Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1298-y
ORIGINAL PAPER
Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste Sheila Romo Sa´nchez • Irene Gil Sa´nchez • Marı´a Are´valo-Villena • Ana Briones Pe´rez
Received: 12 March 2014 / Accepted: 30 September 2014 Springer-Verlag Berlin Heidelberg 2014
Abstract The recovery of by-products from agri-food industry is currently one of the major challenges of biotechnology. Castilla-La Mancha produces around three million tons of waste coming from olive oil and wine industries, both of which have a pivotal role in the economy of this region. For this reason, this study reports on the exploitation of grape skins and olive pomaces for the production of lignocellulosic enzymes, which are able to deconstruct the agroindustrial waste and, therefore, reuse them in future industrial processes. To this end, solid-state fermentation was carried out using two local fungal strains (Aspergillus niger—113 N and Aspergillus fumigatus—3). In some trials, a wheat supplementation with a 1:1 ratio was used to improve the growth conditions, and the particle size of the substrates was altered through milling. Separate fermentations were run and collected after 2, 4, 6, 8, 10 and 15 days to monitor enzymatic activity (xylanase, cellulase, b-glucosidase, pectinase). The highest values were recorded after 10 and 15 days of fermentation. The use of A. niger on unmilled grape skin yielded the best outcomes (47.05 U xylanase/g by-product). The multi-enzymatic extracts obtained were purified, freeze dried, and
S. Romo Sa´nchez I. Gil Sa´nchez M. Are´valo-Villena (&) A. Briones Pe´rez Food Science and Technology, University of Castilla-La Mancha (UCLM), Av. Camilo Jose´ Cela 10, Edificio Marie Curie, 13071 Ciudad Real, Spain e-mail: [email protected] S. Romo Sa´nchez e-mail: [email protected] I. Gil Sa´nchez e-mail: [email protected] A. Briones Pe´rez e-mail: [email protected]
immobilized on chitosan by adsorption to assess the possible advantages provided by the different techniques. Keywords Enzyme Fungal strain Waste Solid-state fermentation Immobilization
Introduction Considering the great deal of green waste in nature, some microorganisms show great potential in biotechnology because of their ability to metabolize lignocellulosic compounds. Agroindustrial waste, such as bagasse of sugar cane, wheat, corn, rice husk, fruit skin, and olive pomace (concentrated waste coming from alcohol and oil mill industries), is on the increase because of industrialization. This poses a problem in terms of space available and environmental pollution. In Spain, olive oil and winemaking companies figure prominently in the agri-food sector as some of the leading production and export industries. This extensive production results in approximately three million tons of waste a year (according to AAO, http://aplicaciones.mapa.es/pwAgenciaAO/General. aao?idioma=ING&control_acceso=S). Traditional methods for olive oil and wine waste management include chemical, physical, and biological processes. Hemicelluloses and celluloses represent over 50 % of dry weight of by-products. These may be transformed into soluble sugars by acid or enzymatic hydrolysis [1], which would be an inexpensive and abundant renewable energy source. Solid-state fermentation has been described as a process of insoluble matter transformation that serves both as a physical support and as a source of nutrients in the absence or near absence of free water. However, the substrate should be moist enough to allow
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for growth and metabolism of the microorganisms [2]. Because solid-state fermentation shows great potential in metabolite production [3], this process is particularly useful in the production of microbial products, such as food, fuel oil, and industrial chemicals and pharmaceuticals. Accordingly, the waste produced would be used as substrate in solid-state fermentation (SSF) for enzyme production [4]. Da Silva et al. [5] and Soliman et al. [6] found that Thermoascus auranticus and Aspergillus niger are good producers of lignocellulosic enzymes, that include cellulases (EC 3.2.1.4.), xylanases (EC 3.2.1.8.), and ligninases (EC 1.11.1.14.) which are able to break down the structure of cellulose, hemicellulose, and lignin of plant materials. Enzymes of the cellulolytic system that degrade cellulose altogether are exo-b-1,4-glucanase, endo-b-1,4-glucanase, and b-glucosidase [5]. Hemicellulose, the second most abundant renewable biomass in nature, is largely made up of xylan (25–30 % of dry weight of plant cell wall). Xylanases are the enzymes that can hydrolyze xylan, and they are of considerable interest for their industrial application [6]. Pectinases, another group of enzymes, degrade pectins, which are structural polysaccharides formed by units of polygalacturonic acid and inclusions of other monosaccharides located in the middle lamella and in the primary wall of higher plants [7]. Both molds and yeasts are able to thrive in this type of substrate, thanks to their enzymatic systems. Molds are particularly interesting because they have a set of advantages: (1) they play a major role in a number of food industries, (2) they excrete enzymes of a different nature that allow molds to metabolize complex mixtures of organic compounds found in most residues [8], (3) their filamentous morphology or pellet guarantees enzyme separation and production at a low cost [5, 9]. At present, the enzymes’ biotechnology is a common point among different sectors of the industry. A promising approach to retain their stability is the immobilization. Many are the supports (inorganics—bentonite, ceramics, silica…—or organics—chitosan, alginate, proteins…) and immobilization technics (encapsulation, adsorption, reticulation among others) used depending on the end use of the enzyme. Among others, immobilization on chitosan by adsorption is one of the most used. The aim of this study is to produce b-glucosidase, cellulase, xylanase, and pectinase by solid-state fermentation of by-products, considering factors such as composition of the medium, milling degree, and microbial strain. Enzymes were purified, lyophilized, and immobilized for industrial application of the enzymatic extract obtained.
