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Manganese peroxidase production in submerged cultures by free and immobilized mycelia of Nematoloma frowardii
Bioresource Technology 97 (2006) 469–476
Manganese peroxidase production in submerged cultures by free and immobilized mycelia of Nematoloma frowardii J. Rogalski a, J. Szczodrak a b
b,* ,
G. Janusz
a
Department of Biochemistry, Maria Curie-Skłodowska University, 3 M.C.-Skłodowska Square, 20-031 Lublin, Poland Department of Industrial Microbiology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland Received 16 March 2004; received in revised form 19 January 2005; accepted 4 March 2005 Available online 20 April 2005
Abstract The agaric basidiomycete Nematoloma frowardii has been suggested as a good alternative for production of the extracellular ligninolytic enzyme, manganese-dependent peroxidase (MnP). Some cultural and environmental factors influencing the enzymatic activity in shaken flasks and aerated fermenter cultures were evaluated to improve the yields of the process. A low nitrogen medium (1.36 mM N added as ammonium tartrate), containing 16 g/l glucose (C/N ratio = 65.3), 2 mM Mn2+ and inoculated with immobilized polyurethane foam mycelium, made it possible to obtain a MnP yield of 2304 nkat/l in 8 days. Under these operational conditions, the enzyme productivity in the immobilized cells of N. frowardii was 1.4 times higher than that obtained with the free fungus. In the procedure with the reusable immobilized mycelium (semi-continuous culture) as many as three subsequent 10 day batches could be fermented by using the same carrier with no loss of MnP activity. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Nematoloma frowardii; Manganese peroxidase; Shake cultures; Fermenter cultures; Recycling strategy; Optimization
1. Introduction During recent years, there has been a great interest in potential application of manganese peroxidase (MnP) in biopulping and biobleaching, as well as in bioremediation processes (Harazono et al., 1996; Hofrichter et al., 1998; Breen and Singleton, 1999). MnP is an extracellular heme-containing glycoprotein produced only by ligninolytic (wood-rotting and litter-degrading) basidiomycetes, especially during the secondary metabolism (Gold et al., 2000). This enzyme catalyzes the H2O2dependent oxidation of Mn2+ to a highly reactive Mn3+. The latter, stabilized by chelating dicarboxylic acids, is a low-molecular-mass diffusible mediator, which nonspecifically oxidizes a variety of phenolic and non-pheno-
*
Corresponding author. Tel.: +48 81 5375909; fax: +48 81 5375959. E-mail address: [email protected] (J. Szczodrak).
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.03.002
lic substances, including lignin and toxic pollutants (Perez and Jeffries, 1992; Moreira et al., 1997a). The aromatic structures are depolymerized via formation of phenoxy or aryl cation radicals, which finally result in the breakdown of the molecule (Hatakka, 1994). Due to the important degradative potential of Mnperoxidase, there is general interest in producing the enzyme biotechnologically. An intensive research program was embarked upon to define nutritional, physiological and environmental factors related to the activation of the MnP system and development of an efficient production system for large-scale operations. Production of MnP by white-rot fungi is highly regulated by nutrients. In Phanerochaete chrysosporium––the most-investigated white-rot fungus––MnP is synthesized in response to nitrogen, carbon or sulfur limitation. Particularly, manganese and nutrient nitrogen have been shown to produce strong regulating effects (Jeffries et al., 1981; Bonnarme and Jeffries, 1990; Gold and Alic, 1993).
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Additionally, environmental factors—such as nutrient feed rate, agitation rate, use of oxygen versus air, and fungus immobilization—have also been proven to promote MnP synthesis (Moreira et al., 1997b). Immobilization of fungal cells is an attractive technique for accomplishing high cell densities in order to achieve a rapid extracellular enzyme production. Entrapment is a commonly used immobilization procedure, but surface adsorption has two advantages—namely its simplicity and better physiological conditions. In fixed-bed bioreactors, operating with immobilized mycelia, colonization of the mycelia in a large surface area biofilm is desirable to avoid mass transfer restrictions (Moreira et al., 1998). Nematoloma frowardii—the South American agaric white-rot fungus—has been intensively investigated in Germany as a producer of Mn-peroxidase and degrader of lignin and humic substances (Hofrichter and Fritsche, 1997; Hofrichter et al., 1999). MnP from this fungus is also capable of mineralizing different aromatics directly, including xenobiotics and polycyclic hydrocarbons (Sack et al., 1997; Hofrichter et al., 1998). These features suggest this organism to be a good candidate for further applications at a larger scale and make it an interesting objective to scale-up ligninolytic production. However, the application of this fungus requires a complete knowledge of the fermentation pattern in shaken flask and fermenter cultures, under conditions similar to those existing in industrial size reactors. This paper describes the optimization of some im-portant culture parameters for overproduction of Mn-peroxidase by free and immobilized mycelia of N. frowardii cultured in batch or semi-continuous operations. In the immobilized cultures, a recycling strategy was proven to reactivate MnP production and to maintain MnP activity for extended periods of time. For this purpose, a fixed-net bioreactor with cubes of polyurethane foam as adsorptive mycelium support was designed.
S9C, Polpren, Zgierz, Poland) with a density of 0.035354 g/cm3 and porosity of 91.93%. Polyurethane foam cubes were washed twice in methanol, then three times in distilled water to ensure a thorough leaching of all impurities. Washed carriers were added to 250 ml conical flasks (15 cubes per flask) or to a 2 l bioreactor (60 cubes), autoclaved with the culture medium and inoculated with the fungal homogenate. 2.3. Media compositions The basal medium contained 13 g glucose, 2 g KH2PO4, 0.5 g MgSO4 Æ 7H2O, 0.1 g CaCl2, 0.5 g ammonium tartrate, 0.25 g yeast extract and 1.8 g sodium succinate per litre; this medium was adjusted to pH 4.5 prior to sterilization. The culture production medium was identical with the basal one, except that Mn2+ (as MnCl2) was supplemented at an appropriate concentration to stimulate MnP production. It was optimized during experiments with respect to manganese, carbon and nitrogen concentration. The optimized culture medium used for the production of MnP had the same composition as the culture production medium, except that glucose, ammonium tartrate and Mn2+ were added at concentrations of 16 g/l, 0.25 g/l and 2 mM (1 mM of Mn2+ added on the third day of cultivation), respectively. All the media were autoclaved for 20 min at 121 °C. 2.4. Preparation of the inocula Conical flasks (250 ml) containing 100 ml of basal medium were inoculated with mycelia on GPY from slant tubes (one tube per flask), and incubated at 28 °C for 21 days. Mycelial mats in cultures were broken in a Waring blender (three times for 10 s at 6000 rpm), and homogenates were used as inocula in shaken flasks and aerated bioreactor cultures. 2.5. Shake cultures
2. Methods 2.1. Organism Stock cultures of N. frowardii (Horak) b19 DSM 11239 (= ATCC 201144; family Strophariaceae) were maintained, through periodic transfer, at 4 °C on GPY agar slants (per litre, 1.0 g of glucose, 0.5 g of peptone, 0.1 g of yeast extract, 20 g of agar) and used for inoculations. 2.2. Preparation of foam for immobilization The fungus was immobilized on cube-shaped carriers (1 cm3 volume) consisting of polyurethane foam (type
Cultivation of free and immobilized mycelia was conducted in 250 ml conical flasks containing 100 ml of basal medium or the optimized culture production medium, and 15 cubes per flask of polyurethane foam (only for immobilization experiments). Each flask was seeded with 5 ml homogenate of active mycelium and placed on a rotary shaker at 150 rpm and 23 °C for 34 days (unless otherwise stated). 2.6. Bioreactor cultures Experiments were performed in 2 l batches in a 3 l glass fermenter (BioFlo III, New Brunswick Scientific, Edison, NY, USA) equipped with pH, temperature and pO2-sensors. Unless otherwise stated, fermentation
