- Email: [email protected]
The effect of morphology and oxygen uptake on penicillin production by Aspergillus nidulans in submerged culture
Mycol. Res. 101 (10) : 1237–1241 (1997)
1237
Printed in the United Kingdom
The effect of morphology and oxygen uptake on penicillin production by Aspergillus nidulans in submerged culture
J. M O O R E A N D M. E. B U S H E L L Microbial Products Laboratory, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, U.K.
Penicillin was not detected in Aspergillus nidulans liquid cultures with pelleted morphology (pellets 1–5 mm diam.) but production was observed in cultures with homogeneous filamentous morphologies. Adenylate Energy Charge (AEC) and Oxygen Uptake Rates (OUR) were measured in cultures exhibiting the two morphologies and it was shown that the values of both parameters were lowest in pelleted cultures and highest in cultures with a filamentous morphology. In addition, a third morphological form, ‘ micropellets ’ with intermediate properties between the other two types was encountered. We propose that oxygen limitation brought about by the mass transfer restrictions of pelleted growth inhibits penicillin production.
The development of pelleted morphology by liquid cultures of some fungal species has been observed for many years (first reference, Ray, 1897). Superficially, pelleted growth appears to resemble that of colony growth on agar surfaces (Georgio & Shuler, 1986), both containing cells whose growth dynamics range between exponentially accreting and senescent, nongrowing. Whitaker & Long (1973) classified pelleted morphologies according to shape. Pellets may arise via a number of mechanisms, including spore aggregates or from entangled hyphae. Recent work has also described instances of individual pellets each arising from a single spore (Thomas, 1992). A gradient of dissolved nutrient availability occurs along the radius of each individual pellet (Kobayshi, van Dedeem & Moo-Young, 1973). Thus, the pellet may contain hyphae at different stages of nutrient limitation. This may account for the lysis, sometimes observed at the centre of the pellet, resulting in hollow structures (Trinci, 1970). It has been established that pelleted cultures are oxygenlimited, due to insufficient oxygen transfer from the exterior of the pellet to the interior (Yano, Kodama & Yamada, 1961). Using a miniature dissolved oxygen electrode Huang & Bungay (1973) proved that the oxygen tension decreases progressively between the exterior and the centre of the pellet. This observation was confirmed by Cronenberg et al. (1994) who used miniature electrodes to measure both glucose and oxygen levels inside pellets of P. chrysogenum. This effect has important consequences in several areas of commercial fungal product formation. Oxygen limitation inhibits complete activity of the TCA cycle, allowing intermediates to accumulate (Gray et al., 1966). It has been proposed that these effects account for the production of citric acid by Aspergillus niger cultures only when in a pelleted form (Papagianni, Mattey & Kristansen, 1994). A connection between internal pellet dissolved oxygen concentration and pellet size has been established by Kobayshi
et al. (1973), who also demonstrated that the rate of respiration decreased with an increase in pellet size. In Aspergillus nidulans, penicillin G production is growth dissociated. Penicillin synthesis is inhibited by high glucose uptake rates (Espesso, 1992), which presumably result in correspondingly high growth rates. This effect can be circumvented in antibiotic production process by controlling growth rate using appropriate nutrient feeding strategies (Lynch & Bushell, 1995). This primary control of culture physiology may, however, be eclipsed by secondary, morphology-dependent effects. A. nidulans liquid cultures may be manipulated to control the morphology. Under appropriate conditions, pellets (1–5 mm diam.), micropellets (less than 1 mm diam.), and filamentous morphologies may be obtained (Mitard & Riba, 1988). Reports concerning the effect of pellet formation on antibiotic production have pointed out that it can aid overall reactor mass transfer as it creates a two-phase system. Confining the mycelium to the solid, pellet phase avoids mycelium-induced viscosity increases in the liquid phase of the culture (e.g. Vechtlifshitz et al., 1992). However this advantage may be illusory since the availability of oxygen to the hyphae per se may be more important than culture-vessel mass transfer. However reports to support this latter hypothesis are rare (e.g. Dion et al., 1954) ; therefore, the present study was undertaken. This paper describes the relationship between culture morphology, oxygen uptake rate, adenylate energy charge and penicillin production rate, in Aspergillus nidulans. MATERIALS AND METHODS Strains and culture media A penicillin G-producing Aspergillus nidulans (Fidam) G. Winter paba A1 (wild-type) strain, obtained from Dr H. N.
Oxygen uptake and penicillin yield in A. nidulans Arst, Royal Postgraduate Medical School, was used throughout. Antibiotic concentrations were measured routinely by bioassay using a strain of Micrococcus luteus ATC9341R obtained by Dr M. A. Haxell, Pfizer Central Research, Sandwich, Kent. The chemically defined antibiotic production medium for A. nidulans contained the following major nutrients : (g l−" in water purified by reverse osmosis) glucose 10, NaNO 7±0, $ KH PO 2±8, K HPO 7±0, Na HPO 4±2 ; and the following # % # %− # % trace components : (g l ") MgSO \7H O 0±2, FeSO \7H O % # % # 1±0, CuCl 0±025, CaCl \2H O 0±1, ZnSO \7H O 0±2, # # # % # MnSO \4H O 1, (NH ) Mo O 0±019 and vitamin mix ; % # %' ( #( biotin 0±01, pyridoxin 0±01, thiamine 0±01, riboflavin 0±01, nicotinic acid 0±01, p-aminobenzoic acid 0±01). Glucose, salts, vitamin and trace solution were autoclaved separately and then mixed. The pH was adjusted to 7±0 with 5 KOH prior to autoclaving. This formulation resulted in a glucose-limited batch culture. Nitrate limitation was achieved by decreasing the sodium nitrate concentration to 2±0 g l−" and increasing that of glucose of 30 g l−", and phosphate limitation by increasing the glucose concentration to 30 g l−", decreasing the concentration of potassium dihydrogen phosphate to 0±1 g l−", that of dipotassium hydrogen phosphate to zero and adding MOPS buffer at 20 g l−". All shake flask cultures were carried out in 250-ml baffled Erlenmeyer flasks containing 25 ml of medium, shaken at 250 rpm. A. nidulans and M. luteus were routinely maintained on Malt-yeast agar, and grown at 30 °C. A malt–yeast broth starter medium was inoculated at 10) spores ml−". After 48 h agitation, 2 ml was removed and used (A. nidulans) to inoculate preculture flasks containing the defined medium. After 24 h further incubation, the precultures were used at approximately 5 % (v}v) as an inoculum for bioreactors or experimental flask cultures.