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Materials and methods Microorganisms After initial experiments for strain screening, two local fungal strains, Aspergillus niger (strain N 113 N) and Aspergillus fumigatus (strain N 3), were selected (both deposited in the GenBank Database with accession numbers FJ499449 and FJ499462) due to their high enzymatic activity. These strains were isolated from olive paste and olives, respectively, in the Yeast Biotechnology Laboratory at the University of Castilla-La Mancha (Spain) [10]. Molds were maintained using the method suggested by Castellani [11]. A. niger and A. fumigatus were deposited in the Spanish Type Culture Collection (CECT) whose accession numbers are 20828 and 20827, respectively (http://www.cect.org). Natural substrates Grape skin (S) and olive pomace (P) were the by-products collected locally from agroindustries, as well as the wheat (W) used as a medium supplement. The grape skins and olive pomace were air dried at 40 C/24 h, and separated into two series, one of which was milled with an ultracentrifugal mill (Retsch ZM200) using a sieve with a pore size of 0.5 lm. The other series was not milled. The chemical composition of S and P was analyzed, quantifying moisture, starch, lignin, cellulose, hemicellulose, pectin, fat, and ash percentage (Van Soest; PNTLACC/FQ 002; Gravimetry; AOAC 1997, 985.29; Regulation 152/2009 Annex II H Proceeding B; Regulation (EC) 152/2009 Annex III A). Solid-state fermentation and enzyme production To obtain the precultures, the A. niger and A. fumigatus strains were grown in 250 mL Erlenmeyer flasks containing 50 mL PDA medium (agar slant) at 30 C/65 % moisture for 7 days. Then, 100 mL of the basal medium (0.3 % KH2PO4; 0.35 % (NH4)2SO4; 0.05 % MgSO47H2O; 0.05 % CaCl2) was added to culture, and slightly scratched to obtain mycelial suspension. The design of SSF medium was prepared according to da Silva et al. [5] with slight modifications. 5 mL of suspension of each mycelium was transferred to 100 mL Erlenmeyer flasks containing 5 g of solid substrate to be tested (S or P). As many as eight different fermentations were run for each of the molds, depending on whether or not wheat was added and the by-products were milled: S [unmilled grape skin]; SW [unmilled grape skin (2.5 g) ? wheat (2.5 g)];
Bioprocess Biosyst Eng
mS [milled grape skin]; mSW [milled grape skin (2.5 g) ? wheat (2.5 g)]; P [olive pomace]; PW [olive pomace (2.5 g) ? wheat (2.5 g)]; mP [milled olive pomace]; mPW [milled olive pomace (2.5 g) ? wheat (2.5 g)]. Enzyme production was sampled after 2, 4, 6, 10, and 15 days for assessment over time. The flasks were incubated at 25 C/65 % moisture during the time period set. Afterwards, 100 mL of distilled water was added to the flasks and they were maintained under shaking for 300 at room temperature for extracellular enzyme extraction. The content of the flasks was centrifuged (4,500 rpm/150 /4 C) and the supernatant was maintained at -20 C until analysis.
Table 1 Raw material composition (%) in grape skins and olive pomace
Enzyme assays
The purified extract, quick frozen at -80 C, was subsequently lyophilized at pressures of 2 9 10-2 and -53 C without cryoprotectant and with 20 % skimmed milk (Nestle Sveltesse, Spain) [19]. The qualitative analysis of proteins of the purified and lyophilized extract was monitored by SDS-PAGE electrophoresis in sulfate–polyacrylamide gel (Bio-Rad CriterionTM XT Precast Gel 12 % Bis–Tris) using an 8–220 kDa molecular weight marker (MW Sigma ColorBurst). The staining was performed with Coomassie Blue.
Activity of the enzymes pectinase, xylanase, cellulase, and b-glucosidase was assessed. 0.1 mL of the extract (supernatant) was incubated at 37 C/300 with 0.4 mL of the corresponding substrate in 0.1 M acetate buffer, pH 5: 0.5 % (w/v) xylan from beechwood (Sigma-Aldrich Corporation, St Louis) for xylanase; 1 % (w/v) carboxymethylcellulose (CMC—Panreac AppliChem, Germany) for cellulase; 0.5 % (w/v) pectin from apple (Sigma) for pectinase; and 1 % (w/v) D-(?)-cellobiose (Fluka Chemical Corporation) for b-glucosidase. Following the incubation period, the reducing sugars released were measured using the 3,5-dinitrosalicylic acid (DNSA) method [12]. Assessment of b-glucosidase activity was based on the protocol suggested by Are´valo-Villena et al. [13]. Parallely, blank absorbance was measured in the same way as the samples but with no incubation (at 0 time). For reducing sugar measurement, standard curves of galacturonic acid, xylose, and glucose were used, depending on the enzyme to be assessed. Assays were performed in triplicate in all cases and outcomes were calculated as enzyme units (U) defined as lmol of released product per minute under reaction conditions.
Composition (%) Humidity
Grape skins 5.6
Olive pomace 3.4
Starch
2.5
1.3
Lignin
22.5
22.6
Cellulose
25.9
22.9
Hemicellulose
3.6
5.9
Pectin
1.3
5.8
Fat Ash
9.8 6.5
15.9 3.3
Multi-enzymatic extract immobilization The purified and lyophilized enzymes (with and without cryoprotectant) were immobilized on chitosan by physical adsorption. The immobilization conditions, which were established based on previous research [20], involved an enzymatic concentration of 0.048 g/mL in acetate buffer pH 5, 50 mM in a final volume of 50 mL. The reaction was maintained for 2 h at 10 C under gentle shaking. Afterwards, up to 4 washes were performed with the same buffer to eliminate the unintegrated protein and cryoprotectant. Xylanase activity of the enzymatic extracts was monitored in triplicate prior to and after each biotechnological process following the previously described protocol.