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conditions were the following: temperature was 23 °C; pH was not regulated; agitation rate was 200 rpm. The bioreactor was aerated with air through internal aeration loops at a constant flow rate of one litre per min. Antifoam B emulsion (Sigma–Aldrich Fine Chemicals, St. Louis, MO, USA) was added periodically to the fermenter cultures in order to break the foam. The free mycelium was grown as pellets in the optimized culture production medium. The medium was inoculated with 10% (v/v) of the fungal homogenate, and the culture was run for 24 days. In the bioreactor operating with the immobilized mycelium, a set used for the immobilization was designed. It consisted of a Teflon net with four combined pockets. The immobilization set was filled with 1 cm3 cubes of polyurethane foam (15 cubes per pocket) and placed into the fermenter at its wall. The bioreactor with the optimized culture production medium was inoculated with 10% (v/v) of the fungal homogenate, and the culture was run for 44 days. Manganese peroxidase was produced in a semi-continuous mode by applying three alternate feeding cycles, in which the culture fluid was replaced periodically by a fresh medium every 10 days. This procedure led to a repeated MnP production at high levels of activity. 2.7. Assays Samples of culture fluids were withdrawn periodically and analyzed for MnP activity and pH. The activity of MnP was measured by a modified method of Wariishi et al. (1992). The final volume of 1 ml reaction mixture contained 50 mM sodium malonate buffer (pH 4.5), 0.5 mM MnCl2, 0.2 mM hydrogen peroxide, and the enzyme solution suitably diluted to maintain linearity. The reaction was initiated at 25 °C by addition of H2O2, and the initial rate of Mn3+-malonate chelate complex formation was monitored spectrophotometrically by following the initial increase in absorbance at 270 nm (e270 = 11,590 M 1 cm 1). One unit of MnP activity
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(U) was calculated as 1 lmol of oxidized substrate per minute, and expressed as nanokatals per litre of culture (1 U corresponds to 16.67 nkat). Spectrophotometric measurements were obtained with a UV–visible light spectrophotometer (model UV-160A; Shimadzu, Tokyo, Japan). Pellet size distribution was determined on an aliquot spread onto Petri plates, according to Moreira et al. (2000a). 2.8. Statistics Submerged cultures were performed in triplicate, and analyses were carried out at least in duplicate. The values reported here are the means, and standard deviations were less than 1% in all cases. Other methodological details are given in tables and figures.
3. Results and discussion 3.1. Effect of cultural and environmental conditions on MnP production It has already been reported that secretion of MnP enzymes by different white-rot fungi is strongly dependent on growth conditions (Martinez et al., 1996). Therefore, different manganese, nitrogen and carbon concentrations, as well as agitation rate and immobilization of the fungus, were investigated to determine optimal physiological and environmental conditions to maximize MnP production by N. frowardii in shaken flask cultures. The influence of Mn2+ concentration and mode of its introduction into the culture on production of extracellular MnP in 34 day shaken flask cultures of N. frowardii is given in Table 1. Total Mn2+ concentrations were varied in a range from 0 to 6 mM, and 1 mM of Mn2+ was introduced into the medium every 3 days. In the absence of Mn2+, N. frowardii produced relatively small amounts of the enzyme (up to 505 nkat/l after 15 days). Almost
Table 1 Effect of Mn2+ concentration on the production of MnP by N. frowardii in 34 day shaken flask culturesa Mn2+ in the medium
Daysb
Concentration (mM) Total
Mode of introduction into the culture
–
Control without Mn2+ 2+
Mn added on day 3 Mn2+ (1 mM) added at start 1 mM added on day 3 1 mM added on days 3 and 6 1 mM added on days 3, 6 and 9 1 mM added on days 3, 6, 9 and 12 1 mM added on days 3, 6, 9, 12 and 15
1 1 2 3 4 5 6 a b
MnP activity (nkat/l)
15
504.8 ± 4.9
18 15 15 15 15 15 15
507.6 ± 2.8 1009.6 ± 7.7 1209.6 ± 10.8 822.9 ± 6.1 808.8 ± 5.6 755.5 ± 6.4 718.1 ± 5.6
Composition of the medium was the same as that of the basal one, except for the Mn2+, its concentration was varied as indicated. Incubation period for maximum activity.
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the same level of MnP was detected when 1 mM of Mn2+ was added to the culture medium on day 3, but the maximum activity was delayed until day 18. Cultivation of the fungus on the complete medium, containing 1 mM of Mn2+ as a regular medium component, doubled the enzyme activity as compared with Mn-free medium. However, the maximum MnP activity (1210 nkat/l) for this fungus was measured in the presence of 2 mM Mn2+, of which 1 mM was added on the third day of cultivation. Subsequent addition of Mn2+ to the complete medium at regular 3-day intervals decreased enzyme productivity (even by 41% at 6 mM Mn2+), but the time of maximum MnP production (15 days) was unaffected by the increasing Mn2+ concentration. Manganese is reported to be a potent regulator of ligninolytic activity (Brown et al., 1990; Perez and Jeffries, 1992). It stimulates mostly the production of MnP, so that up to 24 times higher levels of the enzyme were achieved in cultures containing Mn2+ compared with Mn-free cultures (Bonnarme and Jeffries, 1990). In the present study, a Mn2+ concentration of 2 mM was found to be the best for synthesis of MnP by N. frowardii in shaken flask cultures. Thus, about 2.4 times higher yields of the enzyme were obtained in cultures containing Mn2+, compared with non-induced cultures. The optimum Mn2+ concentration was much lower than that (5 mM) reported by Moreira et al. (1998) for continuous immobilized cultures of P. chrysosporium, but still much higher than that (0.73 mM) established by Perez and Jeffries (1992) and Ben Hamman et al. (1999) for batch cultures of P. chrysosporium and Phanerochaete flavido-alba. Also, Giardina et al. (2000) showed that MnP production by Pleurotus ostreatus increased 3–10 times during solid-state fermentation on different kinds of wood sawdust supplemented with 0.1 mM MnSO4. It has also been reported (Kerem and Hadar, 1995; Camarero et al., 1996) that addition of Mn2+ enhances
the degradation of lignin during solid-state fermentation with different Pleurotus species, without increasing MnP levels. Manganese was not required for MnP production and biobleaching by Bjerkandera sp. strain BOS55 (Moreira et al., 1999) or by the coprophilic fungus Stropharia semiglobata (Steffen et al., 2000). Furthermore, a repression of MnP activity in Pleurotus eryngii liquid culture supplemented with MnSO4 was also reported (Martinez et al., 1996). In the present work, all Mn2+-containing cultures of N. frowardii showed higher MnP activity than Mn2+-free cultures. However, an increase in the concentration of extra manganese (from 2 to 6 mM Mn2+) repressed MnP production, as compared with that where the optimum dose of Mn2+ was applied. In a further study, an adequate C/N ratio was selected to enhance MnP production. For this purpose, different culture media were assayed. The concentration of carbon (added as glucose) varied from 5 to 16 g/l (27.78–88.88 mM) and that of nitrogen (added as ammonium tartrate) from 0.25 to 1.0 g/l (1.36– 5.43 mM). C/N ratio in the respective media ranged from 5.1 to 65.3. Titres of MnP were measured for 34 day in shaken flask cultures with the optimized Mn2+ dose. The results indicated that MnP activity reached maximum levels (2500 nkat/l on day 15) in cultures with the highest C/N ratio (65.3), i.e., containing 16 g/l glucose (88.88 mM) and 0.25 g/l ammonium tartrate (1.36 mM), respectively (Table 2). Hence, the selected C and N concentrations were used in further studies. Under these nutritional conditions (a strict Nlimitation), the enzyme productivity was over two times higher than that obtained on the initial basal medium used as a control (C/N ratio = 26.5). It should be stressed here that in all cultures with 1 g/l ammonium tartrate (5.43 mM), the increase in nitrogen concentration significantly suppressed MnP activities. Contras-
Table 2 Effects of C/N ratio on MnP production by N. frowardii in 34 day shaken flask culturesa Glucose (g/l)
Ammonium tartrate (g/l)
C/N ratiob (mM/mM)
Daysc
MnP activity (nkat/l)
5
0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00 0.50d
20.4 10.2 5.1 40.8 20.4 10.2 65.3 32.7 16.4 26.5
12 12 15 15 15 15 15 15 15 15
905.1 ± 7.1 955.4 ± 5.7 644.4 ± 4.6 951.5 ± 8.0 1586.9 ± 12.3 430.1 ± 2.9 2500.0 ± 17.5 2050.0 ± 12.2 675.0 ± 4.2 1149.1 ± 9.6
10
16
13d
a Composition of the medium was the same as that of the basal one, but glucose and ammonium tartrate concentrations varied as indicated. A total Mn2+ concentration was 2 mM (1 mM of Mn2+ added on the third day of cultivation). b Calculated as C (glucose)/N (ammonium tartrate) ratio. c Incubation period for maximum activity. d Original basal medium (control).