1238 phenomenon as precisely as with the two extreme cases ; therefore, properties of micropelleted cultures are only described in qualitative terms. Culture biomass concentration determination Five millilitre biomass samples were collected on membrane filters (Gelman 0±45 µm), pre-dried to a constant weight. Filtrates were collected and frozen for further assays (erythromycin, vancomycin, glucose, nitrate and phosphate), and the filter was rinsed with distilled water (3¬10 ml) prior to drying in a Hitachi microwave oven (high power, 5 min). Dry weights and, hence, biomass concentrations were estimated after cooling and desiccation. Specific biomass and antibiotic production rates Specific growth rates were calculated using the partial cubic spline interpolation methodology described by us previously (Bushell et al., 1993 ; McDermott et al., 1993). The idea of using cubic spline interpolation of fermentation data was first proposed by Oner et al. (1986). Antibiotic assays A bioassay employing Micrococcus luteus growing in brain heart infusion agar was performed using procedures described previously (Huck et al., 1991). Challenge strain seed cultures and assays plates were incubated at 30°, and the diameters of the zones of inhibition were recorded after 24 h. Examination of a number of samples using high-performance liquid chromatography (Pfizer Laboratories, Sandwich) confirmed that the bioassay corresponded to the appropriate antibiotics. Residual glucose, nitrate and phosphate
Bioreactor culture The Electrolab bioreactor used had a working volume of 3±5 l. Agitation was provided by disc turbine impellers, rotating at 600 rpm and sterile air was supplied through a sparger (for rates, see below). The temperature was controlled at 30°. Dissolved oxygen concentration in the bioreactor was monitored with an Ingold polarographic dissolved oxygen electrode and maintained above 80 % of air saturation by varying the airflow rate automatically. The pH was controlled at 6±9 using automatic additions of 0±1 HCl and 0±1 NaOH. Foaming was eliminated by including 0±01 % (v}v) Breox FMT30 antifoam (Water Management and Gamlen) in the culture medium. Defined medium for bioreactor cultures contained 10 g l−" glucose. Agitation and aeration conditions were manipulated in order to induce cultures consisting of : pellets 1–3 mm diam. (200 rpm agitation, 2 vvm aeration), and filamentous mycelium (800 rpm agitation, 1 vvm aeration). It was also possible to induce morphological states with intermediate properties between these two extremes, for example ‘ micropellets ’ 0±1–0±4 mm diam. were observed at 500 rpm agitation with 1 vvm aeration. Similar observations were reported by Mitard & Riba (1988). However, it was not possible to reproduce this
A glucose oxidase-based assay kit (Trinder system, Sigma) and a nitrate reductase-based assay kit (Boehringer Mannheim) were employed. The colorimetric procedure described previously (McDermott et al., 1993) was used for phosphate determination. Adenylate energy charge determination Adenylate nucleotide concentrations were measured using the Lumac AEC Kit (Sonc Ltd, Cat. No. 9281-0) and a Biocounter}3M 2010A, using the procedure described by Simpson et al. (1990). Culture biomass was collected on nylon filters (Gelman, 0±45 µm) and resuspended in modified HEPES buffer (m : HEPES 25, MgSO 7±5, EDTA 1). Cell extracts, % obtained using an X-Press Cell (Life Sciences Ltd) were diluted 1 : 100 prior to assay. Reproducibility and replication of experiments All experimental data were obtained from single cultures. Experiments were carried out in triplicate to ensure that the trends and relationships observed in the culture parameters measured were reproducible. Individual assays were replicated four-fold except where stated. Experiments were rejected
J. Moore and M. E. Bushell
1239
where a χ# test indicated significant differences between replicates.
RESULTS AND DISCUSSION
Culture morphology
Manipulation of culture conditions The production medium was reformulated so that cultures could be carried out with glucose, nitrate and phosphate as growth limiting substrates. The glucose-limited batch culture formulation supported the greatest penicillin production (Table 1) and so this medium was used for all subsequent experiments. Cultures exhibited a homogeneous morphological appearance during bioreactor culture, forming pellets (1–3 mm diam.), filamentous mycelium, and intermediate morphological forms under appropriate conditions as described in Materials and Methods.