Purification and lyophilization of enzymes Results A purified extract was obtained after solid-state fermentation of the mold–substrate pair that yielded the best results at the previous stages of the study. The multi-enzymatic extract underwent a variety of technological processes, and its activity was monitored at each of the stages. Supernatant was purified by size exclusion using filter cartridges for the concentration and purification of proteins (Pall, New York, USA). The molecule size selected was 100 kDa, considered best suitable for excreted protein [14– 17]. Samples were run at 4 C for 1 h at 4,500 rpm, and the enzymatic activity was monitored [18].
Natural substrates Table 1 shows the composition of each of the lignocellulosic substrates used in this study. The main constituents in both cases were lignins and celluloses, which are a potential adequate source of carbon for polysaccharidehydrolyzing microorganisms. However, the values of nitrogenized content were low, which would a priori hinder the development of the molds. For this reason, some fermentations were supplemented with wheat. The differences
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Fig. 1 Enzymatic activity of A. fumigatus and A. niger in solid-state fermentation over time (days). S grape skins, SW grape skins and wheat, mS milled grape skins, mSW milled grape skins and wheat
between both by-products lay in the high fat content and the higher percentage of pectins found in olive pomace. Solid-state fermentation and enzyme production Once their composition was determined, the by-products were used for mold growth and enzyme production by SSF using the combinations described in the ‘‘Materials and methods’’ section. Figure 1 shows the values of the enzymatic activities (cellulase, pectinase, b-glucosidase, and xylanase) obtained on grape skins with or without wheat supplement during SSF. Fermentation on unmilled substrates was beneficial both for A. niger and A. fumigatus. A significant increase in activity was observed from the 6th day of fermentation, reaching its maximum activity on the 10th and 15th day. This is likely due to the very metabolic development of the microorganisms. Upon comparison of the enzymatic activities of both molds, A. niger was found to show the
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best results in all cases, even showing a significant activity in grape skins with no wheat supplement. Xylanase reached 47.05 U per g of substrate on the 15th day of fermentation on S. b-glucosidase also showed a rich activity on the 10th day (29.47 U/g), although this only occurred when the grape skins were supplemented with wheat (SW). The enzymatic activity of A. fumigatus was significantly weaker in all cases. Even though the highest activity rate was reported on b-glucosidase, a great difference was found between this rate and that reported on A. niger (10.02 vs 4.17 U). This only occurred when wheat was added, which might be indicative of a greater need of A. fumigatus for nitrogenized material. Figure 2 includes values of maximum enzymatic activity of both molds which were smaller than that of grape skins, particularly in the case of A. niger. The maximum activity values were reported on A. fumigatus on the 4th day of growth for xylanase production (8 U). Virtually all cases required wheat supplementation and unmilled substrates, even for mold growth (none of the molds was able
Bioprocess Biosyst Eng
Fig. 2 Enzymatic activity of A. fumigatus and A. niger in solid-state fermentation over time (days). P olive pomace, PW olive pomace and wheat
to grow on milled olive pomace fermentation; data not shown). Enzyme purification, lyophilization and immobilization Based on the previous results, the microorganism–substrate pair selected was A. niger-unmilled grape skins for SSF for 15 days. The enzymatic extract obtained was purified and lyophilized according to the protocols described in the materials and methods section. Protein size and concentration and xylanase activity were checked because this was the most excreted enzyme, as shown in Fig. 1. Protein electrophoresis of the raw and purified-lyophilized enzymatic extracts of A. niger showed that the protein
patterns of both extracts were similar. The band intensity of the purified-lyophilized extract showed a lower activity of the enzymes in both cases (data not shown). This fact preluded almost total loss of proteins, and thus, of the xylanase activity. For data confirmation, the xylanase activity was measured using xylan as substrate (Table 2). Protein purification held back 83 % of enzymatic activity, which proved the cut size selected was right. Nevertheless, the process of lyophilization had a devastating impact on the activity (almost 90 % of it was lost). The use of the cryoprotectant could not prevent denaturalization, as shown by the low percentages reported (16.4 and 13.0 % with and without cryoprotectant, respectively). Finally, immobilization of the lyophil led to almost total loss of
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Bioprocess Biosyst Eng Table 2 Xylanase activity percentage (%) of the enzymatic extracts of raw, purified, lyophilized, and immobilized A. niger
Raw extract
100.0 ± 1.3
Purified extract
Lyophilized extract
Immobilized extract
With cryoprotectant
Without cryoprotectant
With cryoprotectant
Without cryoprotectant
82.8 ± 0.3
16.4 ± 2.6
13.0 ± 0.1
5.4 ± 0.10
0.03 ± 0.0
the activity (less than 10 % in both cases, with and without cryoprotectant).
