Simultaneous valorization and detoxification of the hemicellulose rich liquor from the organosolv fractionation

1 1 2 3 4 Simultaneous valorization and detoxification of the hemicellulose rich 5 liquor from the organosolv fractionation 6 7 María García-Torreiro, José Carlos Martínez-Patiñoa, Beatriz Gullón, Thelmo A. Lú-8 Chau, María Teresa Moreira, Juan M. Lema, Gemma Eibes* 9 10 Dept. of Chemical Engineering, Institute of Technology, Universidade de Santiago de 11 Compostela, 15782 Santiago de Compostela, Spain 12 13 aPresent address: Department of Chemical, Environmental and Materials Engineering, 14 Agrifood Campus of International Excellence (ceiA3), Universidad de Jaén, Jaén, Spain 15 16 *Corresponding author: Gemma Eibes 17 e-mail address: [email protected] 18 Tel: +34-881816020, Fax: +34-881816702 19 Postal address: Rúa Constantino Candeira s/n, 15782, Santiago de Compostela, Spain 20 21 22 2 Abstract 23 Fractionation of lignocellulosic biomass with solvents (organosolv process) generates a 24 hemicellulose-rich liquor with a high content of phenolics which is particularly toxic. 25 This work addresses the utilization of this stream as a potential carbon source for the 26 production of ligninolytic enzymes (LE). Among six basidiomycetes species, Irpex 27 lacteus and Ganoderma lucidum presented the highest activities of manganese peroxidase 28 (646±122 U L-1) and laccase (1,497±161 U L-1), respectively, growing on a medium 29 composed mainly of cellulose fibers, lignin and hemicellulose. The influence of each 30 lignocellulosic fraction on the LE production mechanisms was studied in more detail. The 31 high concentration of phenolic compounds in the hemicellulose-rich stream acted as 32 inducer of LE production, with levels even greater than those of xylose. Acute toxicity 33 tests on Vibrio fischeri revealed a substantial reduction of the toxicity after the fungal 34 treatment (by ca. 12–21 times). The proposed valorization and detoxification of this 35 currently non-exploited and abundant by-product stream is a promising strategy to 36 enhance the industrial feasibility of the organosolv fractionation process. 37 38 Keywords: manganese peroxidase, laccase, lignocellulose, white-rot fungi, organosolv 39 fractionation 40 41 42 3 1. Introduction 43 White-rot fungi are the only group of microorganisms currently known that degrade all 44 basic wood polymers, i.e., cellulose, hemicellulose and lignin into low-molecular weight 45 compounds that can be assimilated for growth (Camarero et al., 2014). This is achieved 46 by their ability to produce several hydrolytic enzymes (cellulases and hemicellulases) and 47 their unique network of oxidative (ligninolytic) enzymes. The study of the extracellular 48 ligninolytic enzyme (LE) system of Phanerochaete chrysosporium demonstrated that 49 lignin peroxidase (LiP, EC 1.11.1.14), manganese peroxidase (MnP, EC 1.11.1.13) and 50 laccase (EC 1.10.3.2) are the primary enzymes associated with the degradation of lignin 51 (Kuwahara et al., 1984). 52 The LE system plays a fundamental role in the bioconversion of lignocellulose. 53 Furthermore, LE can also be applied for other purposes, such as the production of second-54 generation biofuels, organic synthesis (antibiotics, polymers, building blocks), cosmetics 55 (skin-lightening agents), nanobiotechnology (biofuel cells and biosensors for biomedical 56 applications), bioremediation, biopulping and biobleaching in paper industry as well as 57 the food and textile industry (Alcalde, 2015; Yadav and Yadav, 2015). 58 However, to meet market demands, the large-scale production of these enzymes at 59 low cost is mandatory. Although significant efforts have been devoted to enhance LE 60 production by heterologous protein expression (Alcalde, 2015), the levels of enzymatic 61 production are still rather limited (Eibes et al., 2009) and still have to be obtained from 62 wild strains (Elisashvili and Kachlishvili, 2009). 