper and Fennell, 1965) were used. A spore suspension was prepared in a solution of Tween-40 (Facciotti et al., 1990), and the spore concentration in this suspension was measured using a Neubauer counting chamber (Dacie and Lewis, 1968). This spore suspension was used to inoculate the shaken flasks.
Culture Media
The medium for inoculum preparation (shaker cultivation) contained cassava flour syrup as the main carbon source (Facciotti et al., 1990). The initial total reducing sugar concentration (TRS0) was 20 g/L. The medium was complemented by the following nutrients: (NH4)2SO4 (5.0 g/L); Na2HPO4.12H2O (3.78 g/L); KH2PO4 (3.50 g/L); MgSO4.7H2O (0.50 g/L) and yeast extract (0.1 g/L). The pH of the medium was adjusted according to Table 1. Fermenter cultivations were carried out, using initial total reducing sugar concentrations (TRS0) of 20 g/L (group E20) and 80 g/L (group E80). In group E20, the culture medium had the same composition as that of the shaker medium. In group E80, the components of the culture medium were increased proportionally to the TRS0.
Inoculum Preparation
Flasks containing 200 mL of the culture medium were inoculated with the spore suspension, using a spore concentration (Cspore) of 9.2 x 105 spores/mL (Pamboukian, 1997). The flasks were incubated in a rotary shaker at 35oC and 200 rev/min. Cultivation period and inoculum pH are shown in Table 1. After this cultivation period, the fermenter containing 9 L of culture medium was inoculated with 1 L of the cell suspension.
Culture Conditions
The batch runs were performed in 15-L fermenters, under the following conditions: working volume = 10 L; agitation rate = 700 rev/min; air flow rate = 10 L/min; internal pressure = 1.2 atm; pH = 4.0 and temperature = 35oC.
Analytical Techniques
Samples collected periodically from the fermenter were evaluated for dry cell mass (X) (Facciotti et al., 1990), total reducing sugar (TRS) (Schmidell and Fernandes, 1977), glucoamylase activity (A) (Schmidell and Menezes, 1986) and pH. One glucoamylase activity unit (U) was defined as the quantity of enzyme that releases 1 g of glucose per hour from a 4% (w/v) soluble starch solution at a pH of 4.2 and 60oC. Rheological properties (consistency index (K) and flow behavior index (n) from the "Power Law") were evaluated by off-line measurement, using a Brookfield LVDV-III viscosimeter (Queiroz et al., 1997). Microorganism morphology was observed using an optical microscope (Pamboukian, 1997).
Table 2: Inoculum morphology and Aspergillus morphology in fermenter
RESULTS AND DISCUSSION
The cell suspensions used as inoculum for the fermenter, obtained by shaker cultivation, showed different morphologies, as indicated in Table 2.
In runs E20-1 and E80-1, the inocula were cultivated in the shaker for 24 hours so that the resulting cell suspensions would be in the exponential growth phase. These inocula grew mainly in pellet form. In runs E20-2 and E80-2, the inocula were cultivated in the shaker for 7 hours (the period of time necessary for the beginning of spore germination), at a pH of 2.5 so that spore agglomeration could be avoided. These inocula contained dispersed spores at the beginning of germination process. In runs E20-3 and E80-3, the inocula were cultivated in the shaker for 7 hours, at a higher pH (5.5), which caused spore agglomeration.
These distinct inoculum morphologies led to distinct growth morphologies in the process carried out in the fermenter, as indicated in Table 2. In runs from group E20, the presence of dispersed spores in the inoculum led to a filamentous growth in the fermenter (run E20-2), whereas the presence of either spore agglomerates or pellets in the inoculum led to pellet formation (runs E20-3 and E20-1). These facts can also be observed in Figures 1 and 2, which indicate broth rheological properties (consistency index, K, and flow behavior index, n), in runs E20-1, E20-2 and E20-3. The flow behavior index (n) was near 1.0 at the beginning of fermentation, indicating a Newtonian behavior at the beginning of cultivation. As biomass increased, the broth became pseudoplastic, with a sharp decrease in n and a simultaneous increase in K, as previously observed in other works (Pedersen et al., 1993; Queiroz et al., 1997). The maximum consistency index (K) in run E20-2 was about 2.00 Pa.sn, significantly higher than the maximum values in runs E20-1 (1.20 Pa.sn) and E30-3 (0.75 Pa.sn).
In runs from group E80, growth occurred primarily in the filamentous form, in spite of the distinct inocula morphologies. Nevertheless, the presence of pellets at the beginning of fermenter cultivations was observed in runs E80-1 and E80-3. Figures 3 and 4 present the broth rheological properties obtained in these runs. The decrease in n was sharper in runs from group E80 than in runs from group E20. Regarding the maximum consistency index (K), the values reached were about 7.00 Pa.sn (E80-1), 7.20 Pa.sn (E80-2) and 6.00 Pa.sn (E80-3).