Discussion This study relied on two molds in the species of Aspergillus (A. niger and A. fumigatus), which were isolated from oleic environments. Solid-state fermentations were run on two substrate types, i.e. grape skins and olive pomace, for industrial-use enzyme production. The analysis of the composition of both substrates showed that their main constituents are lignins and celluloses, which is consistent with the data reported in the literature. According to Georgieva and Ahring [21], olive pomace is largely made of lignin (22.8 %), a percentage that is very close to that reported in our study (22.6 %). Cara et al. [1] determined the composition of raw materials of olive trees, reporting dry biomass percentages slightly higher than those reported by our study (25 % in cellulose and 15.8 % in hemicellulose). This difference in percentage may be due to the mixture of substrates with other types of materials from the trees themselves. As for grape skins, the data found in the literature does not differ significantly with respect to pectin percentage from those reported by the present study (cellulose 27–37 %) [22]. Enzyme production using filamentous molds for solidstate fermentation or submerged fermentation is a widely used biotechnological method. Selection of a fermentation type depends on the physiological adaptation of the organism. The growth pattern of filamentous molds in submerged culture usually ranges between pellet-like and filamentous, which may affect enzymatic production [23]. Previous studies conducted in our laboratory with submerged culture did not report any pectinase and glucosidase activity [10]. In contrast, the same molds cultured in SSF in the present study did show activity. This is due to the fact that SSF provides the molds with a closed environment similar to their natural habitat, which stimulates them to produce more hemicellulolytic enzymes [5]. For all four enzymes examined, wheat supplement had an impact both on A. niger and A. fumigatus because of the easily assimilable nitrogen that wheat encouraged the production of enzymes. This increase is associated with the particle size of substrates. According to Salihu et al. [9],
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this is one of the factors that most affect fermentation, together with pre-treatment and water retention capacity. In resume, the study provides evidence of the appropriateness of grape skins and olive pomace for enzyme production, and thus, the validity of these by-products for this purpose is reinforced. The combination A. niger–grape skin (strain–substrate) yielded optimal results, being xylanase the enzyme with the highest concentration. Milling of both substrates did not improve enzyme production in any case. While not necessary for grape skins, wheat supplement stimulated microbial growth and enzyme production in olive pomace. Selection of cut size for purification (100 kDa) was based on the size of enzymes under examination: b-glucosidase, 100 kDa [14]; xylanase, 31 kDa [16]; pectinase, between 25 and 50 kDa [15]; and cellulase, 21 kDa [17]. The outcomes were consistent with previous research, since the purification process yielded an enzymatic extract with almost 83 % of residual activity. The lyophilization process, however, did not meet expectations, not even with the help of a cryoprotectant. There are many compounds that can be used to this aim (glycerol, skimmed milk, dimethyl sulfoxide, and carbohydrates, such as glucose, lactose, saccharose, and inositol). Further research with other cryoprotectant agents is thus needed for yield improvement. The results yielded in our study for adsorption-induced multi-enzymatic extract immobilization on chitosan are not representative, nor do they prove this process right or wrong. The reason for this is that the residual activity of the lyophil used was much too low (activity dropped more than 80 %). Although there is no research addressing immobilization of multi-enzymatic systems, there are studies dealing with coupling between two enzymes. The objective of such studies is either to obtain a final product or to eliminate a by-product of one of the two enzymes for being contaminating or detrimental to the process [24]. Accordingly, some of these biological catalysts would be able to prevent others from coupling on the substrate. Currently, there is an interest in the development of techniques that guarantee enzyme immobilization on a support on a specific area, revealing significant zones for stabilization. All these data pave the way for future studies including lyophilization assays with different cryoprotectants and/or assays involving immobilization of the purified extract
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without prior lyophilisation, which might be the key to the biotechnological advance of this topic. Acknowledgments The authors wish to express their gratitude to Dr. Mario Canales for protein electrophoresis assistant and to Dr. Hector L. Ramirez for immobilization process.
References 1. Cara C, Ruiz E, Oliva JM, Sa´ez F, Castro E (2008) Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification. Bioresour Technol 99:1869–1876 2. Singhania RR, Patel AK, Soccol CR, Pandey A (2009) Recent advances in solid-state fermentation. Biochem Eng J 44:13–18 3. Holker U, Hofer M, Lenz J (2004) Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl Microbiol Biotechnol 64:175–186 4. Couto SR, Sanroma´n MA (2006) Application of solid-state fermentation to food industry—a review. J Food Eng 76:291–302 5. da Silva R, Lago ES, Merheb CW, Macchione MM, Park YK, Gomes E (2005) Production of xylanase and CMCase on solid state fermentation in different residues by Thermoascus aurantiacus Miehe. Braz J Microbiol 36:235–241 6. Soliman HM, Sherief A-DA, El-Tanash AB (2012) Production of xylanase by Aspergillus niger and Trichoderma viride using some agriculture residues. Int J Agric Res 7:46–57 7. Kashyap DR, Vohra PK, Chopra S, Tewari R (2001) Applications of pectinases in the commercial sector: a review. Bioresour Technol 77:215–227 8. Jin B, Yan XQ, Yu Q, van Leeuwen JH (2002) A comprehensive pilot plant system for fungal biomass protein production and wastewater reclamation. Adv Environ Res 6:179–189 9. Salihu A, MdZ Alam, AbdulKarima MI, Salleh HM (2012) Lipase production: an insight in the utilization of renewable agricultural residues. Resour Conserv Recycl 58:36–44 ´ beda-Iranz J, Briones-Pe´rez 10. Alves-Baffi M, Romo-Sa´nchez S, U A (2012) Fungi isolated from olive ecosystems and screening of their potential biotechnological use. New Biotechnol 29:451–456 11. Castellani A (1939) Viability of some pathogenic fungi in distilled water. J Trop Med Hyg 42:225–226 12. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428
