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Cost analysis in laccase production
Journal of Environmental Management 92 (2011) 2907e2912
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Cost analysis in laccase production Johann F. Osma a, b, José L. Toca-Herrera c, Susana Rodríguez-Couto d, e, * Centro de Microelectrónica (CMUA), Department of Electrical and Electronics Engineering, Universidad de los Andes, Carrera 1 N 18A e 12, Bogota, Colombia Departament d’Enginyeria Quimica, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Spain c Department of NanoBiotechnology, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 11, 1190 Vienna, Austria d CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spain e IKERBASQUE, Basque Foundation for Science, Alameda de Urquijo 36, 48011 Bilbao, Spain a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2011 Received in revised form 10 June 2011 Accepted 27 June 2011 Available online 19 July 2011
In this paper the cost of producing the enzyme laccase by the white-rot fungus Trametes pubescens under both submerged (SmF) and solid-state fermentation (SSF) conditions was studied. The fungus was cultured using more than 45 culture medium compositions. The cost of production was estimated by analyzing the cost of the culture medium, the cost of equipment and the operating costs. The cost of the culture medium represented, in all cases, the highest contribution to the total cost, while, the cost of equipment was significantly low, representing less than 2% of the total costs. The cultivation under SSF conditions presented a final cost 50-fold lower than the one obtained when culturing under SmF conditions at flask scale. In addition, the laccase production under SSF conditions in tray bioreactors reduced the final cost 4-fold compared to the one obtained under SSF conditions at flask scale, obtaining a final price of 0.04 cent V/U. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Cost Laccase Pricing Solid-state fermentation Submerged fermentation Trametes pubescens
1. Introduction Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are enzymes with considerable interest in industrial and environmental biotechnology due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases.
Abbreviations: ABTS, 2,20 -azino-di-[3-ethyl-benzo-thiazolin-sulphonate]; ActLac, maximum laccase activity of the extracted crude enzyme; Cap, capacity of the equipment; CCM, cost of the culture medium; CEq A, cost of the autoclave per cultivation; CEq I, cost of the incubator per cultivation; CEq, cost of equipment; COp A, cost of operation of the autoclave per cultivation; COp I, cost of operation of the incubator per cultivation; COp, operating costs; CostLac, cost of producing laccase; Dmax, cultivation time in days; EA, energy consumption of autoclaving processes to sterilize the total number of cultures per cycle of cultivation; Gxx, glucose xx concentration in the culture medium; LT, lifetime of the equipment; MEA, malt extract agar; Myy, mandarin peelings concentration (yy) in the culture medium; P, price of equipment divided by the number of cycles; PDA, potato dextrose agar; SmF, submerged fermentation; SS, sunflower-seed shells; SSF, solid-state fermentation; TA, tannic acid; VolLac, volume of the extracted crude laccase; Yzz, yeast concentration (zz) in the culture medium. * Corresponding author. CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spain. Tel.: þ34 943 212800x2239; fax: þ34 943 213076. E-mail address: [email protected] (S. Rodríguez-Couto). 0301-4797/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2011.06.052
The cost analysis of the laccase production is an important feature for its industrial exploitation, however most papers neglect this aspect. Several authors have defined their cultivation methods or culture media as low-cost (Mukherjee et al., 2008; Ng et al., 2010), however, in few cases the real cost of production has been calculated. Different approaches at laboratory scale have proved their suitability for industrial scale production but in general terms, few of them have included a cost analysis to give support to this statement. Numerous reports on strategies to improve the production of laccase enzymes, such as the isolation of new fungal strains, heterologous expression of laccase genes, optimization of growth conditions and use of inducers and stimulators, have been recently published. However, the utilization of inducers can cause problems of both cost-effectiveness and environmental pollution. Also, the enzyme yields obtained by recombinant expression of laccase genes (Hong et al., 2002, 2006; Li et al., 2007) are relatively low for the demands in industrial applications. Therefore, the development of approaches for the production of laccase with high efficiency, environmental friendliness and cost-effectiveness is a top priority. One potential solution for the above-mentioned issues would be to decrease the production cost of laccases by using cheap growth substrates such as agricultural and food wastes or wastewater from the food or pulp and paper industries. In addition, the reutilization
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of such wastes would help to alleviate smog pollution. Thus, such an approach is being subject of increased research. However, economical studies regarding the production of laccase enzymes using synthetic media and waste materials are lacking. The present paper provides an estimation of the production cost of laccase enzymes under different culture conditions. 2. Materials and methods 2.1. Microorganism Trametes pubescens MB 89 (CBS 696.94; Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was obtained from the Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences (Vienna, Austria) and was maintained on malt extract agar (MEA) or potato dextrose agar (PDA) plates at 4 C and sub-cultured every three months. 2.2. Culture conditions T. pubescens was cultured in both submerged (SmF) and solidstate fermentation (SSF) conditions using different culture media. Under SmF conditions cultures were performed in cotton-plugged Erlenmeyer flasks (250 mL) containing 100 mL of culture medium. The composition of the culture medium was prepared according to Rodríguez-Couto et al. (2006). Three different variables were taken into consideration in our analysis: carbon and organic nitrogen sources and inducers of laccase activity. Three carbon sources were tested: glucose (10 g/L), glycerol (10 g/L) and mandarin peelings (30 g/L). Also, the addition of yeast extract as an organic nitrogen source was studied by the combination in different proportions of glucose, mandarin peelings and yeast extract (GxxeMyyeYzz cultures). The concentration of glucose (Gxx) was varied between 0 and 30 g/L, the concentration of mandarin peelings (Myy) between 0 and 60 g/L and the concentration of yeast extract (Yzz) between 0.5 and 25 g/L. Finally, the addition of different inducers in the culture with glucose (10 g/L) was analyzed. Cultures were incubated statically, under an air atmosphere, at 30 C and in complete darkness. Under SSF conditions flask-scale cultures were performed in cotton-plugged Erlenmeyer flasks (250 mL) containing 20 mL of culture medium. The compositions of the culture medium were prepared according to Osma et al. (2007) when using banana skin, Osma et al. (2006) when using wheat bran and Rodríguez-Couto et al. (2009) when using sunflower-seed shells as support. Tray bioreactors were used when cultures were scaled-up as described in Rodríguez-Couto et al. (2009). All cultures were incubated statically, under an air atmosphere, at 30 C and in complete darkness. 2.3. Analytical determinations Laccase activity was determined spectrophotometrically as described by Niku-Paavola et al. (1990) with ABTS (2,20 -azino-di[3-ethyl-benzo-thiazolin-sulphonate]) as a substrate. One activity unit was defined as the amount of enzyme that oxidized 1 mmol ABTS per min. The activities were expressed in U/L. 2.4. Cost analysis In this study, the cost of the laccase production was divided in three terms: CCM, CEq and COp. In order to determine the costs related with equipment and operation (CEq and COp), an Isotemp Standard Lab incubator and a 2340-M Heidolph/Tuttnauer Tabletop autoclave from Fisher Scientific Inc. (Suwanee, USA) were selected as model equipments. Their lifetime (LT) was determined equal to
their warranty time, one year for the incubator and two years for the autoclave. Power consumption and capacity of both equipments were calculated upon manufacturer description. For the CCM, most of the market prices for reagents or compounds were obtained from SIGMAeAldrichÒ (St. Louis, USA). In some special cases, such as agricultural materials, the cost was assumed to be equal to the lowest one of any commercialized product with similar characteristics (E-Food Depot, 2010; Index Mundi, 2010; Czech Direct, 2010; Vita Cost, 2010). Thus, in the case of the mandarin peelings, the price of a cooking product of fresh mandarin peelings was used, whereas for the sunflower-seed shells the price of bricks, made of sunflower hulls, for heating was used. Unfortunately, the price of banana skin could not be estimated and had to be assumed as null. The CEq was represented in all cases as the price of an incubator (considering the number of cultures that can be grown simultaneously) and an autoclave. For COp, the energy spent by the use of the autoclave and the incubator was taken into consideration (Esplugas et al., 2002). Nevertheless, the cost of manpower was not considered in the analysis, since it will depend on the automation and monitoring of the process, the experience of the worker and the volume of laccase to be produced. The cost of producing laccase (CostLac) was calculated as the ratio between the sum of the culture medium, equipment and operating costs and the produced enzyme activity (Equation (1)). The latter was calculated as the maximum laccase activity (ActLac) multiplied by the volume of the extracted crude enzyme (VolLac) that represents the number of total units of laccase produced.