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tingly, the increase in carbon concentration enhanced MnP yields. Having taken this fact into account, it is postulated that the development of ligninolytic activity by N. frowardii is the result of a nitrogen-based regulatory mechanism rather than that of C limitation. Similarly to P. chrysosporium and related lignin-degrading fungi, this fungus requires a strict N-limitation to maximize MnP production (Kirk et al., 1986; Hatakka, 1994; Buswell et al., 1995; Moilanen et al., 1996). Meanwhile, Clitocybula dusenii did not need such a strict Nlimitation for producing Mn peroxidase (Ziegenhagen and Hofrichter, 2000), and MnP peroxidase titres in cultures of Bjerkandera sp. BOS55 and P. flavido-alba were stimulated by a high N medium (Mester et al., 1996; Ben Hamman et al., 1997; Moreira et al., 2000a). The effect of increasing the agitation rate of the rotary shaker from 100 to 300 rpm on MnP production by free and immobilized mycelia of N. frowardii is shown in Table 3. In free pellet cultures, the enzyme activity attained its maximum (about 2880 nkat/l) after 15 days of incubation at an agitation speed of 200 rpm. At 150 rpm, and in the same time period, MnP activity achieved a slightly higher value in comparison with the one recorded at 100 rpm, but the use of the shaker with a revolution rate of 100 rpm required a longer period of incubation (18 days) for the maximum enzyme activity. Increase of the agitation rate up to 300 rpm, however, had an undesirable inactivation effect on the secreted enzyme, reducing its enzymatic activity by about 24% compared with the maximum reached at 200 rpm. The drop in MnP activity at an excessively high agitation rate (300 rpm) was probably caused by the effect of mechanical (shear) stress on the fungal pellets. This follows from the distribution of the pellet sizes at the different agitation rates. Initially, on day 2, the pellet size was distributed between those smaller than 0.5 mm and those greater than 3.5 mm. By days 5 and 7, however, the ratio of pellets in the smaller range (between 0.5 mm and 1.2 mm) for the agitation rate of 300 rpm
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increased significantly. Moreover, under these conditions, a small proportion of pellets (about 2–5% of the total population) disintegrated into hyphal branches. These facts indicate that shear stress affected the secretion and/or stability of the N. frowardii enzyme and resulted in a decrease in MnP activity. Different results in this respect were obtained by Moreira et al. (2000a) in shaken flask cultures of Bjerkandera sp. BOS55. Though the distribution of the pellet size was similar to N. frowardii, the level of MnP activity was independent of the rate of agitation (from 150 to 275 rpm) and reached similar values in the range of 3334 nkat/l after 4–6 days of cultivation. These authors found the increase in the agitation rate to imply a higher consumption of nutrients and an earlier appearance of MnP activity in pellets of smaller size, but observed no significant effect on MnP activity. The results obtained when working with the immobilized polyurethane-foam mycelium are clearly better than those achieved with the free fungus. For example, at agitation rate of 200 rpm the volume activity of MnP was over 38% higher than that achieved by free pellets under the same agitation conditions. However, the process was slower and the maximum activity was delayed until day 18 (Table 3). At lower agitation rate (150 rpm), the production of MnP from the immobilized mycelium was also higher (by 16.4%) in comparison with the experiments performed with the free fungus, but the enzyme achieved its maximum 3 days earlier, i.e. on day 12. It should be noted that N. frowardii grew rapidly into the polyurethane-foam cubes. After a few days of incubation, the carrier was completely penetrated and its surface covered with a dense layer of fungal hyphae. The immobilized fungus and the support were highly stable during agitation. Neither fragments of the mycelium nor fragments of the support sheared off into the culture liquid. Thus, polyurethane-foam cubes of 1.0 cm3 can be considered a good immobilization matrix for N. frowardii b19. 3.2. Enzyme production in fermenter cultures
Table 3 Effect of agitation rate and immobilization of the mycelium on MnP production by N. frowardii in shaken flask cultures on the optimized culture production medium Shaker speed (rpm)
Daysa
MnP activity (nkat/l)
Free mycelium (as pellets)b
100 150 200 300
18 15 15 15
2345.9 ± 14.6 2398.3 ± 14.1 2879.8 ± 13.9 2193.1 ± 10.3
Immobilized mycelium on polyurethane foam (as biofilm)c
150 200
12 18
2792.4 ± 16.3 3981.1 ± 23.5
a b c
Incubation period for maximum activity. Cultivation time was 33 days; temp. 23 °C. Cultivation time was 27 days; temp. 23 °C.
MnP production by the free mycelium of N. frowardii was also carried out in a 3 l fermenter with constant air flow and agitation rates. Typical culture profiles are shown in Fig. 1. Biosynthesis conditions were fixed taking into account the previous results obtained in agitated flask cultures (the optimized culture production medium with the determined Mn2+ dose and C/N ratio, inoculum quantity, initial pH of the medium, agitation rate and incubation temperature), as an initial step to larger scale fermentations. The extracellular concentration of MnP continued to rise with time to reach the maximum activity of 1627 nkat/l on the 12th day. Thereafter, the enzyme activity declined very quickly. The pH of the medium decreased (from 4.8 to 4.1) over the
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Fig. 1. Time course of MnP production, oxygen consumption, and pH during aerated fermenter cultures of free N. frowardii mycelium. The optimized culture production medium with initial pH of 4.8 was used. Symbols: (s) MnP activity; (d) pH; (n) oxygen concentration.