´ beda JF, Navascue´s E, Bri13. Are´valo-Villena M, Dı´ez Pe´rez J, U ones AI (2006) A rapid method for quantifying aroma precursors: application to grape extract, musts and wines made from several varieties. Food Chem 99:183–190 ´ beda-Iranzo JF, Briones-Pe´rez A (2007) b– 14. Are´valo-Villena M, U glucosidase activity in wine yeasts: application in enology. Enzyme Microb Tech 40:420–425 15. Palacios A, Santiago L, Guerrand D Utilizacio´n de enzimas de maceracio´n en vinificacio´n en tinto. Personal communication 16. Ncube T, Howard RL, Abotsi EK, Jansen van Rensburg EL, Ncube I (2012) Jatropha curcas seed cake as substrate for production of xylanase and cellulose by Aspergillus niger FGSCA733 in solid state-fermentation. Ind Crop Prod 37:118–123 17. Krisana A, Rutchadaporn S, Jarupan G, Lily E, Sutina T, Kanyawim K (2005) Endo-1,4-b-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation. J Biochem Mol Biol 38:17–23 ´ beda-Iranzo J, Briones-Pe´rez AI (2006) 18. Are´valo-Villena M, U Relationship between Debaryomyces pseudopolymorphus enzymatic extracts and release of terpenes in wine. Biotechnol Prog 22:375–381 19. Garcı´a-Lo´pez MD, Uruburu-Ferna´ndez F La conservacio´n de cepas microbianas. Coleccio´n espan˜ola de cultivos tipo. Personal communication ´ beda20. Romo S, Camacho C, Go´mez L, Villalonga-Santana R, U Iranzo J, Are´valo-Villena M, Briones-Pe´rez A, Ramı´rez HL (2012) Inmovilizacio´n de celulasa sobre una matriz de quitinaquitosana. Revista Cubana de Quı´mica 24:57–64 21. Georgieva TI, Ahring BK (2007) Potential of agroindustrial waste from olive oil industry for fuel ethanol production. Biotechnol J 2:1547–1555 22. Gonza´lez-Centeno MR, Rosello´ C, Simal S, Garau MC, Lo´pez F, Femenia A (2010) Physico-chemical properties of cell wall materials obtained from ten grape varieties and their byproducts: grape pomaces and stems. LWT Food Sci Technol 43:1580–1586 23. Mitchell DA, Lonsane BK (1992) Definition, characteristics and potential in solid substrate cultivation. In: Doelle HW, Mitchell DA, Rol CE (eds) Solid state fermentation. Elsevier, New York, pp 455–467 24. van de Velde F, Lourenco ND, Bakker M, van Rantwijk F, Sheldon RA (2000) Improved operational stability of peroxidases by coimmobilization with glucose oxidase. Biotechnol Bioeng 69:286–291
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ORIGINAL PAPER
Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste Sheila Romo Sa´nchez • Irene Gil Sa´nchez • Marı´a Are´valo-Villena • Ana Briones Pe´rez
Received: 12 March 2014 / Accepted: 30 September 2014 Springer-Verlag Berlin Heidelberg 2014
Abstract The recovery of by-products from agri-food industry is currently one of the major challenges of biotechnology. Castilla-La Mancha produces around three million tons of waste coming from olive oil and wine industries, both of which have a pivotal role in the economy of this region. For this reason, this study reports on the exploitation of grape skins and olive pomaces for the production of lignocellulosic enzymes, which are able to deconstruct the agroindustrial waste and, therefore, reuse them in future industrial processes. To this end, solid-state fermentation was carried out using two local fungal strains (Aspergillus niger—113 N and Aspergillus fumigatus—3). In some trials, a wheat supplementation with a 1:1 ratio was used to improve the growth conditions, and the particle size of the substrates was altered through milling. Separate fermentations were run and collected after 2, 4, 6, 8, 10 and 15 days to monitor enzymatic activity (xylanase, cellulase, b-glucosidase, pectinase). The highest values were recorded after 10 and 15 days of fermentation. The use of A. niger on unmilled grape skin yielded the best outcomes (47.05 U xylanase/g by-product). The multi-enzymatic extracts obtained were purified, freeze dried, and
S. Romo Sa´nchez I. Gil Sa´nchez M. Are´valo-Villena (&) A. Briones Pe´rez Food Science and Technology, University of Castilla-La Mancha (UCLM), Av. Camilo Jose´ Cela 10, Edificio Marie Curie, 13071 Ciudad Real, Spain e-mail: [email protected] S. Romo Sa´nchez e-mail: [email protected] I. Gil Sa´nchez e-mail: [email protected] A. Briones Pe´rez e-mail: [email protected]
immobilized on chitosan by adsorption to assess the possible advantages provided by the different techniques. Keywords Enzyme Fungal strain Waste Solid-state fermentation Immobilization
Introduction Considering the great deal of green waste in nature, some microorganisms show great potential in biotechnology because of their ability to metabolize lignocellulosic compounds. Agroindustrial waste, such as bagasse of sugar cane, wheat, corn, rice husk, fruit skin, and olive pomace (concentrated waste coming from alcohol and oil mill industries), is on the increase because of industrialization. This poses a problem in terms of space available and environmental pollution. In Spain, olive oil and winemaking companies figure prominently in the agri-food sector as some of the leading production and export industries. This extensive production results in approximately three million tons of waste a year (according to AAO, http://aplicaciones.mapa.es/pwAgenciaAO/General. aao?idioma=ING&control_acceso=S). Traditional methods for olive oil and wine waste management include chemical, physical, and biological processes. Hemicelluloses and celluloses represent over 50 % of dry weight of by-products. These may be transformed into soluble sugars by acid or enzymatic hydrolysis [1], which would be an inexpensive and abundant renewable energy source. Solid-state fermentation has been described as a process of insoluble matter transformation that serves both as a physical support and as a source of nutrients in the absence or near absence of free water. However, the substrate should be moist enough to allow