63 LE production highly depends on the fungal species, source of lignocellulosic 64 substrate and cultivation method (Elisashvili et al., 2008). The presence of lignocellulose 65 was found to be a prerequisite for LE production by white-rot fungi in submerged cultures 66 (Kapich et al., 2004; Gassara et al., 2012) and the various lignocellulosic fractions have 67 4 been reported to influence LE production in a different manner. For example, P. 68 chrysosporium was found to secrete laccase in the presence of cellulose, but not glucose 69 (Srinivasan et al., 1995). T. versicolor produced higher laccase yields in the presence of 70 natural lignocellulose-containing substrates such as wheat straw or wood, rather than with 71 glucose (Schlosser et al., 1997). Toxic aromatic compounds and lignin also have a 72 remarkable influence on LE production with T. versicolor or Phlebia radiata (Rogalski 73 et al., 1991a; Rogalski et al. 1991b). Understanding the physiological mechanisms 74 regulating enzyme synthesis by white rot fungi could therefore be useful for improving 75 the efficient production of LE. 76 The organosolv process, i.e. pulping with ethanol-water, allows the fractionation of 77 the lignocellulosic materials on its main constituents, i.e. cellulose fibers, lignin and 78 hemicellulose fraction (Laure et al., 2014). However, only a few works have studied 79 potential applications of the hemicellulose liquor after lignin recovery (Kautto et al., 80 2013; Hallberg et al., 2011). The presence of degradation products from the 81 carbohydrates, soluble low molecular weight lignin and other possible inhibitors may 82 limit its conversion to biofuel (Kautto et al., 2013). On the other hand, considering this 83 fraction as waste stream, the large costs associated with wastewater treatment would limit 84 the economic viability of the organosolv biorefinery (Laure et al., 2014). 85 Recently, the interest on the valorization of agro-industrial residues to produce LE at 86 lower cost has increased (Palma et al., 2016). Several residual streams, such as apple 87 pomace sludge (Gassara et al., 2012), fishery residue, brewery waste, pulp and paper 88 industry sludge (Gassara et al., 2010), sugarcane residue (Maza et al., 2015), olive oil 89 wastewater (Mann et al., 2015), oat husks, waste from paper process industry (Winquist 90 et al., 2008) have been evaluated as carbon source. However, to our knowledge, the 91 5 application of the hemicelluloses fraction from organosolv process for ligninolytic 92 enzyme production has not been reported yet. 93 This study evaluates the production of LEs in submerged fermentations by different 94 fungal strains using the hemicellulose liquor of an organosolv process as carbon source. 95 With the aim of better analyzing the specific requirements of the fungal strains for LE 96 production, the individual or combined addition of the other fractionation products 97 (cellulose fibers and lignin) was evaluated before using the hemicellulose liquor as the 98 only carbon source. Moreover, the acute toxicity of the treated liquor was assessed with 99 the bioluminescent photobacterium Vibrio fischeri. 100 101 2. Materials and methods 102 2.1. Lignocellulosic substrates 103 The lignocellulosic substrates used as carbon source in this study were cellulose 104 fibers (C6), organosolv lignin (L) and a concentrated C5-sugars liquid fraction (C5), all 105 provided by the Fraunhofer Center for Chemical-Biotechnological Processes (CBP, 106 Leuna, Germany). The physico-chemical characterisation of these substrates is given in 107 Table 1. The fractionation of beech wood in the organosolv process was performed at the 108 Fraunhofer-CBP. Debarked beech wood chips were pulped with a 1:1 ethanol/water 109 mixture at 170°C for 100 minutes using 0.5% of sulphuric acid (based on dry wood) at a 110 liquor-to-wood ratio of 3.2:1 and the pressure was maintained at 20 bar using nitrogen 111 gas (Laure et al., 2014). The solid fraction obtained after the pulping of the beech wood 112 was washed; disintegrated and dewatered, obtaining the cellulose fibers (C6). The liquid 113 phase was composed mainly of lignin, C5-sugars from hemicellulose fraction and 114 degradation products. Lignin (L) was precipitated from this stream by the addition of 115 water and filtered for its separation (Schulze et al., 2016). By the recovery of the ethanol 116 6 from the filtrates of precipitated lignin, a raw hemicellulose fraction was obtained (C5). 