Effect of culture morphology on growth dynamics The growth patterns observed with the two extreme morphological forms were similar and comparable maximum biomass concentrations were observed (Fig. 1) albeit at different times. The specific growth rates of both cultures were estimated using the partial cubic spline procedure, assuming the exponential growth model. The apparent maximum specific growth rate of the pelleted culture was considerably lower than that of the filamentous culture (Fig. 1) presumably
Growth limiting nutrient
Time when penicillin Maximum production was first penicillin yield detected (h) (mg g−" biomass)
Glucose Sodium nitrate K}Na phosphate
30 62 30
4±1 1±9 1±6
10
0·1
8
0·08
6
0·06
4
0·04
2
0·02
0
20
40
60 Time (h)
80
100
0 120
Kr value (cube root growth law) (g l−" h−")
Correlation coefficient for regression of x"/$ on t
µmax value (exponential growth model)* (h−")
Filamentous 0±0139 0±83 0±086 Pelleted 0±0176 0±933 0±0063 * No correlation coefficient is calculated during partial cubic spline analysis since the curve is forced to fit the data points during this technique.
Table 3. Penicillin yields, OUR and AEC in bioreactor cultures of A. nidulans with different morphologies
Culture morphology
Maximum penicillin yield Maximum OUR (mg g−" biomass) ( mol l−" h−")
Maximum AEC (dimensionless)
Filamentous Pelleted
4±1 0
0±90 0±35
0±40 0±26
due to the reported adherence of pelleted A. nidulans cultures to linear growth dynamics following the cube root growth law (Trinci, 1970) : [xt]"/$ ¯ kt[x ]"/$, ! (where xt is the biomass concentration obtained after time t from an initial biomass concentration of x and k is the linear ! growth constant), rather than the exponential model : ln xt ¯ ln x µt, !
Apparent specific growth rate (h–1)
Biomass concentration (g l–1)
Table 1. Effect of reformulation of the culture medium on growth and antibiotic production in bioreactor cultures of A. nidulans
0
Table 2. Kinetic growth rate constants calculated for bioreactor cultures of A. nidulans with different morphologies
Fig. 1. Biomass concentration in bioreactor culture of A. nidulans with filamentous (circles) and pelleted (squares) morphology and specific growth rate, calculated assuming exponential growth, in cultures with filamentous (dashed curve) and pelleted (dotted curve) morphology. The average standard error of the mean of replicate determinations was 5 % of the mean.
(where µ is specific growth rate constant and µ ¯ (dx}dt) (1}x)) used in the partial cubic spline technique. The value of the pellet growth rate constant k was calculated by linear regression of the cube root of biomass concentration on culture time. Comparing the growth rate constants obtained assuming the two different models suggested that the exponential law is the most appropriate to the filamentous culture whereas the cube root model is the most appropriate to the pelleted culture (Table 2). These calculations confirm that our designation of pelleted morphology is consistent with that of previous reports concerning the physiology of mycelial growth in submerged culture. This difference in growth dynamics has been attributed to mass transfer limitation (Pirt, 1970). Cultures with intermediate morphologies were also observed, when the agitation conditions were varied, whose growth kinetics varied between linear and exponential, depending on the degree of pelleting in the culture.
Penicillin production Penicillin production was observed in the filamentous cultures but not in the pelleted cultures (Table 3). Penicillin was also detected in cultures containing intermediate morphological forms but in lower concentration.
1240
0·5
1
0·4
0·8
0·3
0·6
0·2
0·4
0·1
0·2
0
0
20
40
60 Time (h)
80
100
throughout these investigations. J. M. acknowledges receipt of a BBSRC}Pfizer CASE award. AEC units
OUR (mmol l–1 h–1)
Oxygen uptake and penicillin yield in A. nidulans
0 120
Fig. 2. Adenylate energy charge (squares) and oxygen uptake rate (circles) in bioreactor culture of A. nidulans with filamentous (open) and pelleted (filled) morphology. Adenylate energy charge in filamentous, oxygen-limited batch cultures (triangles). The average standard errors of the means of replicate determinations were 7±3 % of the mean (energy charge) and 3 % (oxygen uptake rates).
Oxygen uptake and adenylate energy charge OURs appeared to be affected by the culture morphology (Fig. 2) with the pelleted culture exhibiting a significantly lower rate. Huang & Bungay (1973) used a miniature dissolved oxygen electrode to demonstrate a progressive decrease in oxygen tension towards the centre of the pellet across its radius. Their report is consistent with our observation of a lower specific oxygen uptake rate in pelleted culture as, presumably, only the outer layers of hyphae are able to assimilate oxygen at maximum rate. AEC was also dependent on morphology, the values observed in the pelleted culture being considerably lower throughout the culture (Fig. 2). Intermediate AEC values were also observed in the ‘ micropelleted ’ cultures (those with intermediate morphology between pellets and filamentous mycelium). Antibiotic production is known to be inhibited under conditions that results in a low intra-cellular ATP content throughout growth (Yang & Wang, 1996). When filamentous cultures were subjected to decreased aeration, resulting in oxygen limitation, AEC was less than 0±5 throughout the culture and no penicillin was detected (Fig. 2). Oxygen limitation was defined as the condition prevailing when the biomass concentration was dependent on the dissolved oxygen concentration (for a discussion see Clark, Langley & Bushell, 1995). The air-flow required to effect oxygen limitation (0±1 vvm) was too small to allow accurate OUR measurement. Our results indicate that penicillin production is inhibited by pellet formation and that this effect is probably a result of oxygen limitation. Pelleted cultures exhibit a decrease in AEC value which is likely to be a result of a decrease in oxygen uptake rate occasioned by mass transfer constraints along the radius of the pellet. We would like to thank Dr H. N. Arst (Royal Postgraduate School) for providing the strain of Aspergillus nidulans and Dr M. A. Haxell and other members of Animal Health and Discovery Department, Pfizer Central Research for support