The distinct morphologies affected both growth kinetics and glucoamylase production. Figures 5 and 6 show the profiles of biomass concentration and glucoamylase production in runs from group E20. In runs E20-2 and E20-3, the biomass profiles were similar and the cell concentration reached a maximum (Xf) of about 9.0 g/L after 21 hours of cultivation. The biomass values in run E20-1 were higher than those obtained in runs E20-2 and E20-3 during almost the entire cultivation period, owing to the higher inoculum cell concentration. However, a maximum of 8.40 g/L was obtained in run E20-1, similar to runs E20-2 and E20-3, as shown in Table 3. Thus, both cell productivity (Px) and growth yield (Yx/s) were similar in these runs (about 0.40 g/(L.h) and 0.40 g/g, respectively).
Regarding glucoamylase production, the glucoamylase activity profiles were similar in runs E20-2 and E20-3, up to 20 hours of cultivation. However, after this period, the overall glucoamylase production rate in run E20-3 decreased markedly and glucoamylase activity reached a maximum (Am) of about 945 U/L. The highest glucoamylase activity was obtained in run E20-2 (1,221 U/L) which was about 20% higher than the activity obtained in run E20-1 (1,019 U/L) and 30% higher than that obtained in run E20-3 (945 U/L). Therefore, the highest glucoamylase productivity (Pa) was obtained in run E20-2 (51 U/(L.h)), and it was about 27% and 46% higher than the productivities obtained in runs E20-1 and E20-3, respectively, as shown in Table 3. Also, the glucoamylase yield from substrate (YA/s) was higher in run E20-2 (53 U/g) than in runs E20-1 and E20-3 (about 43 U/g).
Figures 7 and 8 show the profiles of biomass concentration and glucoamylase production obtained in runs from group E80. The biomass profiles were similar, reaching a maximum cell concentration (Xf) of between 13 g/L and 14 g/L after 44 hours of cultivation, as indicated in Table 3. Thus, cell productivity (Px) was similar in all three runs (about 0.30 g/(L.h)), as was growth yield (Yx/s) (between 0.17 g/g and 0.18 g/g).
From Figure 8, it can be observed that glucoamylase activity reached similar maximum values in runs E80-1 (5,434 U/L) and E80-3 (5,636 U/L). On the other hand, glucoamylase activity reached 6,444 U/L in run E80-2, which was about 18% and 14% higher than the activities reached in runs E80-1 and E80-3, respectively. Therefore, the highest glucoamylase productivity (Pa) was obtained in run E80-2 (134 U/(L.h)), which was about 20% and 24% higher than the productivities obtained in runs E80-1 (112 U/(L.h)) and E80-3 (108 U/(L.h)), as shown in Table 3. Also, the glucoamylase yield from substrate (YA/s) was higher in run E80-2 (85 U/g) than in runs E80-1 (69 U/g) and E80-3 (72 U/g).
Thus, in runs from group E80, inoculation techniques did not significantly influence the growth morphology obtained in the fermenter, as occurred in runs from group E20. This fact was probably due to the influence of other factors, which acted simultaneously. For example, for high TRS0, oxygen becomes growth limiting, as observed in previous works (Facciotti et al., 1990). The inoculation technique which produced the highest level of glucoamylase in the fermenter was shaker culture for 7 hours, at a pH of 2.5, both in runs from group E20 and in runs from group E80. This technique produced an inoculum formed by isolated spores at the beginning of germination and avoided pellet formation in the fermenter.
CONCLUSIONS
In Aspergillus awamori cultivations, inoculum preparation plays an important role in microorganism morphology and in glucoamylase production in the fermenter. The inoculum which led to the highest glucoamylase activity was shaker cultivated for 7 hours (the period of time necessary for the beginning of spore germination), at a pH of 2.5. This low pH value avoided spore agglomeration during the germination phase, and the inoculum composed of dispersed spores led to filamentous growth in the fermenter, preventing pellet formation and increasing glucoamylase production. Rheological properties of the fermentation broth were shown to be related to microorganism morphology in this fermentation process.
NOMENCLATURE
A Glucoamylase activity, U/L
Ao Glucoamylase activity at the beginning of cultivation, U/L
Am Maximum glucoamylase activity, U/L
K Broth consistency index, Pa.sn
n Flow behavior index, dimensionless
Pa Glucoamylase productivity, U/(L.h)
Px Cell productivity, g/(L.h)
TRS0 Initial total reducing sugar concentration, g/L
tm Instant of maximum glucoamylase activity, h
tf Instant of end of carbon source, h
U Glucoamylase activity unit
X Biomass concentration, g/L
Xo Biomass concentration at the beginning of cultivation, g/L
Xf Biomass concentration at the end of cultivation, g/L
YA/S Glucoamylase yield from substrate, U/g
Yx/s Growth yield, g/g
ACKNOWLEDGEMENT
The authors are grateful to the undergraduate students of Chemical Engineering Course at the "Escola Politécnica da USP," who took part in the "Semana de Fermentação/1997" of the course entitled "PQI-572 - Laboratório de Engenharia Bioquímica II."