whole period of incubation. Under air supply, the dissolved oxygen was gradually depleted by the free mycelium, and its content declined by 15% after 12 days of cultivation. Afterwards, the oxygen consumption remained stable with a low decrease at the end of the fermentation, after 24 days. It should be noted here that the cultivation of N. frowardii in a bioreactor resulted in much lower enzymatic activities than those achieved by the free fungus in shaken flasks. In fermenter culture, however, the enzyme production reached maximum levels within shorter incubation periods, and relatively high titres of MnP were produced under air atmosphere. This fact offers an economic advantage in the culturing of N. frowardii in comparison with the model fungus P. chrysosporium, which requires the use of oxygen or oxygen enriched air in bioreactors to express ligninolytic activity (Moreira et al., 1998). In order to maximize MnP production and stability in bioreactor cultures, a semi-continuous operation was tested with a reusable immobilized mycelium of N. frowardii. For this purpose, a fixed-net bioreactor aerated with air was designed and filled with the immobilized polyurethane-foam mycelium. The results shown in Fig. 2 indicate that both the stability and productivity of the process can be highly improved by following this strategy. Similarly to the fermenter culture with free mycelium, the production of MnP started shortly after the addition of extra Mn2+ to the optimized culture production medium and reached a level of about 1600 nkat/ l after 14 days of incubation. After the culture liquid was replaced by a fresh medium, this high MnP level was actually reached again within a shorter incubation period, i.e. 8 days. The next two cycles gave even better results: the highest MnP activities of 2078–2304 nkat/l were obtained after 8 days of cultivation. Observation
Fig. 2. Repeated batch production of MnP in fermenter culture using N. frowardii mycelium immobilized on polyurethane foam. The enzyme production was performed on the optimized culture production medium in 10-day cycles. Arrows in respective curves show addition of Mn2+ into the medium. Vertical dash lines separate successive cycles and show the days on which the culture liquid was replaced by fresh medium. Symbols: (s) MnP activity; (d) pH; (n) oxygen concentration.
of the early stages of the operation (first cycle) shows a delay in MnP production, indicating that the mycelium took some time before acclimating to the conditions leading to MnP secretion. Under air supply, the dissolved oxygen was continuously consumed by the immobilized mycelium over the whole period of incubation (44 days), and its content dropped by 17%. However, in respective batches, the depletion of dissolved oxygen by the immobilized fungus was lower than that recorded in the bioreactor for free pellet culture (Fig. 1). Consequently, an 8-day period was assumed to be the optimum time for MnP synthesis by the immobilized N. frowardii mycelium in repeated bioreactor cultures. It is noteworthy that surface cultures of N. frowardii showed an average MnP activity of 5.35 nkat/l, reaching this level after 24 days of cultivation (Sack et al., 1997). In the present study, fermenter cultures of the reusable immobilized mycelium had 299–431 times higher MnP levels, which they reached within three times shorter incubation periods. The immobilized fungus also produced from 1.3 to 1.4 times higher amounts of MnP compared with those obtained in the bioreactor with the free mycelium. Contrary results in this respect were reported by Moreira et al. (2000b). Under the conditions applied (a 2 l fermenter with mycelium immobilized in polyurethane foam cubes; agitation rate, 150 rpm; aeration flow, 0.8 v v 1 min 1; temperature, 30 °C; pH not regulated; inoculum quantity, 10% v/v), relatively low MnP activities (about 417 nkat/l after 8–9 days) were reached in batch cultures of the immobilized Bjerkandera sp. BOS55 mycelium compared with those achieved under the same operational conditions with free pellet cultures (2500 nkat/l after 7–8 days).
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Recycling of the immobilized mycelium enabled repeated use of the biomass for MnP production (semicontinuous culturing). Moreover, the recycling procedure maintained the fungus in its idiophase, in which MnP is normally produced, so that MnP production started immediately after the addition of fresh medium. Ziegenhagen and Hofrichter (2000) reported a similar procedure to produce high amounts of MnP with a reusable immobilized mycelium of the agaric basidiomycete fungus C. dusenii in shaken flask cultures. Also, Rivela et al. (2000), using an immersion bioreactor, obtained a high MnP activity produced under solid-state conditions by the mycelium of P. chrysosporium immobilized on cubes of fibrous nylon sponge.
4. Conclusions Summarizing, the data presented herein show that the wood-decaying fungus N. frowardii is a good producer of extracellular MnP in submerged cultures, either as free or immobilized mycelium, and hence justifies further investigations on the application of the strain to obtain an active ligninolytic preparation for biotechnological purposes. Also, the appropriate combination of operational conditions (the use of N-limited medium, sequential addition of adequate Mn2+, immobilized polyurethane-foam mycelium), recycling strategy and bioreactor design (fixed-net fermenter aerated with air) allowed for semi-continuous and effective MnP production by N. frowardii during the idiophase for prolonged periods.
Acknowledgements This study was financially supported in part by the Polish State Committee for Scientific Research (Grant PBZ-KBN-098/T09/2003) and by the BW/UMCS and BS/UMCS research programmes.
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Brown, J.A., Glenn, J.K., Gold, M.K., 1990. Manganese regulates expression of manganese peroxidase by Phanerochaete chrysosporium. J. Bacteriol. 172, 3125–3140. Buswell, J.A., Cai, Y.J., Chang, S.T., 1995. Effect of nutrient nitrogen on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes. FEMS Microbiol. Lett. 128, 81–88. Camarero, S., Bockle, B., Martinez, M.J., Martinez, A.T., 1996. Manganese-mediated lignin degradation by Pleurotus pulmonarius. Appl. Environ. Microbiol. 62, 1070–1072. Giardina, P., Palmieri, G., Fontanella, B., Rivieccio, V., Sannia, G., 2000. Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust. Arch. Biochem. Biophys. 376, 171–179. Gold, M.H., Alic, M., 1993. Molecular biology of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Microbiol. Rev. 57, 605–622. Gold, M.H., Youngs, H.L., Gelpke, M.D.S., 2000. Manganese peroxidase. In: Sigel, A., Sigel, H. (Eds.), Metal Ions in Biological System. Marcel Dekker Inc., New York, Basel, pp. 559–586. Harazono, K., Kondo, R., Sakai, K., 1996. Bleaching of hardwood Kraft pulp with manganese peroxidase from Phanerochaete sordida YK-624 without addition of MnSO4. Appl. Environ. Microbiol. 62, 913–917. Hatakka, A., 1994. Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13, 125–135. Hofrichter, M., Fritsche, W., 1997. Depolymerization of low-rank coal by extracellular fungal enzyme systems. II. The ligninolytic enzymes of the coal-humic-acid-depolymerizing fungus Nematoloma frowardii b19. Appl. Microbiol. Biotechnol. 47, 419–424. Hofrichter, M., Scheibner, K., Schneegaß, I., Fritsche, W., 1998. Enzymatic combustion of aromatic and aliphatic compounds by manganese peroxidase from Nematoloma frowardii. Appl. Environ. Microbiol. 64, 399–404. Hofrichter, M., Vares, T., Kalsi, M., Galkin, S., Scheibner, K., Fritsche, W., Hatakka, A., 1999. Production of manganese peroxidase and organic acids and mineralization of 14C-labelled lignin (14C-DHP) during solid-state fermentation of wheat straw with the white rot fungus Nematoloma frowardii. Appl. Environ. Microbiol. 65, 1864–1870. Jeffries, T.W., Choi, S., Kirk, T.K., 1981. Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 42, 290–296. Kerem, Z., Hadar, Y., 1995. Effect of manganese on preferential degradation of lignin by Pleurotus ostreatus during solid-state fermentation. Appl. Environ. Microbiol. 61, 3057–3062. Kirk, T.K., Croan, S., Tien, M., Murtagh, E., Farell, R.L., 1986. Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb. Technol. 8, 27–32. Martinez, M.J., Ruiz-Duenaz, F.J., Guillen, F., Martinez, A.T., 1996. Purification and catalytic properties of two manganese peroxidase isoenzymes from Pleurotus eryngii. Eur. J. Biochem. 237, 424– 432. Mester, T., Pena, M., Field, J.A., 1996. Nutrient regulation of extracellular peroxidases in the white-rot fungus Bjerkandera sp. strain BOS55. Appl. Microbiol. Biotechnol. 44, 778–784. Moilanen, A.M., Lundell, T., Vares, T., Hatakka, A., 1996. Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata. Appl. Microbiol. Biotechnol. 45, 792– 799. Moreira, M.T., Palma, C., Feijoo, G., Lema, J.M., 1997a. Decolorisation of ion-exchange effluents derived from sugar-mill operations by Bjerkandera sp. BOS55. Int. Biodeter. Biodegr. 40, 125–129. Moreira, M.H., Feijoo, G., Palma, C., Lema, J.M., 1997b. Continuous production of manganese peroxidase by Phanerochaete
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chrysosporium immobilized on polyurethane foam in a pulsed packed bed bioreactor. Biotechnol. Bioeng. 56, 130–137. Moreira, M.H., Palma, C., Feijoo, G., Lema, J.M., 1998. Strategies for the continuous production of ligninolytic enzymes in fixed and fluidized bed bioreactors. J. Biotechnol. 66, 27–39. Moreira, M.T., Feijoo, G., Sierra-Alvarez, R., Field, J.A., 1999. Reevaluation of the manganese requirement for the biobleaching of kraft pulp by white rot fungi. Biores. Technol. 70, 255–260. Moreira, M.T., Feijoo, G., Lema, J.M., 2000a. Manganese peroxidase production by Bjerkandera sp. BOS55. 1. Regulation of enzymatic production. Bioprocess Eng. 23, 657–661. Moreira, M.T., Torrado, A., Feijoo, G., Lema, J.M., 2000b. Manganese peroxidase production by Bjerkandera sp. BOS55. 2. Operation in stirrer tank reactors. Bioprocess Eng. 23, 657–661. Perez, J., Jeffries, T.W., 1992. Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 58, 2402–2409.