REFERENCES Bushell, M. E., Bell, S. L., Scott, M. F., Snell, K., Speir, R. E., Wardell, J. N. & Sanders, P. G. (1993). A three-phase pattern in growth, monoclonal antibody production and metabolite exchange in a hybridoma bioreactor culture. Biotechnology and Bioengineering 42, 133–139. Carilli, A. (1961). Aeration study. III. Continuous measurement of dissolved oxygen during fermentation in large fermenters. Scientific Report of the Instituto Superiore di Sanita[ (Rome) 1 177–189. Carter, B. L. A. & Bull, A. T. (1971). The effect of oxygen tension in the medium on the morphology and growth kinetics of Aspergillus nidulans. Journal of General Microbiology 66, 265–273. Clark, G. J., Langley, D. & Bushell, M. E. (1995). Oxygen limitation can induce microbial secondary metabolite formation : investigations with miniature electrodes in shaker and bioreactor culture. Microbiology 141, 663–669. Cronenberg, C. C. H., Ottengraf, S. P. P., van den Heuvel, J. C., Pottel, F., Sziele, D., Schu$ gerl, K. & Bellgardt, K. H. (1994). Influence of age and structure of Penicillium chrysogenum pellets on the internal concentration profiles. Bioprocess Engineering 10, 209–216. Dion, V. M., Carilli, A., Sermonti, G. & Chain, E. B. (1954). The effect of mechanical agitation on the morphology of P. chrysogenum in stirred fermentation. Scientific Report of the Instituto Superiore di Sanita[ (Rome) 17, 184–205. Espesso, P. (1992). Carbon catabolite repression can account for the temporal pattern of expression of a penicillin biodefined gene in Aspergillus nidulans. Molecular Microbiology 6, 1457–1465. Georgiou, G. & Shuler, M. L. (1986). A computer model for the growth and differentiation of a fungal colony on solid substrate. Biotechnology and Bioengineering 28, 405–416. Gray, C. T., Wimpenny, J. W. T. & Mossman, M. R. (1966). Regulation of the metabolism in facultative bacteria. Part III. Effects of aerobiosis and anaerobiosis and nutrients on the formation of Krebs cycle enzymes in E. coli. Biochemica et Biophysica Acta 117, 33–41. Huang, M. Y. & Bungay, H. R. (1973). Microprobe measurements of oxygen concentration in mycelial pellets. Biotechnology and Bioengineering 15, 1193–1197. Huck, T. A., Porter, N. & Bushell, M. E. (1991). Positive selection of antibioticproducing soil isolates. Journal of General Microbiology 137, 2321–2329. Kobayshi, H., van Dedeem, G. & Moo-Young, M. (1973). Oxygen transfer into mycelial pellets. Biotechnology and Bioengineering 27, 45–47. Lynch, H. C. & Bushell, M. E. (1995). The physiology of erythromycin biosynthesis in cyclic fed batch culture. Microbiology 141, 3105–3111. Martin, J. F. & Demain, A. L. (1977). Effect of exogenous nucleotides on the candicidin fermentation. Canadian Journal of Microbiology 23, 1334–1339. McDermott, J. F., Lethbridge, G. & Bushell, M. E. (1993). Estimation of the kinetic constants and elucidation of trends in growth and erythromycin production in batch and continuous culture of Saccharopolyspora erythraea using curve fitting techniques. Enzymes and Microbial Technology 15, 657–663. Mitard, A. & Riba, J. P. (1988). Morphology and growth of Aspergillus niger ATCC 26036 cultivated at several different shear rates. Biotechnology and Bioengineering 32, 835–840. Oner, M. D., Erickson, L. E. & Young, S. S. (1986). Utilisation of spline functions for smoothing fermentation data and for estimation of specific rates. Biotechnology and Bioengineering 28, 902–918. Papagianni, M., Mattey, M. & Kristansen, B. (1994). Morphology and citric acid production of A. niger PM1. Biotechnology Letters 16, 929–934. Phillips, D. H. (1966). Oxygen transfer into mycelial pellets. Biotechnology and Bioengineering 8, 456–460. Pirt, S. J. (1970). Principles of Microbe and Cell Cultivation. Blackwell Scientific Publications : Oxford, U.K. Ray, J. (1897). Variations des champignons infe! rieurs sous l’influence du milieu. Thesis. Faculte! des Sciences, Universite! de Paris, Paris, France. Shanahan, J. F. (1991). Effect of carbon dioxide on the rheological behaviour and oxygen transfer in submerged penicillin fermentation. Biotechnology and Bioengineering 38, 1223–1232.
J. Moore and M. E. Bushell Simpson, W. J., Fernendaz, J. L., Hammond, J. R. M., Senior, P. S., McCarthy, B. J., Jago, P. H., Sidorowicz, S., Jassin, J. A. A. & Denyer, S. P. (1990). A highly sensitive assay for adenosine triphosphate employing an improved firefly luciferase reagent. Letters in Applied Microbiology 11, 208–210. Thomas, C. R. (1992). Mycelial morphology. The effect of spore inoculum level. Biotechnology Letters 14, 1071–1074. Trinci, A. J. P. (1970). Kinetics of the growth of mycelial pellets of Aspergillus nidulans. Archives of Microbiology 77, 353–367. Vechtlifshitz, S. E., Sasson, Y. & Braun, S. (1992). Nikkomycin production (Accepted 28 January 1997 )
1241 in pellets of Streptomyces tendae. Journal of Applied Bacteriology 72, 195–200. Whitaker, A. & Long, P. A. (1973). Fungal pelleting. Process Biochemistry 11, 27–31. Yano, T., Kodama, T. & Yamada, K. (1961). Fundamental studies on the aerobic fermentation. Agricultural and Biological Chemistry 25, 580–584. Yang, S. S. & Wang, J. Y. (1996). Morphogenesis, ATP content and oxytetracycline production by Streptomyces rimosus in solid substrate cultivation. Journal of Applied Bacteriology 80, 545–550.