CCM þ CEq þ COp ActLac $VolLac
CostLac ¼
(1)
2.4.1. Cost of equipment The CEq reflects the cost of the incubator and the autoclave. However, it is important to remark that this term was not expressed as the simple sum of the cost of these items but also their LT and capacity were included to determine the real price of the equipment per cultivation. In order to express this cost as detailed as possible, the price of the equipment (P) was divided by the number of cycles of operation that can be carried out depending on the cultivation time. Thus, if the maximum laccase activity of a specific culture is obtained in 7 days (Dmax), then the number of cycles that the incubator can be used is 52 in a year. Therefore, if the LT of the equipment is 2 years 104 cycles can be carried out. Also, the capacity of the equipment (Cap) has to be taken into consideration in order to establish the maximum number of cultures that can take place in each cycle. If the incubator can hold 50 Erlenmeyer flasks (250 mL), then the maximum number of cultures that can be carried out is 5200 during the 2 years of operation of the incubator. In this sense, the following expression (Equation (2)) can be suggested to calculate the cost of the incubator per culture (CEq I).
CEq
I
PI 365 $LTI $CapI Dmax
¼
(2)
The operating capacity of the autoclave is limited by the capacity of the incubator and, thus, the correct expression to calculate the autoclave cost (CEq A) can be expressed as: (Equation (3))
CEq
A
PA 365 $LTA $CapI Dmax
¼
(3)
In general, the CEq can be calculated as the sum of the cost of both equipments per culture (Equation (4)):
J.F. Osma et al. / Journal of Environmental Management 92 (2011) 2907e2912
CEq ¼ CEq
I
þ CEq
A
¼
Dmax=365 CapI
$
PI P þ A LTI LTA
(4)
2.4.2. Operating cost In a similar way to the CEq, the COp has to reflect the culture conditions in its calculation. Therefore, The COp I can be expressed as the price of the energy spent per hour (EI) multiplied by the time of cultivation in hours divided by the capacity of the incubator. In the case of the autoclave, the operating cost (COp A) was calculated using the energy consumption (EA) of the necessary autoclaving processes to sterilize the total number of cultures per cycle of cultivation. The cost of operation of the incubator (COp I) and the autoclave (COp A) per cultivation was calculated as follows (Equations (5) and (6)).
COp
I
COp
A
¼
EI $ð24$Dmax Þ CapI
(5)
¼
EA CapI
(6)
The COp was calculated as the sum of COp I and COp A.
3. Results 3.1. Laccase production 3.1.1. SmF cultivation T. pubescens reached maximum laccase activities of 51 U/L (15th day), 89 U/L (16th day) and 228 U/L (3rd day) when using glucose, glycerol and mandarin peelings as the carbon source, respectively (Table 1). Additionally, yeast extract, an organic nitrogen source, was tested in combination with glucose and mandarin peelings at different concentrations. The combination of these three compounds (glucose, mandarin peelings and yeast extract) led to a maximum laccase activity of 2100 U/L (Table 1). Therefore, the addition of yeast extract in the culture medium led to higher laccase activities than those obtained by just using glucose or mandarin peelings. 3.1.2. SSF cultivation T. pubescens was also cultured under SSF conditions at flask scale, testing three natural supports: banana skin, wheat bran and sunflower-seed shells. The use of sunflower-seed shells considerably increased the laccase production reaching maximum laccase activities of 7750 U/L (10th day), compared to the 2500 U/L (10th day) and the 1600 U/L (20th day) obtained when using wheat bran and banana skin, respectively (Fig. 1) (Osma et al., 2006, 2007; Rodríguez-Couto et al., 2009). The culture medium with sunflower-seed shells was selected as the basal medium to test the effect of different inducers (Rodríguez-Couto et al., 2009). It is known that the addition of inducers considerably influences the activity of laccase (Bollag and Leonowicz, 1984), thus, five inducers were tested at different concentrations and added at different time points of cultivation (inoculation, tropophase e 3rd day and idiophase e 7th day). In almost all cases, laccase activities increased by the addition of the inducer, reaching a maximum of 25,773 U/L when adding 0.5 mM Cu2þ on the 3rd day of cultivation (RodríguezCouto et al., 2009). The joint effect of inducers was also investigated by combining the addition of 0.5 mM Cu2þ with 1 mM of xylidine or 50 mM of tannic acid on the 3rd day or on both the 3rd and the 7th day of cultivation. The use of Cu2þ and tannic acid added on the 3rd day of cultivation reached a maximum laccase activity of 30,272 U/L, while the other combinations led to maximum laccase
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activities similar or lower than the one obtained by just the addition of 0.5 mM Cu2þ on the 3rd cultivation day. Finally, the sunflower-seed shells cultures induced with 0.5 mM Cu2þ and 50 mM of tannic acid were scaled-up to tray bioreactors (Rodríguez-Couto et al., 2009). The surface area of the tray bioreactor was 7.5 times higher than the surface area of the Erlenmeyer flasks used. Due to the technical difficulties of mixing at this scale, the addition of inducers was only studied at inoculation and tropophase (3rd day). The scaled cultures presented a similar behavior to their counterpart at flask scale. Thus, when cultures were not induced, the maximum laccase activity was about 7130 U/L (13th day), while induced cultures reached maximum laccase activities of 31,610 (14th day) and 41,135 U/L (13th day) when induced at inoculation and tropophase, respectively (Rodríguez-Couto et al., 2009). 3.2. Cost analysis The total cost of the culture medium, equipment and operation was compared with the maximum laccase obtained in each culture and the time of cultivation needed. In this sense, the price of the laccase produced was influenced by all three terms (CCM, CEq and COp). Table 1 summarizes the calculation of these three costs, the laccase produced, the time of cultivation and the final price of the laccase produced for each culture performed. 3.2.1. Effect of inducers on the final price of laccase production The addition of most inducers increased the maximum laccase activity in both SmF and SSF cultures. By simply considering the increment on laccase activity, the use of inducers is coherent and reasonable. However, when analyzing the final price of produced enzyme, the use of inducers is not always suitable. Cultures under SmF conditions using glucose as a carbon source and no inducers presented a final price of about 17 cents of Euro per Unit of laccase produced. The maximum laccase activity obtained in this type of culture was 51 U/L after 15 days of cultivation. When adding inducers, the maximum activity was in some cases more than 4-fold higher than that obtained in cultures with no inducers in about the same time of cultivation (16 days). This increment was, however; not reflected in the final price of the produced laccase, which on the contrary increased in 666% its final price compared to the one produced without the addition of inducers. On the other hand, the use of inducers in cultures under SSF conditions presented a decrement in the final price of the produced laccase. Cultures using sunflower-seed shell as support presented a maximum laccase activity of 7750 U/L after 10 days of cultivation. The addition of inducers in this type of culture also increased the laccase activity up to 4-fold in relation to the cultures with no inducers in about the same time of cultivation. In this case, the increment of the laccase activity was reflected in a decrement of the final price of the produced laccase; thus, while cultures without induction presented a final price of 0.3 cents of Euro per Unit, induced cultures presented an average final price of 0.15 cents of Euro per Unit, a reduction of about 49%. 3.2.2. Effect of scaling-up on the final price of laccase production The effect of scaling-up was also analyzed in terms of the final price of the produced laccase. Cultures with sunflower-seed shells with and without inducers were scaled-up to tray bioreactors. Although, the laccase activity profile obtained at flask-scale and reactor-scale cultures was very similar, the final price was reduced when cultures were carried out at larger scale. The final price of the laccase produced in tray bioreactors was 22% lower for cultures without inducers and about 69% lower when induced than their flask-scale counterparts.