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for growth and metabolism of the microorganisms [2]. Because solid-state fermentation shows great potential in metabolite production [3], this process is particularly useful in the production of microbial products, such as food, fuel oil, and industrial chemicals and pharmaceuticals. Accordingly, the waste produced would be used as substrate in solid-state fermentation (SSF) for enzyme production [4]. Da Silva et al. [5] and Soliman et al. [6] found that Thermoascus auranticus and Aspergillus niger are good producers of lignocellulosic enzymes, that include cellulases (EC 3.2.1.4.), xylanases (EC 3.2.1.8.), and ligninases (EC 1.11.1.14.) which are able to break down the structure of cellulose, hemicellulose, and lignin of plant materials. Enzymes of the cellulolytic system that degrade cellulose altogether are exo-b-1,4-glucanase, endo-b-1,4-glucanase, and b-glucosidase [5]. Hemicellulose, the second most abundant renewable biomass in nature, is largely made up of xylan (25–30 % of dry weight of plant cell wall). Xylanases are the enzymes that can hydrolyze xylan, and they are of considerable interest for their industrial application [6]. Pectinases, another group of enzymes, degrade pectins, which are structural polysaccharides formed by units of polygalacturonic acid and inclusions of other monosaccharides located in the middle lamella and in the primary wall of higher plants [7]. Both molds and yeasts are able to thrive in this type of substrate, thanks to their enzymatic systems. Molds are particularly interesting because they have a set of advantages: (1) they play a major role in a number of food industries, (2) they excrete enzymes of a different nature that allow molds to metabolize complex mixtures of organic compounds found in most residues [8], (3) their filamentous morphology or pellet guarantees enzyme separation and production at a low cost [5, 9]. At present, the enzymes’ biotechnology is a common point among different sectors of the industry. A promising approach to retain their stability is the immobilization. Many are the supports (inorganics—bentonite, ceramics, silica…—or organics—chitosan, alginate, proteins…) and immobilization technics (encapsulation, adsorption, reticulation among others) used depending on the end use of the enzyme. Among others, immobilization on chitosan by adsorption is one of the most used. The aim of this study is to produce b-glucosidase, cellulase, xylanase, and pectinase by solid-state fermentation of by-products, considering factors such as composition of the medium, milling degree, and microbial strain. Enzymes were purified, lyophilized, and immobilized for industrial application of the enzymatic extract obtained.
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Materials and methods Microorganisms After initial experiments for strain screening, two local fungal strains, Aspergillus niger (strain N 113 N) and Aspergillus fumigatus (strain N 3), were selected (both deposited in the GenBank Database with accession numbers FJ499449 and FJ499462) due to their high enzymatic activity. These strains were isolated from olive paste and olives, respectively, in the Yeast Biotechnology Laboratory at the University of Castilla-La Mancha (Spain) [10]. Molds were maintained using the method suggested by Castellani [11]. A. niger and A. fumigatus were deposited in the Spanish Type Culture Collection (CECT) whose accession numbers are 20828 and 20827, respectively (http://www.cect.org). Natural substrates Grape skin (S) and olive pomace (P) were the by-products collected locally from agroindustries, as well as the wheat (W) used as a medium supplement. The grape skins and olive pomace were air dried at 40 C/24 h, and separated into two series, one of which was milled with an ultracentrifugal mill (Retsch ZM200) using a sieve with a pore size of 0.5 lm. The other series was not milled. The chemical composition of S and P was analyzed, quantifying moisture, starch, lignin, cellulose, hemicellulose, pectin, fat, and ash percentage (Van Soest; PNTLACC/FQ 002; Gravimetry; AOAC 1997, 985.29; Regulation 152/2009 Annex II H Proceeding B; Regulation (EC) 152/2009 Annex III A). Solid-state fermentation and enzyme production To obtain the precultures, the A. niger and A. fumigatus strains were grown in 250 mL Erlenmeyer flasks containing 50 mL PDA medium (agar slant) at 30 C/65 % moisture for 7 days. Then, 100 mL of the basal medium (0.3 % KH2PO4; 0.35 % (NH4)2SO4; 0.05 % MgSO47H2O; 0.05 % CaCl2) was added to culture, and slightly scratched to obtain mycelial suspension. The design of SSF medium was prepared according to da Silva et al. [5] with slight modifications. 5 mL of suspension of each mycelium was transferred to 100 mL Erlenmeyer flasks containing 5 g of solid substrate to be tested (S or P). As many as eight different fermentations were run for each of the molds, depending on whether or not wheat was added and the by-products were milled: S [unmilled grape skin]; SW [unmilled grape skin (2.5 g) ? wheat (2.5 g)];
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mS [milled grape skin]; mSW [milled grape skin (2.5 g) ? wheat (2.5 g)]; P [olive pomace]; PW [olive pomace (2.5 g) ? wheat (2.5 g)]; mP [milled olive pomace]; mPW [milled olive pomace (2.5 g) ? wheat (2.5 g)]. Enzyme production was sampled after 2, 4, 6, 10, and 15 days for assessment over time. The flasks were incubated at 25 C/65 % moisture during the time period set. Afterwards, 100 mL of distilled water was added to the flasks and they were maintained under shaking for 300 at room temperature for extracellular enzyme extraction. The content of the flasks was centrifuged (4,500 rpm/150 /4 C) and the supernatant was maintained at -20 C until analysis.