1237
Printed in the United Kingdom
The effect of morphology and oxygen uptake on penicillin production by Aspergillus nidulans in submerged culture
J. M O O R E A N D M. E. B U S H E L L Microbial Products Laboratory, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, U.K.
Penicillin was not detected in Aspergillus nidulans liquid cultures with pelleted morphology (pellets 1–5 mm diam.) but production was observed in cultures with homogeneous filamentous morphologies. Adenylate Energy Charge (AEC) and Oxygen Uptake Rates (OUR) were measured in cultures exhibiting the two morphologies and it was shown that the values of both parameters were lowest in pelleted cultures and highest in cultures with a filamentous morphology. In addition, a third morphological form, ‘ micropellets ’ with intermediate properties between the other two types was encountered. We propose that oxygen limitation brought about by the mass transfer restrictions of pelleted growth inhibits penicillin production.
The development of pelleted morphology by liquid cultures of some fungal species has been observed for many years (first reference, Ray, 1897). Superficially, pelleted growth appears to resemble that of colony growth on agar surfaces (Georgio & Shuler, 1986), both containing cells whose growth dynamics range between exponentially accreting and senescent, nongrowing. Whitaker & Long (1973) classified pelleted morphologies according to shape. Pellets may arise via a number of mechanisms, including spore aggregates or from entangled hyphae. Recent work has also described instances of individual pellets each arising from a single spore (Thomas, 1992). A gradient of dissolved nutrient availability occurs along the radius of each individual pellet (Kobayshi, van Dedeem & Moo-Young, 1973). Thus, the pellet may contain hyphae at different stages of nutrient limitation. This may account for the lysis, sometimes observed at the centre of the pellet, resulting in hollow structures (Trinci, 1970). It has been established that pelleted cultures are oxygenlimited, due to insufficient oxygen transfer from the exterior of the pellet to the interior (Yano, Kodama & Yamada, 1961). Using a miniature dissolved oxygen electrode Huang & Bungay (1973) proved that the oxygen tension decreases progressively between the exterior and the centre of the pellet. This observation was confirmed by Cronenberg et al. (1994) who used miniature electrodes to measure both glucose and oxygen levels inside pellets of P. chrysogenum. This effect has important consequences in several areas of commercial fungal product formation. Oxygen limitation inhibits complete activity of the TCA cycle, allowing intermediates to accumulate (Gray et al., 1966). It has been proposed that these effects account for the production of citric acid by Aspergillus niger cultures only when in a pelleted form (Papagianni, Mattey & Kristansen, 1994). A connection between internal pellet dissolved oxygen concentration and pellet size has been established by Kobayshi
et al. (1973), who also demonstrated that the rate of respiration decreased with an increase in pellet size. In Aspergillus nidulans, penicillin G production is growth dissociated. Penicillin synthesis is inhibited by high glucose uptake rates (Espesso, 1992), which presumably result in correspondingly high growth rates. This effect can be circumvented in antibiotic production process by controlling growth rate using appropriate nutrient feeding strategies (Lynch & Bushell, 1995). This primary control of culture physiology may, however, be eclipsed by secondary, morphology-dependent effects. A. nidulans liquid cultures may be manipulated to control the morphology. Under appropriate conditions, pellets (1–5 mm diam.), micropellets (less than 1 mm diam.), and filamentous morphologies may be obtained (Mitard & Riba, 1988). Reports concerning the effect of pellet formation on antibiotic production have pointed out that it can aid overall reactor mass transfer as it creates a two-phase system. Confining the mycelium to the solid, pellet phase avoids mycelium-induced viscosity increases in the liquid phase of the culture (e.g. Vechtlifshitz et al., 1992). However this advantage may be illusory since the availability of oxygen to the hyphae per se may be more important than culture-vessel mass transfer. However reports to support this latter hypothesis are rare (e.g. Dion et al., 1954) ; therefore, the present study was undertaken. This paper describes the relationship between culture morphology, oxygen uptake rate, adenylate energy charge and penicillin production rate, in Aspergillus nidulans. MATERIALS AND METHODS Strains and culture media A penicillin G-producing Aspergillus nidulans (Fidam) G. Winter paba A1 (wild-type) strain, obtained from Dr H. N.
Oxygen uptake and penicillin yield in A. nidulans Arst, Royal Postgraduate Medical School, was used throughout. Antibiotic concentrations were measured routinely by bioassay using a strain of Micrococcus luteus ATC9341R obtained by Dr M. A. Haxell, Pfizer Central Research, Sandwich, Kent. The chemically defined antibiotic production medium for A. nidulans contained the following major nutrients : (g l−" in water purified by reverse osmosis) glucose 10, NaNO 7±0, $ KH PO 2±8, K HPO 7±0, Na HPO 4±2 ; and the following # % # %− # % trace components : (g l ") MgSO \7H O 0±2, FeSO \7H O % # % # 1±0, CuCl 0±025, CaCl \2H O 0±1, ZnSO \7H O 0±2, # # # % # MnSO \4H O 1, (NH ) Mo O 0±019 and vitamin mix ; % # %' ( #( biotin 0±01, pyridoxin 0±01, thiamine 0±01, riboflavin 0±01, nicotinic acid 0±01, p-aminobenzoic acid 0±01). Glucose, salts, vitamin and trace solution were autoclaved separately and then mixed. The pH was adjusted to 7±0 with 5 KOH prior to autoclaving. This formulation resulted in a glucose-limited batch culture. Nitrate limitation was achieved by decreasing the sodium nitrate concentration to 2±0 g l−" and increasing that of glucose of 30 g l−", and phosphate limitation by increasing the glucose concentration to 30 g l−", decreasing the concentration of potassium dihydrogen phosphate to 0±1 g l−", that of dipotassium hydrogen phosphate to zero and adding MOPS buffer at 20 g l−". All shake flask cultures were carried out in 250-ml baffled Erlenmeyer flasks containing 25 ml of medium, shaken at 250 rpm. A. nidulans and M. luteus were routinely maintained on Malt-yeast agar, and grown at 30 °C. A malt–yeast broth starter medium was inoculated at 10) spores ml−". After 48 h agitation, 2 ml was removed and used (A. nidulans) to inoculate preculture flasks containing the defined medium. After 24 h further incubation, the precultures were used at approximately 5 % (v}v) as an inoculum for bioreactors or experimental flask cultures.