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Table 1 Cost analysis of laccase produced under different cultivation conditions. Culture medium (carbon source, support and/or inducer) SmF Glucose Glucose þ ABTS Glucose þ Tween 20 Glucose þ Soy oil Glucose þ Malaquite Green Glucose þ 1 mM Cu2þ Glucose þ 2 mM Cu2þ Glucose þ 50 mM Tannic Acid Glucose þ 100 mM Tannic Acid Glycerol Mandarin peels G30-M30-Y15 G20-M45-Y20 G20-M45-Y10 G20-M15-Y20 G20-M15-Y10 G15-M60-Y15 G15-M30-Y25 G15-M30-Y15 G15-M30-Y05 G15-M0-Y15 G10-M45-Y20 G10-M45-Y10 G10-M15-Y20 G10-M15-Y10 G0-M30-Y15
Max. laccase activity [U/L]
Laccase produced [U]
Time [days]
CCM [V]
COp [V]
CEq [V]
Total cost [V]
Final price [cent V/U]
51 5 110 225 80 7 25 200 105 89 228 442 241 1305 1817 714 291 436 557 282 290 847 2107 1358 66 844
4.1 0.4 8.8 18.0 6.4 0.6 2.0 16.0 8.4 7.1 18.2 35.4 19.3 104.4 145.4 57.1 23.3 34.9 44.6 22.6 23.2 67.8 168.6 108.6 5.3 67.5
15 16 16 16 16 15 10 10 16 16 3 12 6 8 8 8 3 11 10 9 10 3 8 3 10 3
0.5761 3.1116 5.2081 0.5761 0.5761 0.5764 0.5767 0.5770 0.5779 0.6641 0.6421 0.7888 0.8257 0.7639 0.7597 0.6979 0.8143 0.8101 0.7483 0.6865 0.6823 0.7987 0.7369 0.7327 0.6709 0.7078
0.1236 0.1315 0.1315 0.1315 0.1315 0.1236 0.0842 0.0842 0.1315 0.1315 0.0290 0.1000 0.0526 0.0684 0.0684 0.0684 0.0290 0.0921 0.0842 0.0763 0.0842 0.0290 0.0684 0.0290 0.0842 0.0290
0.0070 0.0074 0.0074 0.0074 0.0074 0.0070 0.0046 0.0046 0.0074 0.0074 0.0014 0.0056 0.0028 0.0037 0.0037 0.0037 0.0014 0.0051 0.0046 0.0042 0.0046 0.0014 0.0037 0.0014 0.0046 0.0014
0.7066 3.2505 5.3470 0.7151 0.7150 0.7070 0.6655 0.6659 0.7169 0.8030 0.6724 0.8943 0.8811 0.8360 0.8318 0.7700 0.8446 0.9072 0.8371 0.7669 0.7711 0.8290 0.8090 0.7630 0.7597 0.7381
17.32 812.62 60.76 3.97 11.17 126.24 33.28 4.16 8.53 11.28 3.69 2.53 4.57 0.80 0.57 1.35 3.63 2.60 1.88 3.40 3.32 1.22 0.48 0.70 14.39 1.09
SSF flask scale Banana skin Wheat bran SS SS þ 1% v/v coconut oil SS þ 2% v/v coconut oil SS þ 0.1 mM Cu2þ SS þ 0.3 mM Cu2þ SS þ 0.5 mM Cu2þ SS þ Soy oil 1% (v/v) SS þ Soy oil 2% (v/v) SS þ 25 mM Tannic acid SS þ 50 mM Tannic acid SS þ 1 mM Xylidine SS þ Cu2þ þ tannic Ac. (added 3rd day) SS þ Cu2þ þ xylidine (added 3rd day) SS þ Cu2þ þ tannic Ac. (added 3rd and 7th day) SS þ Cu2þ þ xylidine (added 3rd and 7th day)
1600 2500 7751 10,951 18,382 18,773 23,621 25,773 9708 10,165 10,635 10,064 12,494 30,272 19,085 22,946 26,645
8.0 12.5 77.5 109.5 183.8 187.7 236.2 257.7 97.1 101.7 106.4 100.6 124.9 302.7 190.8 229.5 266.4
20 10 10 8 9 10 10 10 9 10 10 10 10 11 11 11 8
0.1098 0.1098 0.1459 0.1461 0.1464 0.1459 0.1460 0.1460 0.1459 0.1460 0.1461 0.1463 0.1461 0.1463 0.1461 0.1467 0.1464
0.1631 0.0842 0.0842 0.0684 0.0763 0.0842 0.0842 0.0842 0.0763 0.0842 0.0842 0.0842 0.0842 0.0921 0.0921 0.0921 0.0684
0.0093 0.0046 0.0046 0.0037 0.0042 0.0046 0.0046 0.0046 0.0042 0.0046 0.0046 0.0046 0.0046 0.0051 0.0051 0.0051 0.0037
0.2822 0.1986 0.2348 0.2183 0.2268 0.2348 0.2348 0.2348 0.2264 0.2348 0.2350 0.2351 0.2349 0.2435 0.2433 0.2439 0.2185
3.53 1.59 0.30 0.20 0.12 0.13 0.10 0.09 0.23 0.23 0.22 0.23 0.19 0.08 0.13 0.11 0.08
SSF bioreactor scale Tray bioreactor Tray bioreactor þ Cu2þ þ tannic Ac. (added 0 day) Tray bioreactor þ Cu2þ þ tannic Ac. (added 3rd day)
7130 31,610 41,135
570.4 2528.8 3290.8
13 14 13
1.0945 1.0975 1.0975
0.2357 0.2522 0.2357
0.0126 0.0135 0.0126
1.3428 1.3632 1.3458
0.24 0.05 0.04
Fig. 1. Laccase production by T. pubescens grown under solid-state fermentation conditions: (dod) on banana skin; (dΔd) on wheat bran; (dAd) on sunflowerseed shells.