Rivela, I., Rodriguez Couto, S., Sanroma´n, A., 2000. Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor. Biotechnol. Lett. 22, 1443– 1447. Sack, U., Hofrichter, M., Fritsche, W., 1997. Degradation of polycyclic aromatic hydrocarbons by manganese peroxidase of Nematoloma frowardii. FEMS Microbiol. Lett. 152, 227–234. Steffen, K.T., Hofrichter, M., Hatakka, A., 2000. Mineralisation of 14 C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi. Appl. Microbiol. Biotechnol. 54, 819–825. Wariishi, H., Valli, K., Gold, M.H., 1992. Manganese (II) oxidation by manganese peroxidase from the basidiomysete Phanerochaete chrysosporium. J. Biol. Chem. 267, 23688–23695. Ziegenhagen, D., Hofrichter, M., 2000. A simple and rapid method to gain high amounts of manganese peroxidase with immobilized mycelium of the agaric white-rot fungus Clitocybula dusenii. Appl. Microbiol. Biotechnol. 53, 553–557.
Manganese peroxidase production in submerged cultures by free and immobilized mycelia of Nematoloma frowardii J. Rogalski a, J. Szczodrak a b
b,* ,
G. Janusz
a
Department of Biochemistry, Maria Curie-Skłodowska University, 3 M.C.-Skłodowska Square, 20-031 Lublin, Poland Department of Industrial Microbiology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland Received 16 March 2004; received in revised form 19 January 2005; accepted 4 March 2005 Available online 20 April 2005
Abstract The agaric basidiomycete Nematoloma frowardii has been suggested as a good alternative for production of the extracellular ligninolytic enzyme, manganese-dependent peroxidase (MnP). Some cultural and environmental factors influencing the enzymatic activity in shaken flasks and aerated fermenter cultures were evaluated to improve the yields of the process. A low nitrogen medium (1.36 mM N added as ammonium tartrate), containing 16 g/l glucose (C/N ratio = 65.3), 2 mM Mn2+ and inoculated with immobilized polyurethane foam mycelium, made it possible to obtain a MnP yield of 2304 nkat/l in 8 days. Under these operational conditions, the enzyme productivity in the immobilized cells of N. frowardii was 1.4 times higher than that obtained with the free fungus. In the procedure with the reusable immobilized mycelium (semi-continuous culture) as many as three subsequent 10 day batches could be fermented by using the same carrier with no loss of MnP activity. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Nematoloma frowardii; Manganese peroxidase; Shake cultures; Fermenter cultures; Recycling strategy; Optimization
1. Introduction During recent years, there has been a great interest in potential application of manganese peroxidase (MnP) in biopulping and biobleaching, as well as in bioremediation processes (Harazono et al., 1996; Hofrichter et al., 1998; Breen and Singleton, 1999). MnP is an extracellular heme-containing glycoprotein produced only by ligninolytic (wood-rotting and litter-degrading) basidiomycetes, especially during the secondary metabolism (Gold et al., 2000). This enzyme catalyzes the H2O2dependent oxidation of Mn2+ to a highly reactive Mn3+. The latter, stabilized by chelating dicarboxylic acids, is a low-molecular-mass diffusible mediator, which nonspecifically oxidizes a variety of phenolic and non-pheno-
*
Corresponding author. Tel.: +48 81 5375909; fax: +48 81 5375959. E-mail address: [email protected] (J. Szczodrak).
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.03.002
lic substances, including lignin and toxic pollutants (Perez and Jeffries, 1992; Moreira et al., 1997a). The aromatic structures are depolymerized via formation of phenoxy or aryl cation radicals, which finally result in the breakdown of the molecule (Hatakka, 1994). Due to the important degradative potential of Mnperoxidase, there is general interest in producing the enzyme biotechnologically. An intensive research program was embarked upon to define nutritional, physiological and environmental factors related to the activation of the MnP system and development of an efficient production system for large-scale operations. Production of MnP by white-rot fungi is highly regulated by nutrients. In Phanerochaete chrysosporium––the most-investigated white-rot fungus––MnP is synthesized in response to nitrogen, carbon or sulfur limitation. Particularly, manganese and nutrient nitrogen have been shown to produce strong regulating effects (Jeffries et al., 1981; Bonnarme and Jeffries, 1990; Gold and Alic, 1993).
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Additionally, environmental factors—such as nutrient feed rate, agitation rate, use of oxygen versus air, and fungus immobilization—have also been proven to promote MnP synthesis (Moreira et al., 1997b). Immobilization of fungal cells is an attractive technique for accomplishing high cell densities in order to achieve a rapid extracellular enzyme production. Entrapment is a commonly used immobilization procedure, but surface adsorption has two advantages—namely its simplicity and better physiological conditions. In fixed-bed bioreactors, operating with immobilized mycelia, colonization of the mycelia in a large surface area biofilm is desirable to avoid mass transfer restrictions (Moreira et al., 1998). Nematoloma frowardii—the South American agaric white-rot fungus—has been intensively investigated in Germany as a producer of Mn-peroxidase and degrader of lignin and humic substances (Hofrichter and Fritsche, 1997; Hofrichter et al., 1999). MnP from this fungus is also capable of mineralizing different aromatics directly, including xenobiotics and polycyclic hydrocarbons (Sack et al., 1997; Hofrichter et al., 1998). These features suggest this organism to be a good candidate for further applications at a larger scale and make it an interesting objective to scale-up ligninolytic production. However, the application of this fungus requires a complete knowledge of the fermentation pattern in shaken flask and fermenter cultures, under conditions similar to those existing in industrial size reactors. This paper describes the optimization of some im-portant culture parameters for overproduction of Mn-peroxidase by free and immobilized mycelia of N. frowardii cultured in batch or semi-continuous operations. In the immobilized cultures, a recycling strategy was proven to reactivate MnP production and to maintain MnP activity for extended periods of time. For this purpose, a fixed-net bioreactor with cubes of polyurethane foam as adsorptive mycelium support was designed.