1238 phenomenon as precisely as with the two extreme cases ; therefore, properties of micropelleted cultures are only described in qualitative terms. Culture biomass concentration determination Five millilitre biomass samples were collected on membrane filters (Gelman 0±45 µm), pre-dried to a constant weight. Filtrates were collected and frozen for further assays (erythromycin, vancomycin, glucose, nitrate and phosphate), and the filter was rinsed with distilled water (3¬10 ml) prior to drying in a Hitachi microwave oven (high power, 5 min). Dry weights and, hence, biomass concentrations were estimated after cooling and desiccation. Specific biomass and antibiotic production rates Specific growth rates were calculated using the partial cubic spline interpolation methodology described by us previously (Bushell et al., 1993 ; McDermott et al., 1993). The idea of using cubic spline interpolation of fermentation data was first proposed by Oner et al. (1986). Antibiotic assays A bioassay employing Micrococcus luteus growing in brain heart infusion agar was performed using procedures described previously (Huck et al., 1991). Challenge strain seed cultures and assays plates were incubated at 30°, and the diameters of the zones of inhibition were recorded after 24 h. Examination of a number of samples using high-performance liquid chromatography (Pfizer Laboratories, Sandwich) confirmed that the bioassay corresponded to the appropriate antibiotics. Residual glucose, nitrate and phosphate
Bioreactor culture The Electrolab bioreactor used had a working volume of 3±5 l. Agitation was provided by disc turbine impellers, rotating at 600 rpm and sterile air was supplied through a sparger (for rates, see below). The temperature was controlled at 30°. Dissolved oxygen concentration in the bioreactor was monitored with an Ingold polarographic dissolved oxygen electrode and maintained above 80 % of air saturation by varying the airflow rate automatically. The pH was controlled at 6±9 using automatic additions of 0±1 HCl and 0±1 NaOH. Foaming was eliminated by including 0±01 % (v}v) Breox FMT30 antifoam (Water Management and Gamlen) in the culture medium. Defined medium for bioreactor cultures contained 10 g l−" glucose. Agitation and aeration conditions were manipulated in order to induce cultures consisting of : pellets 1–3 mm diam. (200 rpm agitation, 2 vvm aeration), and filamentous mycelium (800 rpm agitation, 1 vvm aeration). It was also possible to induce morphological states with intermediate properties between these two extremes, for example ‘ micropellets ’ 0±1–0±4 mm diam. were observed at 500 rpm agitation with 1 vvm aeration. Similar observations were reported by Mitard & Riba (1988). However, it was not possible to reproduce this
A glucose oxidase-based assay kit (Trinder system, Sigma) and a nitrate reductase-based assay kit (Boehringer Mannheim) were employed. The colorimetric procedure described previously (McDermott et al., 1993) was used for phosphate determination. Adenylate energy charge determination Adenylate nucleotide concentrations were measured using the Lumac AEC Kit (Sonc Ltd, Cat. No. 9281-0) and a Biocounter}3M 2010A, using the procedure described by Simpson et al. (1990). Culture biomass was collected on nylon filters (Gelman, 0±45 µm) and resuspended in modified HEPES buffer (m : HEPES 25, MgSO 7±5, EDTA 1). Cell extracts, % obtained using an X-Press Cell (Life Sciences Ltd) were diluted 1 : 100 prior to assay. Reproducibility and replication of experiments All experimental data were obtained from single cultures. Experiments were carried out in triplicate to ensure that the trends and relationships observed in the culture parameters measured were reproducible. Individual assays were replicated four-fold except where stated. Experiments were rejected
J. Moore and M. E. Bushell
1239
where a χ# test indicated significant differences between replicates.
RESULTS AND DISCUSSION
Culture morphology
Manipulation of culture conditions The production medium was reformulated so that cultures could be carried out with glucose, nitrate and phosphate as growth limiting substrates. The glucose-limited batch culture formulation supported the greatest penicillin production (Table 1) and so this medium was used for all subsequent experiments. Cultures exhibited a homogeneous morphological appearance during bioreactor culture, forming pellets (1–3 mm diam.), filamentous mycelium, and intermediate morphological forms under appropriate conditions as described in Materials and Methods.
Effect of culture morphology on growth dynamics The growth patterns observed with the two extreme morphological forms were similar and comparable maximum biomass concentrations were observed (Fig. 1) albeit at different times. The specific growth rates of both cultures were estimated using the partial cubic spline procedure, assuming the exponential growth model. The apparent maximum specific growth rate of the pelleted culture was considerably lower than that of the filamentous culture (Fig. 1) presumably
Growth limiting nutrient
Time when penicillin Maximum production was first penicillin yield detected (h) (mg g−" biomass)
Glucose Sodium nitrate K}Na phosphate
30 62 30
4±1 1±9 1±6
10
0·1
8
0·08
6
0·06
4
0·04
2
0·02
0
20
40
60 Time (h)
80
100
0 120
Kr value (cube root growth law) (g l−" h−")
Correlation coefficient for regression of x"/$ on t
µmax value (exponential growth model)* (h−")
Filamentous 0±0139 0±83 0±086 Pelleted 0±0176 0±933 0±0063 * No correlation coefficient is calculated during partial cubic spline analysis since the curve is forced to fit the data points during this technique.