3.2.3. Influence of the culture medium cost on the total cost of laccase production The cost of the laccase production was divided in three terms, CCM, CEq and COp, all of them somehow dependent on the inherent characteristics of each culture. However, in all cultures there was a constant tendency, the CCM represented the greatest part of the final cost of production. As shown in Fig. 2, the CCM represents 89% of the total cost for SmF cultures, while this percentage decreases to about 61% for SSF cultures. This shows a higher dependency of SmF cultures on the cost of reagents and materials used in the culture medium than the one presented in SSF. Therefore, the replacement of reagents by low-cost materials is highly desirable. The use of agro-waste materials as the main component in the culture medium highly reduced the CCM, leading to a reduction in the final price of the produced laccase. Jing (2010) pointed out that the initial C/N ratio in the culture medium as well as the kind of C and N sources used were key factors affecting both laccase activity and cost. Thus, he found that a low initial C/N ratio not only stimulated
J.F. Osma et al. / Journal of Environmental Management 92 (2011) 2907e2912
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Fig. 2. Culture medium, equipment and operation costs (CCM, CEq and COp) percentages in SmF cultures, SSF cultures at flask scale and SSF cultures at bioreactor scale.
Table 2 Price of different commercial laccases. Supplier/reference
Description
Price [V]
Amount [g]
Estimated activity [U]
Final price [V/U]
Sigma/L2157 Sigma/53739 Sigma/38429 Jena Bioscience/EN-204
Laccase from Rhus vernificera (>50 units/mg) Laccase from Trametes versicolor, BioChemika (>20 units/mg) Laccase from Trametes versicolor, BioChemika (0.5 units/mg) Laccase EC 1.10.3.2 from Trametes versicolor
153.9 70.4 378.5 300
e 1 10 e
10,000 20,000 5000 5000
0.015 0.004 0.076 0.060
the simultaneous production of laccase and lignin peroxidase (LiP) by Streptomyces lavendulae but also the cost of enzyme production was cut down effectively. However, according to Eggert et al. (1996) the production of ligninolytic enzymes including laccases was stimulated and triggered under N-limitation conditions, i.e. high C/N ratio. We could not find any relationship between the C/N ratio and laccase activity in our cultures. This is in agreement with the results reported by Lebrun et al. (2011), who found no relationships between the C/N ratios and the production of oxidoreductases. One of the major concerns in the production of ligninolytic enzymes is the small amount of enzymes produced that can be sometimes relaxed by scaling them up. However, when SSF cultures were scaled-up to tray bioreactors the final cost of the produced laccase was reduced by reducing the COp, representing only the 18% of the total production costs. Additionally, in all cases the CEq represented less than 2% of the total production costs. 3.2.4. Final price of the produced laccase The price of the produced laccase in each type of cultivation and for different culture conditions was different. In this sense, the average price per type of cultivation was calculated considering the successful cultivation cases (all cultures except SmF cultures induced with ABTS or Cu2þ) showing a final mean price of 7 cents of Euro per Unit for SmF cultures, compared to 0.44 and 0.11 cents of Euro per Unit for SSF cultures at flask scale and at bioreactor scale, respectively. Laccase produced by SSF cultures showed a lower final price than the ones obtained when culturing under SmF conditions. Additionally, the scale-up of the SSF cultures decreased in about 4fold the final price of the laccase produced. This effect was because of the slight decrement in the culture medium costs per volume but mainly because of the reduction in the operating costs. The geometry of the bioreactors led to have more working volume in the incubator and, thus, reducing the operating costs. In comparison with commercially available laccases, the price of the laccase produced under SSF conditions using bioreactors was more than 10-fold lower than those commercialized by SIGMAeAldrichÒ (St. Louis, USA) and Jena Bioscience (Jena, Germany). This, however, does not consider other industrial costs related to the packaging, storage and distribution of the enzyme. Table 2 shows the price of different commercial laccases. 4. Conclusions T. pubescens was cultured under both SmF and SSF conditions using more than 45 different culture medium compositions.
Different variables from the composition of the culture medium were taken into consideration such as carbon source, nitrogen source, supports and inducers. The laccase activity of all cultures was measured and the cost of laccase production was estimated. In this sense, three main terms were defined: cost of the culture medium, cost of equipment and operating costs. In all cases, the cost of the culture medium represented the highest contribution to the total cost, while, the cost of the equipment was significantly low, representing less than 2% of the total costs. Also, the cultivation of the fungus under SSF conditions led to lower costs of the culture medium and operation, presenting a final production cost of about 50-fold lower than the one obtained when culturing under SmF conditions. In addition, the production under SSF conditions was scaled-up to tray bioreactors. In this case the operating costs were clearly reduced, reflecting a final price 4-fold lower than the one obtained at flask scale. In view of the results obtained more efforts in reducing the cost of the culture medium are required to reach economically feasible laccase production systems. Acknowledgments This research was financed by the Spanish Ministry of Education and Science (Project CTQ2007-66541). Appendix. Supplementary material Supplementary data associated with this article can be found, in the on-line version, at doi:10.1016/j.jenvman.2011.06.052. References Bollag, J.M., Leonowicz, A., 1984. Comparative studies of extracellular fungal laccases. Applied and Environmental Microbiology 48, 849e854. Czech Direct, 2010. http://www.czechdirect.ie/ecowoodpellets. E-Food Depot, 2010. http://www.efooddepot.com/products/jiabao_brand/5834/ preserved_mandarin_peel__hypen__0_dot_52oz.html. Eggert, C., Temp, U., Dean, J.F.D., Eriksson, K.-E.L., 1996. A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Letters 391, 144e148. Esplugas, S., Giménez, J., Contreras, S., Pascual, E., Rodríguez, M., 2002. Comparison of different advanced oxidation processes for phenol degradation. Water Research 36, 1034e1042. Hong, F., Meinander, N.Q., Jönsson, L.J., 2002. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnology and Bioengineering 79, 438e449. Hong, Y.Z., Xiao, Y.Z., Zhou, H.M., Fang, W., Zhang, M., Wang, J., Wu, L., Yu, Z., 2006. Expression of a laccase cDNA from Trametes sp. AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation. FEMS Microbiology Letters 258, 96e101.
