Ó Springer 2006
World Journal of Microbiology & Biotechnology (2006) 22:999–1002 DOI 10.1007/s11274-006-9132-6
Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity Nona Mikiashvili1,3, Solomon P. Wasser1,2,*, Eviatar Nevo1 and Vladimir Elisashvili4 1 Institute of Evolution, University of Haifa, Haifa, Israel 2 N. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kiev, Ukraine 3 Department of Biochemistry and Biotechnology, Iv. Javakhishvili State University of Tbilisi, Tbilisi, Georgia 4 Academy of Sciences of Georgia, Institute of Biochemistry and Biotechnology, Tbilisi, Georgia *Author for correspondence: Tel.: +380-9724-8249653, Fax: +380-9724-8288649, E-mail:
[email protected] Received 20 November 2005; accepted 13 January 2006
Keywords: Carbon and nitrogen sources, laccase, manganese peroxidase, peroxidase, Pleurotus ostreatus
Summary The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l)1 and from 55 to 390 U l)1, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5fold, respectively.
Introduction Pleurotus spp. (oyster mushrooms) comprise the group of edible white-rot fungi with important medicinal properties and biotechnological and environmental applications (Cohen et al. 2002). They are highly adaptable to grow and fruit on a wide variety of agro-industrial lignocellulosic wastes due to their capability to synthesize relevant hydrolytic (cellulases and hemicellulases) and unique oxidative (ligninolytic) extracellular enzymes. Various agricultural substrates/ by-products, which may contain significant concentrations of soluble inducers, have been successfully used in submerged and solid-state fermentation for ligninolytic enzyme production (Reddy et al. 2003; Mikiashvili et al. 2004). The data received prove that type and composition of lignocellulosic substrates appear to determine the type and amount of enzyme produced by wood-rotting basidiomycetes (Kapich et al. 2004; Moldes et al. 2004). In spite of the commercial significance of oyster mushrooms, our current knowledge on the physiology of these fungi is limited and therefore further research is needed to realize their potential. Only a few studies of several species basidiomycetes have been conducted on the effect of carbon sources on ligninolytic enzyme
production (Elisashvili et al. 2002; Galhaup et al. 2002; Mikiashvili et al. 2005; Stajic´ et al. 2006). Many previous studies have proved that both the nature and concentration of nitrogen sources are powerful nutrition factors regulating ligninolytic enzyme production by wood-rotting basidiomycetes (Galhaup et al. 2002; Mikiashvili et al. 2005). High nitrogen media gave the highest laccase activity in Lentinus edodes, Rigidoporus lignosus, and Trametes pubescens while the nitrogenlimited conditions enhanced enzyme production in Pycnoporus cinnabarinus, P. sanguineus, and Phlebia radiata (Mester & Field 1997; Gianfreda et al. 1999; Galhaup et al. 2002). The aim of this study was to evaluate the significance of various carbon and nitrogen sources for oxidative enzyme production by new isolates of Pleurotus ostreatus.
Materials and methods Organisms and inoculum preparation Pleurotus ostreatus 98 and P. ostreatus 108, from the Basidiomycetes culture collection of Haifa University
1000 (Wasser et al. 2002), were used in this study. The inoculum was prepared by fungal cultivation on a rotary shaker at 180 rev min)1 in 500-ml flasks containing 100 ml basal medium (g l)1): glucose 10; KH2PO4 0.8; NH4NO3 2; Na2HPO4 0.4; MgSO4 Æ 7H2O 0.5; yeast extract 2. The following microelements were added to the basal medium (g l)1): ZnSO4 Æ 7H2O 0.001; FeSO4 Æ 7H2O 0.005; CaCl2 Æ 2H2O 0.06; CuSO4 Æ 7H2O 0.005; MnSO4 Æ H2O 0.005. After 5 days of fungal cultivation, mycelial pellets were harvested and homogenized with a Waring laboratory blender, three times for 20 s with 1-min intervals.
N. Mikiashvili et al. activity was measured under the same conditions but without manganese. The reaction was terminated by the addition of 40 ll of 2 M NaOH. Absorbance was recorded at 610 nm. All enzyme assays were carried out at 20 °C. One unit of laccase, MnP, and peroxidase activity was defined as an amount of enzyme that transformed 1 lmol substrate per minute.