S9C, Polpren, Zgierz, Poland) with a density of 0.035354 g/cm3 and porosity of 91.93%. Polyurethane foam cubes were washed twice in methanol, then three times in distilled water to ensure a thorough leaching of all impurities. Washed carriers were added to 250 ml conical flasks (15 cubes per flask) or to a 2 l bioreactor (60 cubes), autoclaved with the culture medium and inoculated with the fungal homogenate. 2.3. Media compositions The basal medium contained 13 g glucose, 2 g KH2PO4, 0.5 g MgSO4 Æ 7H2O, 0.1 g CaCl2, 0.5 g ammonium tartrate, 0.25 g yeast extract and 1.8 g sodium succinate per litre; this medium was adjusted to pH 4.5 prior to sterilization. The culture production medium was identical with the basal one, except that Mn2+ (as MnCl2) was supplemented at an appropriate concentration to stimulate MnP production. It was optimized during experiments with respect to manganese, carbon and nitrogen concentration. The optimized culture medium used for the production of MnP had the same composition as the culture production medium, except that glucose, ammonium tartrate and Mn2+ were added at concentrations of 16 g/l, 0.25 g/l and 2 mM (1 mM of Mn2+ added on the third day of cultivation), respectively. All the media were autoclaved for 20 min at 121 °C. 2.4. Preparation of the inocula Conical flasks (250 ml) containing 100 ml of basal medium were inoculated with mycelia on GPY from slant tubes (one tube per flask), and incubated at 28 °C for 21 days. Mycelial mats in cultures were broken in a Waring blender (three times for 10 s at 6000 rpm), and homogenates were used as inocula in shaken flasks and aerated bioreactor cultures. 2.5. Shake cultures
2. Methods 2.1. Organism Stock cultures of N. frowardii (Horak) b19 DSM 11239 (= ATCC 201144; family Strophariaceae) were maintained, through periodic transfer, at 4 °C on GPY agar slants (per litre, 1.0 g of glucose, 0.5 g of peptone, 0.1 g of yeast extract, 20 g of agar) and used for inoculations. 2.2. Preparation of foam for immobilization The fungus was immobilized on cube-shaped carriers (1 cm3 volume) consisting of polyurethane foam (type
Cultivation of free and immobilized mycelia was conducted in 250 ml conical flasks containing 100 ml of basal medium or the optimized culture production medium, and 15 cubes per flask of polyurethane foam (only for immobilization experiments). Each flask was seeded with 5 ml homogenate of active mycelium and placed on a rotary shaker at 150 rpm and 23 °C for 34 days (unless otherwise stated). 2.6. Bioreactor cultures Experiments were performed in 2 l batches in a 3 l glass fermenter (BioFlo III, New Brunswick Scientific, Edison, NY, USA) equipped with pH, temperature and pO2-sensors. Unless otherwise stated, fermentation
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conditions were the following: temperature was 23 °C; pH was not regulated; agitation rate was 200 rpm. The bioreactor was aerated with air through internal aeration loops at a constant flow rate of one litre per min. Antifoam B emulsion (Sigma–Aldrich Fine Chemicals, St. Louis, MO, USA) was added periodically to the fermenter cultures in order to break the foam. The free mycelium was grown as pellets in the optimized culture production medium. The medium was inoculated with 10% (v/v) of the fungal homogenate, and the culture was run for 24 days. In the bioreactor operating with the immobilized mycelium, a set used for the immobilization was designed. It consisted of a Teflon net with four combined pockets. The immobilization set was filled with 1 cm3 cubes of polyurethane foam (15 cubes per pocket) and placed into the fermenter at its wall. The bioreactor with the optimized culture production medium was inoculated with 10% (v/v) of the fungal homogenate, and the culture was run for 44 days. Manganese peroxidase was produced in a semi-continuous mode by applying three alternate feeding cycles, in which the culture fluid was replaced periodically by a fresh medium every 10 days. This procedure led to a repeated MnP production at high levels of activity. 2.7. Assays Samples of culture fluids were withdrawn periodically and analyzed for MnP activity and pH. The activity of MnP was measured by a modified method of Wariishi et al. (1992). The final volume of 1 ml reaction mixture contained 50 mM sodium malonate buffer (pH 4.5), 0.5 mM MnCl2, 0.2 mM hydrogen peroxide, and the enzyme solution suitably diluted to maintain linearity. The reaction was initiated at 25 °C by addition of H2O2, and the initial rate of Mn3+-malonate chelate complex formation was monitored spectrophotometrically by following the initial increase in absorbance at 270 nm (e270 = 11,590 M 1 cm 1). One unit of MnP activity
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(U) was calculated as 1 lmol of oxidized substrate per minute, and expressed as nanokatals per litre of culture (1 U corresponds to 16.67 nkat). Spectrophotometric measurements were obtained with a UV–visible light spectrophotometer (model UV-160A; Shimadzu, Tokyo, Japan). Pellet size distribution was determined on an aliquot spread onto Petri plates, according to Moreira et al. (2000a). 2.8. Statistics Submerged cultures were performed in triplicate, and analyses were carried out at least in duplicate. The values reported here are the means, and standard deviations were less than 1% in all cases. Other methodological details are given in tables and figures.
3. Results and discussion 3.1. Effect of cultural and environmental conditions on MnP production It has already been reported that secretion of MnP enzymes by different white-rot fungi is strongly dependent on growth conditions (Martinez et al., 1996). Therefore, different manganese, nitrogen and carbon concentrations, as well as agitation rate and immobilization of the fungus, were investigated to determine optimal physiological and environmental conditions to maximize MnP production by N. frowardii in shaken flask cultures. The influence of Mn2+ concentration and mode of its introduction into the culture on production of extracellular MnP in 34 day shaken flask cultures of N. frowardii is given in Table 1. Total Mn2+ concentrations were varied in a range from 0 to 6 mM, and 1 mM of Mn2+ was introduced into the medium every 3 days. In the absence of Mn2+, N. frowardii produced relatively small amounts of the enzyme (up to 505 nkat/l after 15 days). Almost
Table 1 Effect of Mn2+ concentration on the production of MnP by N. frowardii in 34 day shaken flask culturesa Mn2+ in the medium
Daysb
Concentration (mM) Total
Mode of introduction into the culture
–
Control without Mn2+ 2+
Mn added on day 3 Mn2+ (1 mM) added at start 1 mM added on day 3 1 mM added on days 3 and 6 1 mM added on days 3, 6 and 9 1 mM added on days 3, 6, 9 and 12 1 mM added on days 3, 6, 9, 12 and 15
1 1 2 3 4 5 6 a b
MnP activity (nkat/l)
15
504.8 ± 4.9
18 15 15 15 15 15 15
507.6 ± 2.8 1009.6 ± 7.7 1209.6 ± 10.8 822.9 ± 6.1 808.8 ± 5.6 755.5 ± 6.4 718.1 ± 5.6
Composition of the medium was the same as that of the basal one, except for the Mn2+, its concentration was varied as indicated. Incubation period for maximum activity.