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Index Mundi, 2010. http://www.indexmundi.com/es/precios-de-mercado/?mercan cia¼aceite-de-soja. Jena Bioscience, 2010. http://www.jenabioscience.com. Jing, D., 2010. Improving the simultaneous production of laccase and lignin peroxidase from Streptomyces lavendulae by medium optimization. Bioresource Technology 101, 7592e7597. Lebrun, J.D., Lamy, I., Mougin, C., 2011. Favouring the bioavailability of Zn and Cu to enhance the production of lignin-modifying enzymes in Trametes versicolor cultures. Bioresource Technology 102, 3103e3109. Li, J.F., Hong, Y.Z., Xiao, Y.Z., Xu, Y.H., Fang, W., 2007. High production of laccase B from Trametes sp. in Pichia pastoris. World Journal of Microbiology & Biotechnology 23, 741e745. Mukherjee, A.K., Adhikari, H., Rai, S.K., 2008. Production of alkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindrica grass and potato peel as low-cost medium: characterization and application of enzyme in detergent formulation. Biochemical Engineering Journal 39, 353e361. Ng, I.-S., Li, C.-W., Chan, S.-P., Chir, J.-L., Chen, P.T., Tong, C.-G., Yu, S.-M., Ho, T.-H.D., 2010. High-level production of a thermoacidophilic b-glucosidase from
Penicillium citrinum YS40-5 by solid-state fermentation with rice bran. Bioresource Technology 101, 1310e1317. Niku-Paavola, M.-L., Raaska, L., Itävaara, M., 1990. Detection of white-rot fungi by a non-toxic stain. Mycological Research 94, 27e31. Osma, J.F., Toca Herrera, J.L., Rodríguez Couto, S., 2006. Laccase production by Trametes pubescens grown on wheat bran under solid-state conditions. In: 6th European Symposium on Biochemical Engineering Science, Salzburg, Austria, August 27e30. Osma, J.F., Toca Herrera, J.L., Rodríguez Couto, S., 2007. Banana skin: a novel waste for laccase production by Trametes pubescens under solid-state conditions. Application to synthetic dye decolouration. Dyes and Pigments 75, 32e37. Rodríguez-Couto, S., Rodríguez, A., Paterson, R.R.M., Lima, N., Teixeira, J.A., 2006. Laccase activity from the fungus Trametes hirsuta using an air-lift bioreactor. Letters in Applied Microbiology 42, 612e616. Rodríguez-Couto, S., Osma, J.F., Toca Herrera, J.L., 2009. Removal of synthetic dyes by an eco-friendly strategy. Engineering in Life Sciences 9, 116e123. SIGMAeAldrichÒ, 2010. http://www.sigmaaldrich.com. Vita Cost, 2010. http://www.vitacost.com/Nutiva-Organic-Extra-Virgin-CoconutOil-15-fl-oz?csrc¼GPF-692752200014.
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Cost analysis in laccase production Johann F. Osma a, b, José L. Toca-Herrera c, Susana Rodríguez-Couto d, e, * Centro de Microelectrónica (CMUA), Department of Electrical and Electronics Engineering, Universidad de los Andes, Carrera 1 N 18A e 12, Bogota, Colombia Departament d’Enginyeria Quimica, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Spain c Department of NanoBiotechnology, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 11, 1190 Vienna, Austria d CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spain e IKERBASQUE, Basque Foundation for Science, Alameda de Urquijo 36, 48011 Bilbao, Spain a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 January 2011 Received in revised form 10 June 2011 Accepted 27 June 2011 Available online 19 July 2011
In this paper the cost of producing the enzyme laccase by the white-rot fungus Trametes pubescens under both submerged (SmF) and solid-state fermentation (SSF) conditions was studied. The fungus was cultured using more than 45 culture medium compositions. The cost of production was estimated by analyzing the cost of the culture medium, the cost of equipment and the operating costs. The cost of the culture medium represented, in all cases, the highest contribution to the total cost, while, the cost of equipment was significantly low, representing less than 2% of the total costs. The cultivation under SSF conditions presented a final cost 50-fold lower than the one obtained when culturing under SmF conditions at flask scale. In addition, the laccase production under SSF conditions in tray bioreactors reduced the final cost 4-fold compared to the one obtained under SSF conditions at flask scale, obtaining a final price of 0.04 cent V/U. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Cost Laccase Pricing Solid-state fermentation Submerged fermentation Trametes pubescens
1. Introduction Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are enzymes with considerable interest in industrial and environmental biotechnology due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases.
Abbreviations: ABTS, 2,20 -azino-di-[3-ethyl-benzo-thiazolin-sulphonate]; ActLac, maximum laccase activity of the extracted crude enzyme; Cap, capacity of the equipment; CCM, cost of the culture medium; CEq A, cost of the autoclave per cultivation; CEq I, cost of the incubator per cultivation; CEq, cost of equipment; COp A, cost of operation of the autoclave per cultivation; COp I, cost of operation of the incubator per cultivation; COp, operating costs; CostLac, cost of producing laccase; Dmax, cultivation time in days; EA, energy consumption of autoclaving processes to sterilize the total number of cultures per cycle of cultivation; Gxx, glucose xx concentration in the culture medium; LT, lifetime of the equipment; MEA, malt extract agar; Myy, mandarin peelings concentration (yy) in the culture medium; P, price of equipment divided by the number of cycles; PDA, potato dextrose agar; SmF, submerged fermentation; SS, sunflower-seed shells; SSF, solid-state fermentation; TA, tannic acid; VolLac, volume of the extracted crude laccase; Yzz, yeast concentration (zz) in the culture medium. * Corresponding author. CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spain. Tel.: þ34 943 212800x2239; fax: þ34 943 213076. E-mail address: [email protected] (S. Rodríguez-Couto). 0301-4797/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2011.06.052
The cost analysis of the laccase production is an important feature for its industrial exploitation, however most papers neglect this aspect. Several authors have defined their cultivation methods or culture media as low-cost (Mukherjee et al., 2008; Ng et al., 2010), however, in few cases the real cost of production has been calculated. Different approaches at laboratory scale have proved their suitability for industrial scale production but in general terms, few of them have included a cost analysis to give support to this statement. Numerous reports on strategies to improve the production of laccase enzymes, such as the isolation of new fungal strains, heterologous expression of laccase genes, optimization of growth conditions and use of inducers and stimulators, have been recently published. However, the utilization of inducers can cause problems of both cost-effectiveness and environmental pollution. Also, the enzyme yields obtained by recombinant expression of laccase genes (Hong et al., 2002, 2006; Li et al., 2007) are relatively low for the demands in industrial applications. Therefore, the development of approaches for the production of laccase with high efficiency, environmental friendliness and cost-effectiveness is a top priority. One potential solution for the above-mentioned issues would be to decrease the production cost of laccases by using cheap growth substrates such as agricultural and food wastes or wastewater from the food or pulp and paper industries. In addition, the reutilization