Results and discussion Effect of the carbon source on ligninolytic enzyme production
Conditions of mushroom cultivation All cultivations were conducted at 25±2 °C on a rotary shaker at 140 rev min)1 in 100-ml Erlenmeyer flasks containing 20 ml of tested medium. The effect of carbon sources on enzyme production was studied using the above-mentioned standard medium with crystalline cellulose (Avicel), soluble carboxymethyl cellulose (CMC), xylan, glucose, cellobiose, maltose, lactose, mannitol, and sodium gluconate at a concentration of 10 g l)1. In addition, two lignocellulosic substrates, grapevine cutting sawdust (GWS) and mandarin peel, at a concentration of 40 g l)1 were fermented by mushrooms. To study the effect of nitrogen sources, the same standard medium containing glucose was used, and all nitrogen-containing inorganic and organic compounds were added to the medium in final concentrations equal to (approximately for organic compounds) 30 mM of nitrogen in addition to yeast extract. A control without a nitrogen source was run in parallel. The initial pH of all media was adjusted to 6.0 by adding 1 M NaOH prior to sterilization. One ml of homogenized mycelia was used to inoculate the flasks containing media. After 5 and 8 days of mushroom cultivation (when cultures reached the end of logarithmic and stationary phases of growth), biomasses were filtered and the solids were separated by centrifugation (6000 rev min)1; 20 min) at 4 °C. All experiments were performed at least two times using three replicates. The data presented in the tables correspond to mean maximal values. Enzyme activity assays The supernatants received after biomass separations were analysed for enzyme activity. Laccase activity was determined by following the oxidation of syringaldazine as a substrate at 525 nm for 1 min (Leonowicz & Grzywnowicz 1981). The reaction mixture in 1 ml contained 100 mM acetate buffer (pH 5.0), 1 mM syringaldazine, and 100 ll appropriately diluted culture filtrate. Manganese-dependent peroxidase (MnP) activity was assayed by the oxidation of Phenol Red (Glenn & Gold, 1985). The reaction mixture in 1 ml contained 50 mM sodium lactate–succinate buffer (pH 4.5), 0.1 mM MnSO4, 0.1 mM H2O2, 3 mM Phenol Red, and 100 ll enzyme filtrate. In addition, peroxidase
In a previous study (Mikiashvili et al. 2004), it was shown that P. ostreatus was a promising producer of both extracellular laccase and MnP in submerged fermentation of mandarin peel. To elucidate the reason by which this substrate enhanced enzyme production and taking into account that mandarin peel contains comparatively high levels of soluble carbohydrates (32– 34%), we studied the role of different carbon sources in P. ostreatus 98 and P. ostreatus 108 ligninolytic enzyme production. All compounds tested ensured good growth of fungi providing the final dry biomass accumulation from 2.5 to 9.1 g l)1 (Table 1). The maximal laccase yield of P. ostreatus 98 varied from 5 to 62 U l)1, and highest enzyme activity was revealed in the presence of sodium gluconate, CMC, glucose, and cellobiose. The medium with CMC provided the highest specific laccase activity (18 U g)1 biomass), which was 2-fold higher than that in medium with sodium gluconate (9.3 U g)1 biomass). None of the pure carbohydrates tested produced laccase levels as high as mandarin peel did. In this case, P. ostreatus 98 laccase level was 38-fold higher than that in fungal cultivation in the presence of sodium gluconate. In contrast, lignified GWS was identified as a poor growth substrate for extracellular laccase secretion. The data received showed that insoluble crystalline cellulose appeared to be a poor substrate for laccase production in P. ostreatus 98, while easily