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the same level of MnP was detected when 1 mM of Mn2+ was added to the culture medium on day 3, but the maximum activity was delayed until day 18. Cultivation of the fungus on the complete medium, containing 1 mM of Mn2+ as a regular medium component, doubled the enzyme activity as compared with Mn-free medium. However, the maximum MnP activity (1210 nkat/l) for this fungus was measured in the presence of 2 mM Mn2+, of which 1 mM was added on the third day of cultivation. Subsequent addition of Mn2+ to the complete medium at regular 3-day intervals decreased enzyme productivity (even by 41% at 6 mM Mn2+), but the time of maximum MnP production (15 days) was unaffected by the increasing Mn2+ concentration. Manganese is reported to be a potent regulator of ligninolytic activity (Brown et al., 1990; Perez and Jeffries, 1992). It stimulates mostly the production of MnP, so that up to 24 times higher levels of the enzyme were achieved in cultures containing Mn2+ compared with Mn-free cultures (Bonnarme and Jeffries, 1990). In the present study, a Mn2+ concentration of 2 mM was found to be the best for synthesis of MnP by N. frowardii in shaken flask cultures. Thus, about 2.4 times higher yields of the enzyme were obtained in cultures containing Mn2+, compared with non-induced cultures. The optimum Mn2+ concentration was much lower than that (5 mM) reported by Moreira et al. (1998) for continuous immobilized cultures of P. chrysosporium, but still much higher than that (0.73 mM) established by Perez and Jeffries (1992) and Ben Hamman et al. (1999) for batch cultures of P. chrysosporium and Phanerochaete flavido-alba. Also, Giardina et al. (2000) showed that MnP production by Pleurotus ostreatus increased 3–10 times during solid-state fermentation on different kinds of wood sawdust supplemented with 0.1 mM MnSO4. It has also been reported (Kerem and Hadar, 1995; Camarero et al., 1996) that addition of Mn2+ enhances
the degradation of lignin during solid-state fermentation with different Pleurotus species, without increasing MnP levels. Manganese was not required for MnP production and biobleaching by Bjerkandera sp. strain BOS55 (Moreira et al., 1999) or by the coprophilic fungus Stropharia semiglobata (Steffen et al., 2000). Furthermore, a repression of MnP activity in Pleurotus eryngii liquid culture supplemented with MnSO4 was also reported (Martinez et al., 1996). In the present work, all Mn2+-containing cultures of N. frowardii showed higher MnP activity than Mn2+-free cultures. However, an increase in the concentration of extra manganese (from 2 to 6 mM Mn2+) repressed MnP production, as compared with that where the optimum dose of Mn2+ was applied. In a further study, an adequate C/N ratio was selected to enhance MnP production. For this purpose, different culture media were assayed. The concentration of carbon (added as glucose) varied from 5 to 16 g/l (27.78–88.88 mM) and that of nitrogen (added as ammonium tartrate) from 0.25 to 1.0 g/l (1.36– 5.43 mM). C/N ratio in the respective media ranged from 5.1 to 65.3. Titres of MnP were measured for 34 day in shaken flask cultures with the optimized Mn2+ dose. The results indicated that MnP activity reached maximum levels (2500 nkat/l on day 15) in cultures with the highest C/N ratio (65.3), i.e., containing 16 g/l glucose (88.88 mM) and 0.25 g/l ammonium tartrate (1.36 mM), respectively (Table 2). Hence, the selected C and N concentrations were used in further studies. Under these nutritional conditions (a strict Nlimitation), the enzyme productivity was over two times higher than that obtained on the initial basal medium used as a control (C/N ratio = 26.5). It should be stressed here that in all cultures with 1 g/l ammonium tartrate (5.43 mM), the increase in nitrogen concentration significantly suppressed MnP activities. Contras-
Table 2 Effects of C/N ratio on MnP production by N. frowardii in 34 day shaken flask culturesa Glucose (g/l)
Ammonium tartrate (g/l)
C/N ratiob (mM/mM)
Daysc
MnP activity (nkat/l)
5
0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00 0.50d
20.4 10.2 5.1 40.8 20.4 10.2 65.3 32.7 16.4 26.5
12 12 15 15 15 15 15 15 15 15
905.1 ± 7.1 955.4 ± 5.7 644.4 ± 4.6 951.5 ± 8.0 1586.9 ± 12.3 430.1 ± 2.9 2500.0 ± 17.5 2050.0 ± 12.2 675.0 ± 4.2 1149.1 ± 9.6
10
16
13d
a Composition of the medium was the same as that of the basal one, but glucose and ammonium tartrate concentrations varied as indicated. A total Mn2+ concentration was 2 mM (1 mM of Mn2+ added on the third day of cultivation). b Calculated as C (glucose)/N (ammonium tartrate) ratio. c Incubation period for maximum activity. d Original basal medium (control).
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tingly, the increase in carbon concentration enhanced MnP yields. Having taken this fact into account, it is postulated that the development of ligninolytic activity by N. frowardii is the result of a nitrogen-based regulatory mechanism rather than that of C limitation. Similarly to P. chrysosporium and related lignin-degrading fungi, this fungus requires a strict N-limitation to maximize MnP production (Kirk et al., 1986; Hatakka, 1994; Buswell et al., 1995; Moilanen et al., 1996). Meanwhile, Clitocybula dusenii did not need such a strict Nlimitation for producing Mn peroxidase (Ziegenhagen and Hofrichter, 2000), and MnP peroxidase titres in cultures of Bjerkandera sp. BOS55 and P. flavido-alba were stimulated by a high N medium (Mester et al., 1996; Ben Hamman et al., 1997; Moreira et al., 2000a). The effect of increasing the agitation rate of the rotary shaker from 100 to 300 rpm on MnP production by free and immobilized mycelia of N. frowardii is shown in Table 3. In free pellet cultures, the enzyme activity attained its maximum (about 2880 nkat/l) after 15 days of incubation at an agitation speed of 200 rpm. At 150 rpm, and in the same time period, MnP activity achieved a slightly higher value in comparison with the one recorded at 100 rpm, but the use of the shaker with a revolution rate of 100 rpm required a longer period of incubation (18 days) for the maximum enzyme activity. Increase of the agitation rate up to 300 rpm, however, had an undesirable inactivation effect on the secreted enzyme, reducing its enzymatic activity by about 24% compared with the maximum reached at 200 rpm. The drop in MnP activity at an excessively high agitation rate (300 rpm) was probably caused by the effect of mechanical (shear) stress on the fungal pellets. This follows from the distribution of the pellet sizes at the different agitation rates. Initially, on day 2, the pellet size was distributed between those smaller than 0.5 mm and those greater than 3.5 mm. By days 5 and 7, however, the ratio of pellets in the smaller range (between 0.5 mm and 1.2 mm) for the agitation rate of 300 rpm
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increased significantly. Moreover, under these conditions, a small proportion of pellets (about 2–5% of the total population) disintegrated into hyphal branches. These facts indicate that shear stress affected the secretion and/or stability of the N. frowardii enzyme and resulted in a decrease in MnP activity. Different results in this respect were obtained by Moreira et al. (2000a) in shaken flask cultures of Bjerkandera sp. BOS55. Though the distribution of the pellet size was similar to N. frowardii, the level of MnP activity was independent of the rate of agitation (from 150 to 275 rpm) and reached similar values in the range of 3334 nkat/l after 4–6 days of cultivation. These authors found the increase in the agitation rate to imply a higher consumption of nutrients and an earlier appearance of MnP activity in pellets of smaller size, but observed no significant effect on MnP activity. The results obtained when working with the immobilized polyurethane-foam mycelium are clearly better than those achieved with the free fungus. For example, at agitation rate of 200 rpm the volume activity of MnP was over 38% higher than that achieved by free pellets under the same agitation conditions. However, the process was slower and the maximum activity was delayed until day 18 (Table 3). At lower agitation rate (150 rpm), the production of MnP from the immobilized mycelium was also higher (by 16.4%) in comparison with the experiments performed with the free fungus, but the enzyme achieved its maximum 3 days earlier, i.e. on day 12. It should be noted that N. frowardii grew rapidly into the polyurethane-foam cubes. After a few days of incubation, the carrier was completely penetrated and its surface covered with a dense layer of fungal hyphae. The immobilized fungus and the support were highly stable during agitation. Neither fragments of the mycelium nor fragments of the support sheared off into the culture liquid. Thus, polyurethane-foam cubes of 1.0 cm3 can be considered a good immobilization matrix for N. frowardii b19. 3.2. Enzyme production in fermenter cultures
Table 3 Effect of agitation rate and immobilization of the mycelium on MnP production by N. frowardii in shaken flask cultures on the optimized culture production medium Shaker speed (rpm)
Daysa
MnP activity (nkat/l)
Free mycelium (as pellets)b
100 150 200 300
18 15 15 15
2345.9 ± 14.6 2398.3 ± 14.1 2879.8 ± 13.9 2193.1 ± 10.3
Immobilized mycelium on polyurethane foam (as biofilm)c
150 200
12 18
2792.4 ± 16.3 3981.1 ± 23.5
a b c
Incubation period for maximum activity. Cultivation time was 33 days; temp. 23 °C. Cultivation time was 27 days; temp. 23 °C.