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of such wastes would help to alleviate smog pollution. Thus, such an approach is being subject of increased research. However, economical studies regarding the production of laccase enzymes using synthetic media and waste materials are lacking. The present paper provides an estimation of the production cost of laccase enzymes under different culture conditions. 2. Materials and methods 2.1. Microorganism Trametes pubescens MB 89 (CBS 696.94; Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was obtained from the Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences (Vienna, Austria) and was maintained on malt extract agar (MEA) or potato dextrose agar (PDA) plates at 4 C and sub-cultured every three months. 2.2. Culture conditions T. pubescens was cultured in both submerged (SmF) and solidstate fermentation (SSF) conditions using different culture media. Under SmF conditions cultures were performed in cotton-plugged Erlenmeyer flasks (250 mL) containing 100 mL of culture medium. The composition of the culture medium was prepared according to Rodríguez-Couto et al. (2006). Three different variables were taken into consideration in our analysis: carbon and organic nitrogen sources and inducers of laccase activity. Three carbon sources were tested: glucose (10 g/L), glycerol (10 g/L) and mandarin peelings (30 g/L). Also, the addition of yeast extract as an organic nitrogen source was studied by the combination in different proportions of glucose, mandarin peelings and yeast extract (GxxeMyyeYzz cultures). The concentration of glucose (Gxx) was varied between 0 and 30 g/L, the concentration of mandarin peelings (Myy) between 0 and 60 g/L and the concentration of yeast extract (Yzz) between 0.5 and 25 g/L. Finally, the addition of different inducers in the culture with glucose (10 g/L) was analyzed. Cultures were incubated statically, under an air atmosphere, at 30 C and in complete darkness. Under SSF conditions flask-scale cultures were performed in cotton-plugged Erlenmeyer flasks (250 mL) containing 20 mL of culture medium. The compositions of the culture medium were prepared according to Osma et al. (2007) when using banana skin, Osma et al. (2006) when using wheat bran and Rodríguez-Couto et al. (2009) when using sunflower-seed shells as support. Tray bioreactors were used when cultures were scaled-up as described in Rodríguez-Couto et al. (2009). All cultures were incubated statically, under an air atmosphere, at 30 C and in complete darkness. 2.3. Analytical determinations Laccase activity was determined spectrophotometrically as described by Niku-Paavola et al. (1990) with ABTS (2,20 -azino-di[3-ethyl-benzo-thiazolin-sulphonate]) as a substrate. One activity unit was defined as the amount of enzyme that oxidized 1 mmol ABTS per min. The activities were expressed in U/L. 2.4. Cost analysis In this study, the cost of the laccase production was divided in three terms: CCM, CEq and COp. In order to determine the costs related with equipment and operation (CEq and COp), an Isotemp Standard Lab incubator and a 2340-M Heidolph/Tuttnauer Tabletop autoclave from Fisher Scientific Inc. (Suwanee, USA) were selected as model equipments. Their lifetime (LT) was determined equal to
their warranty time, one year for the incubator and two years for the autoclave. Power consumption and capacity of both equipments were calculated upon manufacturer description. For the CCM, most of the market prices for reagents or compounds were obtained from SIGMAeAldrichÒ (St. Louis, USA). In some special cases, such as agricultural materials, the cost was assumed to be equal to the lowest one of any commercialized product with similar characteristics (E-Food Depot, 2010; Index Mundi, 2010; Czech Direct, 2010; Vita Cost, 2010). Thus, in the case of the mandarin peelings, the price of a cooking product of fresh mandarin peelings was used, whereas for the sunflower-seed shells the price of bricks, made of sunflower hulls, for heating was used. Unfortunately, the price of banana skin could not be estimated and had to be assumed as null. The CEq was represented in all cases as the price of an incubator (considering the number of cultures that can be grown simultaneously) and an autoclave. For COp, the energy spent by the use of the autoclave and the incubator was taken into consideration (Esplugas et al., 2002). Nevertheless, the cost of manpower was not considered in the analysis, since it will depend on the automation and monitoring of the process, the experience of the worker and the volume of laccase to be produced. The cost of producing laccase (CostLac) was calculated as the ratio between the sum of the culture medium, equipment and operating costs and the produced enzyme activity (Equation (1)). The latter was calculated as the maximum laccase activity (ActLac) multiplied by the volume of the extracted crude enzyme (VolLac) that represents the number of total units of laccase produced.
CCM þ CEq þ COp ActLac $VolLac
CostLac ¼
(1)
2.4.1. Cost of equipment The CEq reflects the cost of the incubator and the autoclave. However, it is important to remark that this term was not expressed as the simple sum of the cost of these items but also their LT and capacity were included to determine the real price of the equipment per cultivation. In order to express this cost as detailed as possible, the price of the equipment (P) was divided by the number of cycles of operation that can be carried out depending on the cultivation time. Thus, if the maximum laccase activity of a specific culture is obtained in 7 days (Dmax), then the number of cycles that the incubator can be used is 52 in a year. Therefore, if the LT of the equipment is 2 years 104 cycles can be carried out. Also, the capacity of the equipment (Cap) has to be taken into consideration in order to establish the maximum number of cultures that can take place in each cycle. If the incubator can hold 50 Erlenmeyer flasks (250 mL), then the maximum number of cultures that can be carried out is 5200 during the 2 years of operation of the incubator. In this sense, the following expression (Equation (2)) can be suggested to calculate the cost of the incubator per culture (CEq I).
CEq
I
PI 365 $LTI $CapI Dmax
¼
(2)
The operating capacity of the autoclave is limited by the capacity of the incubator and, thus, the correct expression to calculate the autoclave cost (CEq A) can be expressed as: (Equation (3))
CEq
A
PA 365 $LTA $CapI Dmax
¼
(3)
In general, the CEq can be calculated as the sum of the cost of both equipments per culture (Equation (4)):
J.F. Osma et al. / Journal of Environmental Management 92 (2011) 2907e2912
CEq ¼ CEq
I
þ CEq
A
¼
Dmax=365 CapI
$
PI P þ A LTI LTA
(4)
2.4.2. Operating cost In a similar way to the CEq, the COp has to reflect the culture conditions in its calculation. Therefore, The COp I can be expressed as the price of the energy spent per hour (EI) multiplied by the time of cultivation in hours divided by the capacity of the incubator. In the case of the autoclave, the operating cost (COp A) was calculated using the energy consumption (EA) of the necessary autoclaving processes to sterilize the total number of cultures per cycle of cultivation. The cost of operation of the incubator (COp I) and the autoclave (COp A) per cultivation was calculated as follows (Equations (5) and (6)).
COp
I
COp
A
¼
EI $ð24$Dmax Þ CapI
(5)
¼
EA CapI
(6)
The COp was calculated as the sum of COp I and COp A.
3. Results 3.1. Laccase production 3.1.1. SmF cultivation T. pubescens reached maximum laccase activities of 51 U/L (15th day), 89 U/L (16th day) and 228 U/L (3rd day) when using glucose, glycerol and mandarin peelings as the carbon source, respectively (Table 1). Additionally, yeast extract, an organic nitrogen source, was tested in combination with glucose and mandarin peelings at different concentrations. The combination of these three compounds (glucose, mandarin peelings and yeast extract) led to a maximum laccase activity of 2100 U/L (Table 1). Therefore, the addition of yeast extract in the culture medium led to higher laccase activities than those obtained by just using glucose or mandarin peelings. 3.1.2. SSF cultivation T. pubescens was also cultured under SSF conditions at flask scale, testing three natural supports: banana skin, wheat bran and sunflower-seed shells. The use of sunflower-seed shells considerably increased the laccase production reaching maximum laccase activities of 7750 U/L (10th day), compared to the 2500 U/L (10th day) and the 1600 U/L (20th day) obtained when using wheat bran and banana skin, respectively (Fig. 1) (Osma et al., 2006, 2007; Rodríguez-Couto et al., 2009). The culture medium with sunflower-seed shells was selected as the basal medium to test the effect of different inducers (Rodríguez-Couto et al., 2009). It is known that the addition of inducers considerably influences the activity of laccase (Bollag and Leonowicz, 1984), thus, five inducers were tested at different concentrations and added at different time points of cultivation (inoculation, tropophase e 3rd day and idiophase e 7th day). In almost all cases, laccase activities increased by the addition of the inducer, reaching a maximum of 25,773 U/L when adding 0.5 mM Cu2þ on the 3rd day of cultivation (RodríguezCouto et al., 2009). The joint effect of inducers was also investigated by combining the addition of 0.5 mM Cu2þ with 1 mM of xylidine or 50 mM of tannic acid on the 3rd day or on both the 3rd and the 7th day of cultivation. The use of Cu2þ and tannic acid added on the 3rd day of cultivation reached a maximum laccase activity of 30,272 U/L, while the other combinations led to maximum laccase