metabolizable compounds ensured comparatively high enzyme activity. Analogically, the replacement of crystalline cellulose by cellobiose increased laccase activity of Cerrena unicolor IBB 62 by 21-fold, while mannitol, cellobiose, and glucose ensured highest laccase activity of P. ostreatus IBB 191 (Elisashvili et al. 2002). Significant laccase formation by Trametes pubescens was shown in the presence of cellobiose and glucose, while poorly utilized lactose and a-cellulose resulted in lowlaccase levels (Galhaup et al. 2002). Recently, Stajic´ et al. (2006) observed the highest laccase activity in the presence of mannitol, glucose, and sodium gluconate in two strains of P. ostreatus. The highest MnP and peroxidase activities were revealed when P. ostreatus 98 was grown in medium with the polysaccharide substrate, xylan. Mannitol and crystalline cellulose also ensured comparatively high MnP activity, while lactose was the appropriate growth
Pleurotus carbon and nitrogen sources
1001
Table 1. Effect of carbon sources on P. ostreatus 98 and P. ostreatus 108 oxidative enzyme activity. Carbon source
Avicel CMC Xylan Glucose Cellobiose Maltose Mannitol Na Gluconate GWS MP
P. ostreatus 98
P. ostreatus 108
Biomass (g l)1)
Laccase (U l)1)
MnP (U l)1)
Peroxidase (U l)1)
Biomass (g l)1)
Laccase (U l)1)
MnP (U l)1)
Peroxidase (U l)1)
ND* 2.8 5.1 8.8 8.2 8.0 9.1 6.7 ND ND
5±0.3(5) 51±6.7(8) 42±1.8(8) 58±3.5(8) 55±2.4(8) 39±2.5(5) 27±0.9(5) 62±8.3(8) 82±4.7(5) 2375±39.8(5)
1.5±0.21(8) 0.3±0.01(8) 3.6±0.20(8) 1.0±0.09(8) 0.9±0.03(8) 0.2±0.01(5) 2.0±0.16(5) 0.4±0.02(5) 2.1±0.09(8) 18.6±1.18(8)
1.6±0.07(8) 0.9±0.03(8) 11.7±1.14(8) 2.7±0.27(8) 2.5±0.11(8) 0.9±0.03(5) 0.5±0.03(8) 0.7±0.02(5) 4.2±0.21(8) 32.4±1.43(8)
ND 2.5 4.8 7.8 8.0 6.7 8.0 7.3 ND ND
203±7.6(5) 148±11.9(8) 390±11.6(8) 90±3.0(8) 128±6.3(8) 55±6.7(8) 79±13.6(5) 284±25.2(8) 52±3.1(5) 1082±60.0(5)
2.6±0.09(8) 10.3±0.32(8) 40.5±12.9(8) 1.4±0.12(8) 21.0±0.88(8) 0.4±0.02(5) 0.6±0.15(8) 0.5±0.04(5) 21.3±0.97(8) 17.8±1.23(8)
11.0±0.34(8) 23.7±1.69(5) 60.5±7.47(8) 0.9±0.18(8) 59.5±3.14(8) 0.9±0.18(5) 0.5±0.09(8) 1.6±0.07(5) 33.1±2.08(8) 28.1±0.66(8)
*Not determined. The numbers in parentheses indicate the day of maximal activity.
substrate for peroxidase accumulation in culture liquid. It is worth noting that mandarin peel provided excellent mushroom growth and extracellular peroxidase secretion. P. ostreatus 108 showed different responses on various carbon source supplementations in culture medium. This strain appeared to be a much better producer of ligninolytic enzymes (Table 1). Of all carbon sources, xylan, followed by sodium gluconate and crystalline cellulose, ensured the highest laccase activity whereas mannitol, glucose, and maltose, which provided excellent mycelia growth, gave low levels of enzyme activity. Xylan and cellobiose exerted the most positive effect on peroxidase accumulation in P. ostreatus 108. Moreover, the use of these compounds resulted in higher enzyme activity than both lignocellulosic substrates used for submerged fermentation. It is interesting that GWS, a poorer growth substrate for laccase production than mandarin peel, ensured rather higher MnP and peroxidase yields. In many cases, basidiomycetous fungi cultivation in the presence of some lignocellulosic residues significantly stimulated ligninolytic enzyme secretion without supplementation of the culture medium with specific inducers (Kapich et al. 2004; Mikiashvili et al. 2005). The positive effect of mandarin peel on laccase production is probably due to the presence of