REFERENCES
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Pamboukian, C.R.D., Influência das Condições de Preparo do Inóculo na Morfologia do Microrganismo e na Síntese de Glicoamilase por Aspergillus awamori. Master's Thesis, Escola Politécnica da Universidade de São Paulo (1997).
Pedersen, A.G.; Bundgaard-Nielsen, M.; Nielsen, J.; Villadsen, J. and Hassager, O., Rheological Characterization of Media Containing Penicillium chrysogenum, Biotechnology and Bioengineering, 41, 162 (1993).
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Raper, K.B. and Fennell, D.I., The Genus Aspergillus. The Williams and Wilkins Co., Baltimore (1965).
Ruohang, W. and Webb, C., Effect of Cell Concentration on the Rheology of Glucoamylase Fermentation Broth, Biotechnology Techniques, 9, No 1, 55 (1995).
Schmidell, W. and Fernandes, M.V., Comparação entre Hidrólise Ácida e Enzimática de Amido para Determinação de Açúcares Redutores Totais, Revista de Microbiologia, 8, No 3, 98 (1977).
Schmidell, W. and Menezes, J.R.G., Influência da Glicose na Determinação da Atividade da Amiloglicosidase. Revista de Microbiologia, 17, No 3, 194 (1986).
Wainwright, M.P.; Trinci, A.P.J. and Moore, D., Aggregation of Spores and Biomass of Phanerochaete chrysosporium in Liquid Culture and the Effect of Anionic Polymers on This Process, Mycological Research, 97, No 7, 801 (1993).
Whitaker, A. and Long, P.A., Fungal Pelleting, Process Biochemistry, 8, 27 (1973).
- Carlsen, M.; Spohr, A.B.; Nielsen, J. and Villadsen, J., Morphology and Physiology of an a -Amylase Producing Strain of Aspergillus oryzae during Batch Cultivations, Biotechnology and Bioengineering, 49, 266 (1996).
- Facciotti, M.C.R.; Schmidell, W. and Aguero, J.M.Z., Glucoamylase Production by Semicontinuous Cultivation of Aspergillus awamori NRRL 3112, Arquivos de Biologia e Tecnologia, 33, No 4, 797 (1990).
- Pamboukian, C.R.D., Influęncia das Condiçőes de Preparo do Inóculo na Morfologia do Microrganismo e na Síntese de Glicoamilase por Aspergillus awamori Master's Thesis, Escola Politécnica da Universidade de Săo Paulo (1997).
- Pedersen, A.G.; Bundgaard-Nielsen, M.; Nielsen, J.; Villadsen, J. and Hassager, O., Rheological Characterization of Media Containing Penicillium chrysogenum, Biotechnology and Bioengineering, 41, 162 (1993).
- Queiroz, M.C.R.; Facciotti, M.C.R. and Schmidell, W., Rheological Changes of Aspergillus awamori Broth during Amyloglucosidase Production, Biotechnology Letters, 19, No 2, 167 (1997).
- Raper, K.B. and Fennell, D.I., The Genus Aspergillus. The Williams and Wilkins Co., Baltimore (1965).
- Ruohang, W. and Webb, C., Effect of Cell Concentration on the Rheology of Glucoamylase Fermentation Broth, Biotechnology Techniques, 9, No 1, 55 (1995).
- Schmidell, W. and Fernandes, M.V., Comparaçăo entre Hidrólise Ácida e Enzimática de Amido para Determinaçăo de Açúcares Redutores Totais, Revista de Microbiologia, 8, No 3, 98 (1977).
- Schmidell, W. and Menezes, J.R.G., Influęncia da Glicose na Determinaçăo da Atividade da Amiloglicosidase. Revista de Microbiologia, 17, No 3, 194 (1986).
- Wainwright, M.P.; Trinci, A.P.J. and Moore, D., Aggregation of Spores and Biomass of Phanerochaete chrysosporium in Liquid Culture and the Effect of Anionic Polymers on This Process, Mycological Research, 97, No 7, 801 (1993).
- Whitaker, A. and Long, P.A., Fungal Pelleting, Process Biochemistry, 8, 27 (1973).
Publication Dates
-
Publication in this collection
30 Oct 1998 -
Date of issue
Sept 1998
History
-
Accepted
18 May 1998 -
Received
05 Mar 1998