Table 3. Penicillin yields, OUR and AEC in bioreactor cultures of A. nidulans with different morphologies
Culture morphology
Maximum penicillin yield Maximum OUR (mg g−" biomass) ( mol l−" h−")
Maximum AEC (dimensionless)
Filamentous Pelleted
4±1 0
0±90 0±35
0±40 0±26
due to the reported adherence of pelleted A. nidulans cultures to linear growth dynamics following the cube root growth law (Trinci, 1970) : [xt]"/$ ¯ kt[x ]"/$, ! (where xt is the biomass concentration obtained after time t from an initial biomass concentration of x and k is the linear ! growth constant), rather than the exponential model : ln xt ¯ ln x µt, !
Apparent specific growth rate (h–1)
Biomass concentration (g l–1)
Table 1. Effect of reformulation of the culture medium on growth and antibiotic production in bioreactor cultures of A. nidulans
0
Table 2. Kinetic growth rate constants calculated for bioreactor cultures of A. nidulans with different morphologies
Fig. 1. Biomass concentration in bioreactor culture of A. nidulans with filamentous (circles) and pelleted (squares) morphology and specific growth rate, calculated assuming exponential growth, in cultures with filamentous (dashed curve) and pelleted (dotted curve) morphology. The average standard error of the mean of replicate determinations was 5 % of the mean.
(where µ is specific growth rate constant and µ ¯ (dx}dt) (1}x)) used in the partial cubic spline technique. The value of the pellet growth rate constant k was calculated by linear regression of the cube root of biomass concentration on culture time. Comparing the growth rate constants obtained assuming the two different models suggested that the exponential law is the most appropriate to the filamentous culture whereas the cube root model is the most appropriate to the pelleted culture (Table 2). These calculations confirm that our designation of pelleted morphology is consistent with that of previous reports concerning the physiology of mycelial growth in submerged culture. This difference in growth dynamics has been attributed to mass transfer limitation (Pirt, 1970). Cultures with intermediate morphologies were also observed, when the agitation conditions were varied, whose growth kinetics varied between linear and exponential, depending on the degree of pelleting in the culture.
Penicillin production Penicillin production was observed in the filamentous cultures but not in the pelleted cultures (Table 3). Penicillin was also detected in cultures containing intermediate morphological forms but in lower concentration.
1240
0·5
1
0·4
0·8
0·3
0·6
0·2
0·4
0·1
0·2
0
0
20
40
60 Time (h)
80
100
throughout these investigations. J. M. acknowledges receipt of a BBSRC}Pfizer CASE award. AEC units
OUR (mmol l–1 h–1)
Oxygen uptake and penicillin yield in A. nidulans
0 120
Fig. 2. Adenylate energy charge (squares) and oxygen uptake rate (circles) in bioreactor culture of A. nidulans with filamentous (open) and pelleted (filled) morphology. Adenylate energy charge in filamentous, oxygen-limited batch cultures (triangles). The average standard errors of the means of replicate determinations were 7±3 % of the mean (energy charge) and 3 % (oxygen uptake rates).
Oxygen uptake and adenylate energy charge OURs appeared to be affected by the culture morphology (Fig. 2) with the pelleted culture exhibiting a significantly lower rate. Huang & Bungay (1973) used a miniature dissolved oxygen electrode to demonstrate a progressive decrease in oxygen tension towards the centre of the pellet across its radius. Their report is consistent with our observation of a lower specific oxygen uptake rate in pelleted culture as, presumably, only the outer layers of hyphae are able to assimilate oxygen at maximum rate. AEC was also dependent on morphology, the values observed in the pelleted culture being considerably lower throughout the culture (Fig. 2). Intermediate AEC values were also observed in the ‘ micropelleted ’ cultures (those with intermediate morphology between pellets and filamentous mycelium). Antibiotic production is known to be inhibited under conditions that results in a low intra-cellular ATP content throughout growth (Yang & Wang, 1996). When filamentous cultures were subjected to decreased aeration, resulting in oxygen limitation, AEC was less than 0±5 throughout the culture and no penicillin was detected (Fig. 2). Oxygen limitation was defined as the condition prevailing when the biomass concentration was dependent on the dissolved oxygen concentration (for a discussion see Clark, Langley & Bushell, 1995). The air-flow required to effect oxygen limitation (0±1 vvm) was too small to allow accurate OUR measurement. Our results indicate that penicillin production is inhibited by pellet formation and that this effect is probably a result of oxygen limitation. Pelleted cultures exhibit a decrease in AEC value which is likely to be a result of a decrease in oxygen uptake rate occasioned by mass transfer constraints along the radius of the pellet. We would like to thank Dr H. N. Arst (Royal Postgraduate School) for providing the strain of Aspergillus nidulans and Dr M. A. Haxell and other members of Animal Health and Discovery Department, Pfizer Central Research for support