117 This raw hemicellulose stream was subsequently concentrated. 118 2.2. Microorganisms and inocula preparation 119 Irpex lacteus (Fr. 238 617/93), Lentinus tigrinus (PW94-2), Stereum hirsutum 120 (PW93-4) and Phanerochaete chrysosporium (BKM-F-1767) were obtained from the 121 culture collection of the Department of Chemical Engineering of the University of 122 Santiago de Compostela (Spain). Bjerkandera sp. R1 was isolated from a Chilean forest 123 in Temuco and identified as a new anamorph of Bjerkandera sp. (Taboada-Puig et al., 124 2011) Ganoderma lucidum was isolated from mushroom spent substrate, kindly provided 125 by Hifas da Terra S.L. (Pontevedra, Spain). All fungal strains were maintained on MEA 126 agar at 4ºC. 127 Mycelia were produced in static cultures for fungal inocula preparation. Five plugs 128 of fungal mycelium from fresh agar plates were inoculated with 200 mL of glucose-129 peptone medium (Kimura et al., 1990) in Fernsbach flasks and incubated at 30ºC. After 130 7 days, fungal cultures were homogenized in a sterilized blender for 30 s and used as 131 inoculum. 132 2.3. Culture conditions 133 Submerged fermentations were performed in 250 mL Erlenmeyer flasks containing 90 134 mL of culture medium and 10 mL of inoculum. The fungal cultures were incubated on a 135 rotary shaker at 150 rpm, initial pH 4.5 and 27ºC. All culture media were prepared with 136 distilled water and contained 1 g L-1 peptone (as nitrogen source), 0.5 mM MnSO4 and 137 0.15 mM CuSO4 (as inducers for enzyme production). Six different combinations of 138 cellulose fiber (C6), organosolv lignin (L), concentrated hemicellulose fraction (C5), 139 and/or xylose (X) were evaluated as carbon source. The concentration of these 140 components in each culture medium is shown in Table 2. Cellulose and lignin 141 7 concentration was calculated considering the humidity and concentration of each of the 142 components in both streams (Table 1). Hemicellulose concentration was expressed in 143 equivalents of TRS, considering that the concentrated hemicellulose fraction had a TRS 144 concentration of 225 ± 5.6 g L-1. 145 2.4. Analytical methods 146 An adapted protocol from NREL (National Renewable Energy Laboratory, Golden, 147 USA) was used for the determination of the biomass composition in glucan, xylan and 148 lignin (Sluiter et al., 2007). Acid hydrolysis allows lignin separation from the sugar 149 fraction, and the amount of lignin recovered is determined by weight. The concentrations 150 of xylan and glucan were calculated from the concentration of the corresponding 151 monomeric sugars using a correction of 0.88 and 0.90 for C5 and C6 sugars, respectively. 152 The Folin-Ciocalteu’s method was used for the quantification of the total phenolic 153 compounds concentration (Singleton and Rossi, 1965). 154 Total reducing sugars (TRS) were determined by the dinitrosalicylic acid (DNS) 155 method (Miller, 1959). Glucose, xylose, rhamnose and acetic acid concentrations in the 156 C5-sugars fraction were determined by HPLC (Hewlett Packard chromatograph), 157 equipped with an ION-300 column (López et al., 2011). The concentration of the xylose 158 oligomers was determined by measuring the released xylose after post-hydrolysis 159 catalyzed with sulphuric acid. The liquid sample from the C5-sugar fraction or the fungal 160 culture supernatant was diluted and H2SO4 was added to reach a final concentration of 161 4% (v/v). The final pH of the sample was 0.7. It was autoclaved at 121ºC for 20 min in a 162 sealed flask. Flasks were