Table 1 Raw material composition (%) in grape skins and olive pomace
Enzyme assays
The purified extract, quick frozen at -80 C, was subsequently lyophilized at pressures of 2 9 10-2 and -53 C without cryoprotectant and with 20 % skimmed milk (Nestle Sveltesse, Spain) [19]. The qualitative analysis of proteins of the purified and lyophilized extract was monitored by SDS-PAGE electrophoresis in sulfate–polyacrylamide gel (Bio-Rad CriterionTM XT Precast Gel 12 % Bis–Tris) using an 8–220 kDa molecular weight marker (MW Sigma ColorBurst). The staining was performed with Coomassie Blue.
Activity of the enzymes pectinase, xylanase, cellulase, and b-glucosidase was assessed. 0.1 mL of the extract (supernatant) was incubated at 37 C/300 with 0.4 mL of the corresponding substrate in 0.1 M acetate buffer, pH 5: 0.5 % (w/v) xylan from beechwood (Sigma-Aldrich Corporation, St Louis) for xylanase; 1 % (w/v) carboxymethylcellulose (CMC—Panreac AppliChem, Germany) for cellulase; 0.5 % (w/v) pectin from apple (Sigma) for pectinase; and 1 % (w/v) D-(?)-cellobiose (Fluka Chemical Corporation) for b-glucosidase. Following the incubation period, the reducing sugars released were measured using the 3,5-dinitrosalicylic acid (DNSA) method [12]. Assessment of b-glucosidase activity was based on the protocol suggested by Are´valo-Villena et al. [13]. Parallely, blank absorbance was measured in the same way as the samples but with no incubation (at 0 time). For reducing sugar measurement, standard curves of galacturonic acid, xylose, and glucose were used, depending on the enzyme to be assessed. Assays were performed in triplicate in all cases and outcomes were calculated as enzyme units (U) defined as lmol of released product per minute under reaction conditions.
Composition (%) Humidity
Grape skins 5.6
Olive pomace 3.4
Starch
2.5
1.3
Lignin
22.5
22.6
Cellulose
25.9
22.9
Hemicellulose
3.6
5.9
Pectin
1.3
5.8
Fat Ash
9.8 6.5
15.9 3.3
Multi-enzymatic extract immobilization The purified and lyophilized enzymes (with and without cryoprotectant) were immobilized on chitosan by physical adsorption. The immobilization conditions, which were established based on previous research [20], involved an enzymatic concentration of 0.048 g/mL in acetate buffer pH 5, 50 mM in a final volume of 50 mL. The reaction was maintained for 2 h at 10 C under gentle shaking. Afterwards, up to 4 washes were performed with the same buffer to eliminate the unintegrated protein and cryoprotectant. Xylanase activity of the enzymatic extracts was monitored in triplicate prior to and after each biotechnological process following the previously described protocol.
Purification and lyophilization of enzymes Results A purified extract was obtained after solid-state fermentation of the mold–substrate pair that yielded the best results at the previous stages of the study. The multi-enzymatic extract underwent a variety of technological processes, and its activity was monitored at each of the stages. Supernatant was purified by size exclusion using filter cartridges for the concentration and purification of proteins (Pall, New York, USA). The molecule size selected was 100 kDa, considered best suitable for excreted protein [14– 17]. Samples were run at 4 C for 1 h at 4,500 rpm, and the enzymatic activity was monitored [18].
Natural substrates Table 1 shows the composition of each of the lignocellulosic substrates used in this study. The main constituents in both cases were lignins and celluloses, which are a potential adequate source of carbon for polysaccharidehydrolyzing microorganisms. However, the values of nitrogenized content were low, which would a priori hinder the development of the molds. For this reason, some fermentations were supplemented with wheat. The differences
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Fig. 1 Enzymatic activity of A. fumigatus and A. niger in solid-state fermentation over time (days). S grape skins, SW grape skins and wheat, mS milled grape skins, mSW milled grape skins and wheat
between both by-products lay in the high fat content and the higher percentage of pectins found in olive pomace. Solid-state fermentation and enzyme production Once their composition was determined, the by-products were used for mold growth and enzyme production by SSF using the combinations described in the ‘‘Materials and methods’’ section. Figure 1 shows the values of the enzymatic activities (cellulase, pectinase, b-glucosidase, and xylanase) obtained on grape skins with or without wheat supplement during SSF. Fermentation on unmilled substrates was beneficial both for A. niger and A. fumigatus. A significant increase in activity was observed from the 6th day of fermentation, reaching its maximum activity on the 10th and 15th day. This is likely due to the very metabolic development of the microorganisms. Upon comparison of the enzymatic activities of both molds, A. niger was found to show the
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best results in all cases, even showing a significant activity in grape skins with no wheat supplement. Xylanase reached 47.05 U per g of substrate on the 15th day of fermentation on S. b-glucosidase also showed a rich activity on the 10th day (29.47 U/g), although this only occurred when the grape skins were supplemented with wheat (SW). The enzymatic activity of A. fumigatus was significantly weaker in all cases. Even though the highest activity rate was reported on b-glucosidase, a great difference was found between this rate and that reported on A. niger (10.02 vs 4.17 U). This only occurred when wheat was added, which might be indicative of a greater need of A. fumigatus for nitrogenized material. Figure 2 includes values of maximum enzymatic activity of both molds which were smaller than that of grape skins, particularly in the case of A. niger. The maximum activity values were reported on A. fumigatus on the 4th day of growth for xylanase production (8 U). Virtually all cases required wheat supplementation and unmilled substrates, even for mold growth (none of the molds was able
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Fig. 2 Enzymatic activity of A. fumigatus and A. niger in solid-state fermentation over time (days). P olive pomace, PW olive pomace and wheat
to grow on milled olive pomace fermentation; data not shown). Enzyme purification, lyophilization and immobilization Based on the previous results, the microorganism–substrate pair selected was A. niger-unmilled grape skins for SSF for 15 days. The enzymatic extract obtained was purified and lyophilized according to the protocols described in the materials and methods section. Protein size and concentration and xylanase activity were checked because this was the most excreted enzyme, as shown in Fig. 1. Protein electrophoresis of the raw and purified-lyophilized enzymatic extracts of A. niger showed that the protein
patterns of both extracts were similar. The band intensity of the purified-lyophilized extract showed a lower activity of the enzymes in both cases (data not shown). This fact preluded almost total loss of proteins, and thus, of the xylanase activity. For data confirmation, the xylanase activity was measured using xylan as substrate (Table 2). Protein purification held back 83 % of enzymatic activity, which proved the cut size selected was right. Nevertheless, the process of lyophilization had a devastating impact on the activity (almost 90 % of it was lost). The use of the cryoprotectant could not prevent denaturalization, as shown by the low percentages reported (16.4 and 13.0 % with and without cryoprotectant, respectively). Finally, immobilization of the lyophil led to almost total loss of
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Bioprocess Biosyst Eng Table 2 Xylanase activity percentage (%) of the enzymatic extracts of raw, purified, lyophilized, and immobilized A. niger
Raw extract
100.0 ± 1.3
Purified extract
Lyophilized extract
Immobilized extract
With cryoprotectant
Without cryoprotectant
With cryoprotectant
Without cryoprotectant
82.8 ± 0.3
16.4 ± 2.6
13.0 ± 0.1
5.4 ± 0.10
0.03 ± 0.0
the activity (less than 10 % in both cases, with and without cryoprotectant).