REFERENCES Bushell, M. E., Bell, S. L., Scott, M. F., Snell, K., Speir, R. E., Wardell, J. N. & Sanders, P. G. (1993). A three-phase pattern in growth, monoclonal antibody production and metabolite exchange in a hybridoma bioreactor culture. Biotechnology and Bioengineering 42, 133–139. Carilli, A. (1961). Aeration study. III. Continuous measurement of dissolved oxygen during fermentation in large fermenters. Scientific Report of the Instituto Superiore di Sanita[ (Rome) 1 177–189. Carter, B. L. A. & Bull, A. T. (1971). The effect of oxygen tension in the medium on the morphology and growth kinetics of Aspergillus nidulans. Journal of General Microbiology 66, 265–273. Clark, G. J., Langley, D. & Bushell, M. E. (1995). Oxygen limitation can induce microbial secondary metabolite formation : investigations with miniature electrodes in shaker and bioreactor culture. Microbiology 141, 663–669. Cronenberg, C. C. H., Ottengraf, S. P. P., van den Heuvel, J. C., Pottel, F., Sziele, D., Schu$ gerl, K. & Bellgardt, K. H. (1994). Influence of age and structure of Penicillium chrysogenum pellets on the internal concentration profiles. Bioprocess Engineering 10, 209–216. Dion, V. M., Carilli, A., Sermonti, G. & Chain, E. B. (1954). The effect of mechanical agitation on the morphology of P. chrysogenum in stirred fermentation. Scientific Report of the Instituto Superiore di Sanita[ (Rome) 17, 184–205. Espesso, P. (1992). Carbon catabolite repression can account for the temporal pattern of expression of a penicillin biodefined gene in Aspergillus nidulans. Molecular Microbiology 6, 1457–1465. Georgiou, G. & Shuler, M. L. (1986). A computer model for the growth and differentiation of a fungal colony on solid substrate. Biotechnology and Bioengineering 28, 405–416. Gray, C. T., Wimpenny, J. W. T. & Mossman, M. R. (1966). Regulation of the metabolism in facultative bacteria. Part III. Effects of aerobiosis and anaerobiosis and nutrients on the formation of Krebs cycle enzymes in E. coli. Biochemica et Biophysica Acta 117, 33–41. Huang, M. Y. & Bungay, H. R. (1973). Microprobe measurements of oxygen concentration in mycelial pellets. Biotechnology and Bioengineering 15, 1193–1197. Huck, T. A., Porter, N. & Bushell, M. E. (1991). Positive selection of antibioticproducing soil isolates. Journal of General Microbiology 137, 2321–2329. Kobayshi, H., van Dedeem, G. & Moo-Young, M. (1973). Oxygen transfer into mycelial pellets. Biotechnology and Bioengineering 27, 45–47. Lynch, H. C. & Bushell, M. E. (1995). The physiology of erythromycin biosynthesis in cyclic fed batch culture. Microbiology 141, 3105–3111. Martin, J. F. & Demain, A. L. (1977). Effect of exogenous nucleotides on the candicidin fermentation. Canadian Journal of Microbiology 23, 1334–1339. McDermott, J. F., Lethbridge, G. & Bushell, M. E. (1993). Estimation of the kinetic constants and elucidation of trends in growth and erythromycin production in batch and continuous culture of Saccharopolyspora erythraea using curve fitting techniques. Enzymes and Microbial Technology 15, 657–663. Mitard, A. & Riba, J. P. (1988). Morphology and growth of Aspergillus niger ATCC 26036 cultivated at several different shear rates. Biotechnology and Bioengineering 32, 835–840. Oner, M. D., Erickson, L. E. & Young, S. S. (1986). Utilisation of spline functions for smoothing fermentation data and for estimation of specific rates. Biotechnology and Bioengineering 28, 902–918. Papagianni, M., Mattey, M. & Kristansen, B. (1994). Morphology and citric acid production of A. niger PM1. Biotechnology Letters 16, 929–934. Phillips, D. H. (1966). Oxygen transfer into mycelial pellets. Biotechnology and Bioengineering 8, 456–460. Pirt, S. J. (1970). Principles of Microbe and Cell Cultivation. Blackwell Scientific Publications : Oxford, U.K. Ray, J. (1897). Variations des champignons infe! rieurs sous l’influence du milieu. Thesis. Faculte! des Sciences, Universite! de Paris, Paris, France. Shanahan, J. F. (1991). Effect of carbon dioxide on the rheological behaviour and oxygen transfer in submerged penicillin fermentation. Biotechnology and Bioengineering 38, 1223–1232.
J. Moore and M. E. Bushell Simpson, W. J., Fernendaz, J. L., Hammond, J. R. M., Senior, P. S., McCarthy, B. J., Jago, P. H., Sidorowicz, S., Jassin, J. A. A. & Denyer, S. P. (1990). A highly sensitive assay for adenosine triphosphate employing an improved firefly luciferase reagent. Letters in Applied Microbiology 11, 208–210. Thomas, C. R. (1992). Mycelial morphology. The effect of spore inoculum level. Biotechnology Letters 14, 1071–1074. Trinci, A. J. P. (1970). Kinetics of the growth of mycelial pellets of Aspergillus nidulans. Archives of Microbiology 77, 353–367. Vechtlifshitz, S. E., Sasson, Y. & Braun, S. (1992). Nikkomycin production (Accepted 28 January 1997 )
1241 in pellets of Streptomyces tendae. Journal of Applied Bacteriology 72, 195–200. Whitaker, A. & Long, P. A. (1973). Fungal pelleting. Process Biochemistry 11, 27–31. Yano, T., Kodama, T. & Yamada, K. (1961). Fundamental studies on the aerobic fermentation. Agricultural and Biological Chemistry 25, 580–584. Yang, S. S. & Wang, J. Y. (1996). Morphogenesis, ATP content and oxytetracycline production by Streptomyces rimosus in solid substrate cultivation. Journal of Applied Bacteriology 80, 545–550.