weighed before and after the thermal treatment to allow 163 correction for any possible evaporation losses. Finally, samples were filtered through 0.45 164 μm cellulose acetate membranes and analysed by HPLC for glucose, xylose, and 165 arabinose using a 1100 series Hewlett-Packard chromatograph fitted with a refractive 166 8 index detector operated at 50°C. Other analysis conditions were as follows: Aminex 167 HPX-87H column (BioRad, Hercules, CA); mobile phase, 0.003 mol/L H2SO4; flow, 0.6 168 mL/min. 169 2.5. Enzymatic activity assays 170 Laccase activity was determined by measuring the oxidation of 5 mM 2,2’-azino-171 bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in 100 mM sodium acetate buffer, 172 pH 5 (ε436=29.3 mM-1 cm-1) (Taboada-Puig et al., 2011). MnP activity was measured by 173 following the oxidation of 1 mM 2,6-dimethoxyphenol (DMP) at 468 nm in a 50 mM 174 sodium malonate buffer (pH 4.5) containing 1 mM MnSO4 after starting the reaction with 175 0.4 mM H2O2 (ε468=49.6 mM-1 cm-1) (Taboada-Puig et al., 2011). LiP activity was 176 determined by measuring the oxidation of 2 mM veratryl alcohol to veratraldehyde at 310 177 nm in 50 mM of tartaric acid (pH 3) and 0.4 mM of H2O2 (ε310=9.3 mM-1 cm-1) (Taboada-178 Puig et al., 2011). All enzymatic activities were expressed as international units (U), 179 defined as the amount of enzyme that transforms 1 µmol substrate/min or the amount of 180 enzyme that releases 1 µmol product/min at 30ºC. 181 2.6. Microtox® toxicity assays 182 Microtox® toxicity assays were performed by using a Microtox® model 500 183 Analyzer. The luminescent marine bacterium Vibrio fischeri was the bioassay 184 microorganism used for these experiments. The results were expressed as EC50 at 5, 15 185 and 30 min, which corresponds to the volume percentage that causes a reduction in the 186 light output of the Microtox® test organism by 50% in 5, 15 and 30 min of contact, 187 estimated according to the “Basic Test” protocol of the software (Microbics, 1992). 188 2.7. Data analysis 189 A statistical analysis was conducted for a correct comparison of results using the 190 software R v.2.12.0 (The R Foundation for Statistical Computing). First, a one-way 191 9 analysis of variance (ANOVA) was carried out to determine if the results obtained at 192 different conditions were significantly different. Then, if the ANOVA confirms the 193 existence of a significant difference (p < 0.05), a post-hoc analysis (Tukey’s HSD) was 194 performed for a level of significance (α) of 0.05. 195 196 3. Results and discussion 197 3.1. Fungal screening 198 Six different white-rot fungi: I. lacteus, Bjerkandera sp. R1, S. hirsutum, L. tigrinus, G. 199 lucidum and P. chrysosporium, were screened for ligninolytic enzyme production in a 200 culture medium composed of cellulose fibers, organosolv lignin and hemicellulose 201 fraction (C6+L+C5) (Table 2). This medium promoted the growth of all tested fungi in the 202 form of pellets, except P. chrysosporium, which did not produce any ligninolytic enzyme. 203 Inhibition of LE production by P. chrysosporium was probably caused by a non-identified 204 component from the hemicellulose stream. Components such as total phenolics, acetic 205 acid, Na+ and SO42- were not expected to inhibit LE production (Koutrotsios and Zervakis, 206 2014). 207 Different sugar consumption profiles were observed (Fig. 1A and 1B), but the 208 common trend among all the fungal strains was the unconsumed sugar fraction that 209 remained at the end of the fermentations (approximately 1 g L-1 of TRS). L. tigrinus and 210 G. lucidum were the strains with the fastest sugars consumption rates; in both cases, the 211 TRS concentration was reduced to 1 g L-1 at day 4. All fungal cultures presented a lag 212 phase of two days, with the exception of S. hirsutum, which had the longest lag phase; it 213 extended up to four days, but thereafter, sugars were depleted in 3d. An increase of TRS 214 at day 5 was detected in the case of I. lacteus, probably due to the hydrolysis of the 215 16 compounds and long-chain fatty acids that are toxic to microorganisms and plants (Mann 365 et al., 2015). 