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activities similar or lower than the one obtained by just the addition of 0.5 mM Cu2þ on the 3rd cultivation day. Finally, the sunflower-seed shells cultures induced with 0.5 mM Cu2þ and 50 mM of tannic acid were scaled-up to tray bioreactors (Rodríguez-Couto et al., 2009). The surface area of the tray bioreactor was 7.5 times higher than the surface area of the Erlenmeyer flasks used. Due to the technical difficulties of mixing at this scale, the addition of inducers was only studied at inoculation and tropophase (3rd day). The scaled cultures presented a similar behavior to their counterpart at flask scale. Thus, when cultures were not induced, the maximum laccase activity was about 7130 U/L (13th day), while induced cultures reached maximum laccase activities of 31,610 (14th day) and 41,135 U/L (13th day) when induced at inoculation and tropophase, respectively (Rodríguez-Couto et al., 2009). 3.2. Cost analysis The total cost of the culture medium, equipment and operation was compared with the maximum laccase obtained in each culture and the time of cultivation needed. In this sense, the price of the laccase produced was influenced by all three terms (CCM, CEq and COp). Table 1 summarizes the calculation of these three costs, the laccase produced, the time of cultivation and the final price of the laccase produced for each culture performed. 3.2.1. Effect of inducers on the final price of laccase production The addition of most inducers increased the maximum laccase activity in both SmF and SSF cultures. By simply considering the increment on laccase activity, the use of inducers is coherent and reasonable. However, when analyzing the final price of produced enzyme, the use of inducers is not always suitable. Cultures under SmF conditions using glucose as a carbon source and no inducers presented a final price of about 17 cents of Euro per Unit of laccase produced. The maximum laccase activity obtained in this type of culture was 51 U/L after 15 days of cultivation. When adding inducers, the maximum activity was in some cases more than 4-fold higher than that obtained in cultures with no inducers in about the same time of cultivation (16 days). This increment was, however; not reflected in the final price of the produced laccase, which on the contrary increased in 666% its final price compared to the one produced without the addition of inducers. On the other hand, the use of inducers in cultures under SSF conditions presented a decrement in the final price of the produced laccase. Cultures using sunflower-seed shell as support presented a maximum laccase activity of 7750 U/L after 10 days of cultivation. The addition of inducers in this type of culture also increased the laccase activity up to 4-fold in relation to the cultures with no inducers in about the same time of cultivation. In this case, the increment of the laccase activity was reflected in a decrement of the final price of the produced laccase; thus, while cultures without induction presented a final price of 0.3 cents of Euro per Unit, induced cultures presented an average final price of 0.15 cents of Euro per Unit, a reduction of about 49%. 3.2.2. Effect of scaling-up on the final price of laccase production The effect of scaling-up was also analyzed in terms of the final price of the produced laccase. Cultures with sunflower-seed shells with and without inducers were scaled-up to tray bioreactors. Although, the laccase activity profile obtained at flask-scale and reactor-scale cultures was very similar, the final price was reduced when cultures were carried out at larger scale. The final price of the laccase produced in tray bioreactors was 22% lower for cultures without inducers and about 69% lower when induced than their flask-scale counterparts.
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Table 1 Cost analysis of laccase produced under different cultivation conditions. Culture medium (carbon source, support and/or inducer) SmF Glucose Glucose þ ABTS Glucose þ Tween 20 Glucose þ Soy oil Glucose þ Malaquite Green Glucose þ 1 mM Cu2þ Glucose þ 2 mM Cu2þ Glucose þ 50 mM Tannic Acid Glucose þ 100 mM Tannic Acid Glycerol Mandarin peels G30-M30-Y15 G20-M45-Y20 G20-M45-Y10 G20-M15-Y20 G20-M15-Y10 G15-M60-Y15 G15-M30-Y25 G15-M30-Y15 G15-M30-Y05 G15-M0-Y15 G10-M45-Y20 G10-M45-Y10 G10-M15-Y20 G10-M15-Y10 G0-M30-Y15
Max. laccase activity [U/L]
Laccase produced [U]
Time [days]
CCM [V]
COp [V]
CEq [V]
Total cost [V]
Final price [cent V/U]
51 5 110 225 80 7 25 200 105 89 228 442 241 1305 1817 714 291 436 557 282 290 847 2107 1358 66 844
4.1 0.4 8.8 18.0 6.4 0.6 2.0 16.0 8.4 7.1 18.2 35.4 19.3 104.4 145.4 57.1 23.3 34.9 44.6 22.6 23.2 67.8 168.6 108.6 5.3 67.5
15 16 16 16 16 15 10 10 16 16 3 12 6 8 8 8 3 11 10 9 10 3 8 3 10 3
0.5761 3.1116 5.2081 0.5761 0.5761 0.5764 0.5767 0.5770 0.5779 0.6641 0.6421 0.7888 0.8257 0.7639 0.7597 0.6979 0.8143 0.8101 0.7483 0.6865 0.6823 0.7987 0.7369 0.7327 0.6709 0.7078
0.1236 0.1315 0.1315 0.1315 0.1315 0.1236 0.0842 0.0842 0.1315 0.1315 0.0290 0.1000 0.0526 0.0684 0.0684 0.0684 0.0290 0.0921 0.0842 0.0763 0.0842 0.0290 0.0684 0.0290 0.0842 0.0290
0.0070 0.0074 0.0074 0.0074 0.0074 0.0070 0.0046 0.0046 0.0074 0.0074 0.0014 0.0056 0.0028 0.0037 0.0037 0.0037 0.0014 0.0051 0.0046 0.0042 0.0046 0.0014 0.0037 0.0014 0.0046 0.0014
0.7066 3.2505 5.3470 0.7151 0.7150 0.7070 0.6655 0.6659 0.7169 0.8030 0.6724 0.8943 0.8811 0.8360 0.8318 0.7700 0.8446 0.9072 0.8371 0.7669 0.7711 0.8290 0.8090 0.7630 0.7597 0.7381
17.32 812.62 60.76 3.97 11.17 126.24 33.28 4.16 8.53 11.28 3.69 2.53 4.57 0.80 0.57 1.35 3.63 2.60 1.88 3.40 3.32 1.22 0.48 0.70 14.39 1.09
SSF flask scale Banana skin Wheat bran SS SS þ 1% v/v coconut oil SS þ 2% v/v coconut oil SS þ 0.1 mM Cu2þ SS þ 0.3 mM Cu2þ SS þ 0.5 mM Cu2þ SS þ Soy oil 1% (v/v) SS þ Soy oil 2% (v/v) SS þ 25 mM Tannic acid SS þ 50 mM Tannic acid SS þ 1 mM Xylidine SS þ Cu2þ þ tannic Ac. (added 3rd day) SS þ Cu2þ þ xylidine (added 3rd day) SS þ Cu2þ þ tannic Ac. (added 3rd and 7th day) SS þ Cu2þ þ xylidine (added 3rd and 7th day)
1600 2500 7751 10,951 18,382 18,773 23,621 25,773 9708 10,165 10,635 10,064 12,494 30,272 19,085 22,946 26,645
8.0 12.5 77.5 109.5 183.8 187.7 236.2 257.7 97.1 101.7 106.4 100.6 124.9 302.7 190.8 229.5 266.4
20 10 10 8 9 10 10 10 9 10 10 10 10 11 11 11 8
0.1098 0.1098 0.1459 0.1461 0.1464 0.1459 0.1460 0.1460 0.1459 0.1460 0.1461 0.1463 0.1461 0.1463 0.1461 0.1467 0.1464
0.1631 0.0842 0.0842 0.0684 0.0763 0.0842 0.0842 0.0842 0.0763 0.0842 0.0842 0.0842 0.0842 0.0921 0.0921 0.0921 0.0684
0.0093 0.0046 0.0046 0.0037 0.0042 0.0046 0.0046 0.0046 0.0042 0.0046 0.0046 0.0046 0.0046 0.0051 0.0051 0.0051 0.0037
0.2822 0.1986 0.2348 0.2183 0.2268 0.2348 0.2348 0.2348 0.2264 0.2348 0.2350 0.2351 0.2349 0.2435 0.2433 0.2439 0.2185
3.53 1.59 0.30 0.20 0.12 0.13 0.10 0.09 0.23 0.23 0.22 0.23 0.19 0.08 0.13 0.11 0.08
SSF bioreactor scale Tray bioreactor Tray bioreactor þ Cu2þ þ tannic Ac. (added 0 day) Tray bioreactor þ Cu2þ þ tannic Ac. (added 3rd day)
7130 31,610 41,135
570.4 2528.8 3290.8
13 14 13
1.0945 1.0975 1.0975
0.2357 0.2522 0.2357
0.0126 0.0135 0.0126
1.3428 1.3632 1.3458
0.24 0.05 0.04
Fig. 1. Laccase production by T. pubescens grown under solid-state fermentation conditions: (dod) on banana skin; (dΔd) on wheat bran; (dAd) on sunflowerseed shells.