MnP production by the free mycelium of N. frowardii was also carried out in a 3 l fermenter with constant air flow and agitation rates. Typical culture profiles are shown in Fig. 1. Biosynthesis conditions were fixed taking into account the previous results obtained in agitated flask cultures (the optimized culture production medium with the determined Mn2+ dose and C/N ratio, inoculum quantity, initial pH of the medium, agitation rate and incubation temperature), as an initial step to larger scale fermentations. The extracellular concentration of MnP continued to rise with time to reach the maximum activity of 1627 nkat/l on the 12th day. Thereafter, the enzyme activity declined very quickly. The pH of the medium decreased (from 4.8 to 4.1) over the
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Fig. 1. Time course of MnP production, oxygen consumption, and pH during aerated fermenter cultures of free N. frowardii mycelium. The optimized culture production medium with initial pH of 4.8 was used. Symbols: (s) MnP activity; (d) pH; (n) oxygen concentration.
whole period of incubation. Under air supply, the dissolved oxygen was gradually depleted by the free mycelium, and its content declined by 15% after 12 days of cultivation. Afterwards, the oxygen consumption remained stable with a low decrease at the end of the fermentation, after 24 days. It should be noted here that the cultivation of N. frowardii in a bioreactor resulted in much lower enzymatic activities than those achieved by the free fungus in shaken flasks. In fermenter culture, however, the enzyme production reached maximum levels within shorter incubation periods, and relatively high titres of MnP were produced under air atmosphere. This fact offers an economic advantage in the culturing of N. frowardii in comparison with the model fungus P. chrysosporium, which requires the use of oxygen or oxygen enriched air in bioreactors to express ligninolytic activity (Moreira et al., 1998). In order to maximize MnP production and stability in bioreactor cultures, a semi-continuous operation was tested with a reusable immobilized mycelium of N. frowardii. For this purpose, a fixed-net bioreactor aerated with air was designed and filled with the immobilized polyurethane-foam mycelium. The results shown in Fig. 2 indicate that both the stability and productivity of the process can be highly improved by following this strategy. Similarly to the fermenter culture with free mycelium, the production of MnP started shortly after the addition of extra Mn2+ to the optimized culture production medium and reached a level of about 1600 nkat/ l after 14 days of incubation. After the culture liquid was replaced by a fresh medium, this high MnP level was actually reached again within a shorter incubation period, i.e. 8 days. The next two cycles gave even better results: the highest MnP activities of 2078–2304 nkat/l were obtained after 8 days of cultivation. Observation
Fig. 2. Repeated batch production of MnP in fermenter culture using N. frowardii mycelium immobilized on polyurethane foam. The enzyme production was performed on the optimized culture production medium in 10-day cycles. Arrows in respective curves show addition of Mn2+ into the medium. Vertical dash lines separate successive cycles and show the days on which the culture liquid was replaced by fresh medium. Symbols: (s) MnP activity; (d) pH; (n) oxygen concentration.
of the early stages of the operation (first cycle) shows a delay in MnP production, indicating that the mycelium took some time before acclimating to the conditions leading to MnP secretion. Under air supply, the dissolved oxygen was continuously consumed by the immobilized mycelium over the whole period of incubation (44 days), and its content dropped by 17%. However, in respective batches, the depletion of dissolved oxygen by the immobilized fungus was lower than that recorded in the bioreactor for free pellet culture (Fig. 1). Consequently, an 8-day period was assumed to be the optimum time for MnP synthesis by the immobilized N. frowardii mycelium in repeated bioreactor cultures. It is noteworthy that surface cultures of N. frowardii showed an average MnP activity of 5.35 nkat/l, reaching this level after 24 days of cultivation (Sack et al., 1997). In the present study, fermenter cultures of the reusable immobilized mycelium had 299–431 times higher MnP levels, which they reached within three times shorter incubation periods. The immobilized fungus also produced from 1.3 to 1.4 times higher amounts of MnP compared with those obtained in the bioreactor with the free mycelium. Contrary results in this respect were reported by Moreira et al. (2000b). Under the conditions applied (a 2 l fermenter with mycelium immobilized in polyurethane foam cubes; agitation rate, 150 rpm; aeration flow, 0.8 v v 1 min 1; temperature, 30 °C; pH not regulated; inoculum quantity, 10% v/v), relatively low MnP activities (about 417 nkat/l after 8–9 days) were reached in batch cultures of the immobilized Bjerkandera sp. BOS55 mycelium compared with those achieved under the same operational conditions with free pellet cultures (2500 nkat/l after 7–8 days).
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Recycling of the immobilized mycelium enabled repeated use of the biomass for MnP production (semicontinuous culturing). Moreover, the recycling procedure maintained the fungus in its idiophase, in which MnP is normally produced, so that MnP production started immediately after the addition of fresh medium. Ziegenhagen and Hofrichter (2000) reported a similar procedure to produce high amounts of MnP with a reusable immobilized mycelium of the agaric basidiomycete fungus C. dusenii in shaken flask cultures. Also, Rivela et al. (2000), using an immersion bioreactor, obtained a high MnP activity produced under solid-state conditions by the mycelium of P. chrysosporium immobilized on cubes of fibrous nylon sponge.
4. Conclusions Summarizing, the data presented herein show that the wood-decaying fungus N. frowardii is a good producer of extracellular MnP in submerged cultures, either as free or immobilized mycelium, and hence justifies further investigations on the application of the strain to obtain an active ligninolytic preparation for biotechnological purposes. Also, the appropriate combination of operational conditions (the use of N-limited medium, sequential addition of adequate Mn2+, immobilized polyurethane-foam mycelium), recycling strategy and bioreactor design (fixed-net fermenter aerated with air) allowed for semi-continuous and effective MnP production by N. frowardii during the idiophase for prolonged periods.
Acknowledgements This study was financially supported in part by the Polish State Committee for Scientific Research (Grant PBZ-KBN-098/T09/2003) and by the BW/UMCS and BS/UMCS research programmes.
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