366 After the fungal treatment and the recovery of the enzyme by ultrafiltration (10 367 kDa), the toxicity of the stream decreased nearly 21 and 12 times for I. lacteus and G. 368 lucidum strains, respectively. The detoxification promoted by I. lacteus seemed more 369 effective than that of G. lucidum, although the removal of phenolic compounds was very 370 similar in both reactors (≈85%). The analysis of the aromatic composition of both treated 371 liquors could help to clarify the different behaviour observed in the detoxification (Asses 372 et al., 2009). Ntougias et al. (2012) evaluated the detoxification of olive mill wastewater 373 by different WRF, and they observed that Ganoderma strains were less efficient than 374 Pleurotus sp. in the reduction of toxicity (by ca. 5–8 and 15 times, respectively). 375 376 4. Conclusions 377 The different lignocellulosic fractions derived from the organosolv fractionation of beech 378 wood influenced the mechanisms associated with LE production between fungal species. 379 Additionally, the residual liquid stream, containing mainly hemicellulosic sugars and 380 phenolic compounds, was observed to be an excellent LE inducer. The utilization of this 381 residue as the sole carbon source provides an opportunity to produce LE in an economical 382 and simple medium and, importantly, the fungal treatment results in a substantial decrease 383 in the liquor toxicity. Furthermore, the valorization and detoxification of this byproduct 384 stream is a promising strategy that may enhance the industrial feasibility of the organosolv 385 process. 386 387 17 Acknowledgments 388 This research was supported by the Spanish Government (Ministry of Economy and 389 Competitiveness) through the ERA-IB2 project 2G-Enzymes (PCIN-2015-031) and 390 ENTIRE and MODENA projects (CTQ2013-44762-R and CTQ2016-79461-R). The 391 authors belong to the Galician Competitive Research Group GRC2013-032 and to the 392 CRETUS Strategic Partnership (AGRUP2015/02). All these programmes are co-funded 393 by FEDER (EU). Authors are grateful to Fraunhofer CBP for provision of the organosolv 394 fractions. 395 396 References 397 Alcalde M, Engineering the ligninolytic enzyme consortium. 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Mean value 532 ± standard error were calculated using triplicate sets 533 534 Parameters Cellulose fibers (C6) Organosolv lignin (L) Parameters Hemicellulose (C5) pH 3.4 2.6 pH 5.5 Humidity (%, w/w) 69.8 ± 0.4 12.7 ± 0.6 TN (mg L-1) 748 ± 55 Ashes (%, w/w) 0.16 ± 0.02 0.14 ± 0.02 Glucose (g L-1) 10.8 Glucan (%, w/w dry basis) 73.5 ± 7.4 0 Xylose (g L-1) 144.3 Xylan (%, w/w dry basis) 11.3 ± 3.9 2.4 ± 0.09 Rhamnose (g L-1) 44.1 Xylose oligomer (g L -1 ) 75.7 Acid soluble lignin (%, w/w dry basis) 2.1 ± 0.2 2.0 ± 0.1 Acetic acid (g L-1) 17.1 Total phenols (g L- 1 ) 40.8 ± 0.8 Total lignin (%, w/w dry basis) 10.4 ± 1.0 92.7 ± 0.9 Na+ (g L-1) 20.7 SO42- (g L-1) 18.8 TN: total nitrogen 535 536 24 537 Table 2: Carbon composition of the different media tested 538 Medium component (g L-1) C 5 +C 6 +L C 5 C 5 +C 6 C 5 +L X X+C 6 +L Cellulose fibers (C 6 ) 10 -- 10 -- -- 10 Organosolv lignin (L) 10 -- -- 10 -- 10 Hemicellulose (C 5 )b 5 5c 5 5 -- -- Xylose (X) -- -- -- -- 5 5 c Unless otherwise stated 539 540 25 541 Table 3: Concentration of sugars and phenols in the C5+C6+L medium 542 Fungi Day Glucose (g L-1) Xylose (g L-1) Rhamnose (g L-1) Xylose oligomers (g L -1 ) Phenolic compounds (g L-1) I. lacteus 3 0.14 ± 0.04 1.53 ± 0.23 0.62 ± 0.07 1.70 0.450 ± 0.010 7 1.59 ± 0.21 0.70 ± 0.13 0.54 ± 0.08 NA NA 9 0.22 ± 0.07 0.55 ± 0.26 0.45 ± 0.21 1.50 0.101 ± 0.002 G. lucidum 3 0.21 ± 0.09 1.37 ± 0.14 0.68 ± 0.02 1.70 0.380 ± 0.010 7 0.07 ± 0.02 0.09 ± 0.02 0.24 ± 0.03 NA NA 9 0.09 ± 0.01 0.09 ± 0.03 0.21 ± 0.02 1.61 0.124 ± 0.002 NA: not available 543 544 545