3.2.3. Influence of the culture medium cost on the total cost of laccase production The cost of the laccase production was divided in three terms, CCM, CEq and COp, all of them somehow dependent on the inherent characteristics of each culture. However, in all cultures there was a constant tendency, the CCM represented the greatest part of the final cost of production. As shown in Fig. 2, the CCM represents 89% of the total cost for SmF cultures, while this percentage decreases to about 61% for SSF cultures. This shows a higher dependency of SmF cultures on the cost of reagents and materials used in the culture medium than the one presented in SSF. Therefore, the replacement of reagents by low-cost materials is highly desirable. The use of agro-waste materials as the main component in the culture medium highly reduced the CCM, leading to a reduction in the final price of the produced laccase. Jing (2010) pointed out that the initial C/N ratio in the culture medium as well as the kind of C and N sources used were key factors affecting both laccase activity and cost. Thus, he found that a low initial C/N ratio not only stimulated
J.F. Osma et al. / Journal of Environmental Management 92 (2011) 2907e2912
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Fig. 2. Culture medium, equipment and operation costs (CCM, CEq and COp) percentages in SmF cultures, SSF cultures at flask scale and SSF cultures at bioreactor scale.
Table 2 Price of different commercial laccases. Supplier/reference
Description
Price [V]
Amount [g]
Estimated activity [U]
Final price [V/U]
Sigma/L2157 Sigma/53739 Sigma/38429 Jena Bioscience/EN-204
Laccase from Rhus vernificera (>50 units/mg) Laccase from Trametes versicolor, BioChemika (>20 units/mg) Laccase from Trametes versicolor, BioChemika (0.5 units/mg) Laccase EC 1.10.3.2 from Trametes versicolor
153.9 70.4 378.5 300
e 1 10 e
10,000 20,000 5000 5000
0.015 0.004 0.076 0.060
the simultaneous production of laccase and lignin peroxidase (LiP) by Streptomyces lavendulae but also the cost of enzyme production was cut down effectively. However, according to Eggert et al. (1996) the production of ligninolytic enzymes including laccases was stimulated and triggered under N-limitation conditions, i.e. high C/N ratio. We could not find any relationship between the C/N ratio and laccase activity in our cultures. This is in agreement with the results reported by Lebrun et al. (2011), who found no relationships between the C/N ratios and the production of oxidoreductases. One of the major concerns in the production of ligninolytic enzymes is the small amount of enzymes produced that can be sometimes relaxed by scaling them up. However, when SSF cultures were scaled-up to tray bioreactors the final cost of the produced laccase was reduced by reducing the COp, representing only the 18% of the total production costs. Additionally, in all cases the CEq represented less than 2% of the total production costs. 3.2.4. Final price of the produced laccase The price of the produced laccase in each type of cultivation and for different culture conditions was different. In this sense, the average price per type of cultivation was calculated considering the successful cultivation cases (all cultures except SmF cultures induced with ABTS or Cu2þ) showing a final mean price of 7 cents of Euro per Unit for SmF cultures, compared to 0.44 and 0.11 cents of Euro per Unit for SSF cultures at flask scale and at bioreactor scale, respectively. Laccase produced by SSF cultures showed a lower final price than the ones obtained when culturing under SmF conditions. Additionally, the scale-up of the SSF cultures decreased in about 4fold the final price of the laccase produced. This effect was because of the slight decrement in the culture medium costs per volume but mainly because of the reduction in the operating costs. The geometry of the bioreactors led to have more working volume in the incubator and, thus, reducing the operating costs. In comparison with commercially available laccases, the price of the laccase produced under SSF conditions using bioreactors was more than 10-fold lower than those commercialized by SIGMAeAldrichÒ (St. Louis, USA) and Jena Bioscience (Jena, Germany). This, however, does not consider other industrial costs related to the packaging, storage and distribution of the enzyme. Table 2 shows the price of different commercial laccases. 4. Conclusions T. pubescens was cultured under both SmF and SSF conditions using more than 45 different culture medium compositions.
Different variables from the composition of the culture medium were taken into consideration such as carbon source, nitrogen source, supports and inducers. The laccase activity of all cultures was measured and the cost of laccase production was estimated. In this sense, three main terms were defined: cost of the culture medium, cost of equipment and operating costs. In all cases, the cost of the culture medium represented the highest contribution to the total cost, while, the cost of the equipment was significantly low, representing less than 2% of the total costs. Also, the cultivation of the fungus under SSF conditions led to lower costs of the culture medium and operation, presenting a final production cost of about 50-fold lower than the one obtained when culturing under SmF conditions. In addition, the production under SSF conditions was scaled-up to tray bioreactors. In this case the operating costs were clearly reduced, reflecting a final price 4-fold lower than the one obtained at flask scale. In view of the results obtained more efforts in reducing the cost of the culture medium are required to reach economically feasible laccase production systems. Acknowledgments This research was financed by the Spanish Ministry of Education and Science (Project CTQ2007-66541). Appendix. Supplementary material Supplementary data associated with this article can be found, in the on-line version, at doi:10.1016/j.jenvman.2011.06.052. References Bollag, J.M., Leonowicz, A., 1984. Comparative studies of extracellular fungal laccases. Applied and Environmental Microbiology 48, 849e854. Czech Direct, 2010. http://www.czechdirect.ie/ecowoodpellets. E-Food Depot, 2010. http://www.efooddepot.com/products/jiabao_brand/5834/ preserved_mandarin_peel__hypen__0_dot_52oz.html. Eggert, C., Temp, U., Dean, J.F.D., Eriksson, K.-E.L., 1996. A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Letters 391, 144e148. Esplugas, S., Giménez, J., Contreras, S., Pascual, E., Rodríguez, M., 2002. Comparison of different advanced oxidation processes for phenol degradation. Water Research 36, 1034e1042. Hong, F., Meinander, N.Q., Jönsson, L.J., 2002. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnology and Bioengineering 79, 438e449. Hong, Y.Z., Xiao, Y.Z., Zhou, H.M., Fang, W., Zhang, M., Wang, J., Wu, L., Yu, Z., 2006. Expression of a laccase cDNA from Trametes sp. AH28-2 in Pichia pastoris and mutagenesis of transformants by nitrogen ion implantation. FEMS Microbiology Letters 258, 96e101.
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