water-soluble aromatic compounds (flavones and flavonols) capable of stimulating biosynthesis of ligninolytic enzymes. Effect of nitrogen sources on ligninolytic enzyme production In the literature, there is contradictory evidence on the effects of nitrogen sources (nature and concentration) on ligninolytic enzyme production. While high nitrogen media gave the highest laccase activity in L. edodes, Rigidoporus lignosus, and T. pubescens, nitrogen-limited conditions enhanced the enzyme production in P. cinnabarinus, P. sanguineus, and Phlebia radiata (Mester & Field 1997; Gianfreda et al. 1999; Galhaup et al. 2002). In this study, all nitrogen sources tested stimulated growth of the mushrooms, increasing the final biomasses (after 8 days of submerged cultivation) by 2–3 times when compared with the control medium (Table 2). In general, the maximal laccase and peroxidase activities were revealed after 5 days of fungal cultivation in media with inorganic compounds, while in supplementation of media with organic nitrogen sources the highest activity of these enzymes accumulated after 8 days of fungal cultivation. Our data show that inorganic and organic
Table 2. Effect of nitrogen source on P. ostreatus 98 and P. ostreatus 108 oxidative enzyme activity. Nitrogen source
Control NH4NO3 NaNO3 NH4Cl NH4H2PO4 (NH4)2SO4 Peptone Casein hydrolysate Corn steep liquor
P. ostreatus 98
P. ostreatus 108
Biomass (g l)1)
Laccase (U l)1)
MnP (U l)1)
Peroxidase (U l)1)
Biomass (g l)1)
Laccase (U l)1)
MnP (U l)1)
Peroxidase (U l)1)
3.5 8.5 6.8 7.3 7.6 8.4 9.7 8.8 10.2
139±10.4(5) 84± 9.0(5) 136±9.7(5) 81±8.0(5) 82±7.2(5) 32±6.1(8) 443±29.0(8) 401±21.2(8) 337±15.9(8)
17.1±2.11(8) 1.9±0.12(5) 5.5±0.41(8) 2.8±0.11(5) 3.6±0.17(5) 0.5±0.05(5) 10.2±0.24(8) 9.2±0.28(8) 14.8±1.36(8)
23.9±3.02(8) 5.6±0.32(5) 8.5±0.36(8) 3.9±0.15(5) 6.0±0.15(5) 0.7±0.08(5) 71.1±3.03(8) 32.1±1.21(8) 36.2±2.12(8)
3.1 8.0 6.1 6.4 7.7 7.9 9.1 8.0 9.5
132±1.2(5) 79±1.0(5) 43±1.3(5) 81±5.1(5) 3±0.2(5) 9±1.2(8) 490±8.7(8) 430±12.3(8) 164±5.3(8)
0.9±0.10(8) 0.2±0.02(5) 0.1±0.01(5) 0.2±0.01(8) 0.2±0.01(5) 0.3±0.04(5) 3.2±0.37(8) 36.1±1.98(8) 1.8±0.02(8)
3.1±0.24(5) 0.4±0.02(5) 0.4±0.04(8) 0.7±0.11(5) 0.8±0.08(5) 0.5±0.04(5) 14.2±2.15(8) 27.6±1.49(8) 10.7±1.02(8)
1002 nitrogen sources at a concentration as high as 30 mM exerted different physiological effects on fungi. Compared to organic compounds, P. ostreatus enzyme activity decreased with supplementation of medium by inorganic N sources. Peptone, followed by casein hydrolysate, appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, in the comparison of tested cultures, specific laccase activities prove that the positive effect of additional nitrogen on enzyme accumulation may be attributed simply to a higher biomass production. For example, the specific laccase activity of P. ostreatus 98 in the control medium was equal to 39.7 U g)1 versus 45.7 U g)1 in medium with peptone. Accordingly, although in culture of P. ostreatus 98 peptone highly favored the extracellular peroxidase accumulation with a maximal value of 71 U l)1, the specific enzyme activity in this medium and the control appeared to be the same. All other tested compounds partially suppressed the production of peroxidases. Concerning P. ostreatus 108, the secretion of MnP and peroxidase by this strain was indeed stimulated by the supplementation of casein hydrolysate to the control medium as shown by the specific MnP and peroxidase activities, which increased 15-fold and 3.5-fold, respectively. In conclusion, the literature data and the results obtained in this work show that the effect of carbon and nitrogen sources depends on the fungal strain and nature of the compound tested. In addition, these major components of nutrition media are factors not only determining the expression of fungal enzyme activity but also the ratio of individual enzymes in enzyme complexes.
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