Discussion This study relied on two molds in the species of Aspergillus (A. niger and A. fumigatus), which were isolated from oleic environments. Solid-state fermentations were run on two substrate types, i.e. grape skins and olive pomace, for industrial-use enzyme production. The analysis of the composition of both substrates showed that their main constituents are lignins and celluloses, which is consistent with the data reported in the literature. According to Georgieva and Ahring [21], olive pomace is largely made of lignin (22.8 %), a percentage that is very close to that reported in our study (22.6 %). Cara et al. [1] determined the composition of raw materials of olive trees, reporting dry biomass percentages slightly higher than those reported by our study (25 % in cellulose and 15.8 % in hemicellulose). This difference in percentage may be due to the mixture of substrates with other types of materials from the trees themselves. As for grape skins, the data found in the literature does not differ significantly with respect to pectin percentage from those reported by the present study (cellulose 27–37 %) [22]. Enzyme production using filamentous molds for solidstate fermentation or submerged fermentation is a widely used biotechnological method. Selection of a fermentation type depends on the physiological adaptation of the organism. The growth pattern of filamentous molds in submerged culture usually ranges between pellet-like and filamentous, which may affect enzymatic production [23]. Previous studies conducted in our laboratory with submerged culture did not report any pectinase and glucosidase activity [10]. In contrast, the same molds cultured in SSF in the present study did show activity. This is due to the fact that SSF provides the molds with a closed environment similar to their natural habitat, which stimulates them to produce more hemicellulolytic enzymes [5]. For all four enzymes examined, wheat supplement had an impact both on A. niger and A. fumigatus because of the easily assimilable nitrogen that wheat encouraged the production of enzymes. This increase is associated with the particle size of substrates. According to Salihu et al. [9],
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this is one of the factors that most affect fermentation, together with pre-treatment and water retention capacity. In resume, the study provides evidence of the appropriateness of grape skins and olive pomace for enzyme production, and thus, the validity of these by-products for this purpose is reinforced. The combination A. niger–grape skin (strain–substrate) yielded optimal results, being xylanase the enzyme with the highest concentration. Milling of both substrates did not improve enzyme production in any case. While not necessary for grape skins, wheat supplement stimulated microbial growth and enzyme production in olive pomace. Selection of cut size for purification (100 kDa) was based on the size of enzymes under examination: b-glucosidase, 100 kDa [14]; xylanase, 31 kDa [16]; pectinase, between 25 and 50 kDa [15]; and cellulase, 21 kDa [17]. The outcomes were consistent with previous research, since the purification process yielded an enzymatic extract with almost 83 % of residual activity. The lyophilization process, however, did not meet expectations, not even with the help of a cryoprotectant. There are many compounds that can be used to this aim (glycerol, skimmed milk, dimethyl sulfoxide, and carbohydrates, such as glucose, lactose, saccharose, and inositol). Further research with other cryoprotectant agents is thus needed for yield improvement. The results yielded in our study for adsorption-induced multi-enzymatic extract immobilization on chitosan are not representative, nor do they prove this process right or wrong. The reason for this is that the residual activity of the lyophil used was much too low (activity dropped more than 80 %). Although there is no research addressing immobilization of multi-enzymatic systems, there are studies dealing with coupling between two enzymes. The objective of such studies is either to obtain a final product or to eliminate a by-product of one of the two enzymes for being contaminating or detrimental to the process [24]. Accordingly, some of these biological catalysts would be able to prevent others from coupling on the substrate. Currently, there is an interest in the development of techniques that guarantee enzyme immobilization on a support on a specific area, revealing significant zones for stabilization. All these data pave the way for future studies including lyophilization assays with different cryoprotectants and/or assays involving immobilization of the purified extract
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without prior lyophilisation, which might be the key to the biotechnological advance of this topic. Acknowledgments The authors wish to express their gratitude to Dr. Mario Canales for protein electrophoresis assistant and to Dr. Hector L. Ramirez for immobilization process.
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