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Chestnut shell is a lignocellulosic biomass highly resistant to decay, owing largely to the presence of lignin. In this work, multiple bioprocesses on chestnut shell by the selected white-rot fungi Dichomitus squalens and Phlebia radiata were evaluated for the potential production of lignin-modifying enzymes (LMEs). The treatment by D. squalens alone at 12 d gained the highest laccase activity (9.42 ± 0.73 U mg-1). H2O2 showed a correlation with laccase and manganese peroxidase activity, accompanied by a rapid consumption of reducing sugar. Gradient ion chromatography analysis revealed only oxalate participated in chestnut shell biodegradation. Furthermore, surface area and total pore volume of chestnut shell were significantly promoted by the fungi according to ESEM and BET. Overall, D. squalens was considered as a suitable fungus for utilization of chestnut shell and laccase production. The roles of LMEs, H2O2 and oxalate provided more understanding for degradation of chestnut shell by the fungi.
Chestnut shell; Dichomitus squalens; Phlebia radiata; laccase; biological process
In recent years, strong attention has been devoted to utilization of agronomic residues, also termed "lignocellulosic biomass", which are the most potential and renewable biological resources. These feed stocks are widely employed in elimination of waste that represents a source of pollution, and production of useful by-products . Chestnut shell, which is a common waste from food processing industry, has recently attracted much attention. It has been used to prepare adsorbent to remove pesticides, copper, lead, cadmium and other heavy metal cations . And the extraction component of chestnut shell has been reported to have antioxidant activity and hepatoprotective effects . In addition, this lignocellulosic biomass was used to produce mono- and oligosaccharides . Though reported above, it is uneasy to fulfill practical application owing to the rigid barrier of chestnut shell that contains a high proportion of lignin. Hereby, a green and sustainable process should be advocated in the utilization of chestnut shell biomass.
The known physiochemical treatments of lignocelluloses biomass result in high-energy consumption and environmental pollution, thus economic and environmental benefits are subjected to restriction. Nevertheless, biological treatment is widely explored and considered as an alternative solution for degradation and utilization of biomass feedstocks , especially pretreatment with the white-rot fungi. Since fungal lignin-modifying enzymes (LMEs), mainly including laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP), play an important role in decomposition of complex lignin structure . Biotreatment of chestnut shell with white-rot fungi should be a feasible and eco-friendly process, but only few works focus on this issue. The earlier study exhibited that co-cultivation of Dichomitus squalens and Phlebia radiata has been selected as a potential strategy in lignocellulolytic biodegradation with high enzyme activity relying on their synergistic effect . Gómez et al. (2005) also compared the potential of chestnut shell and barley bran for laccase production by Coriolopsis rigida under solid-state conditions. The above studies verify that microbial treatment is a considerable and low-cost approach for the utilization of this special biomass.
LMEs such as laccase and MnP, whose molecular weight are greater than 35 kDa, are difficult to pass through the micropore structure of complete timber . Only if cell wall is deeply damaged, LMEs can play a full part in the degradation of lignocelluloses, accordingly some auxiliary media of small molecules are required. In fungal degradation, oxalate might have multiple functions, including an inhibitor of lignin peroxidase, an electron donor of NADH, a source of superoxide, and a chelator for stabilizing manganic ions . MnP was reported being capable of oxidizing oxalate to produce extracellular hydrogen peroxide . Moreover, reactive oxygen species (ROS) produced by basidiomycetes remain unidentified in most cases, but hydroxyl radical (â€¢OH) is likely a participant, which may be the major oxidant that initiate decay within the secondary wood cell wall owing to the great destructiveness to lignocelluloses . It improves the accessibility of ligninolytic enzymes on the internal structure by corroding the lignified cell wall. Extracellular formation of hydrogen peroxide (H2O2) is important not only for catalysis of LMEs, but also for ROS generation such as â€¢OH, superoxide and hydroperoxyls via Fenton reaction (H2O2 + Fe2+ + H+ â†’ H2O + Fe3+ + â€¢OH) . Besides, surface areas and pore size distribution of processed chestnut shell reflect the situation of lignin degradation caused by LMEs.
In this study, D. squalens and P. radiata were employed to pretreat chestnut shell in order to investigate the influence on LMEs production of different processes under the submerged cultivation, and H2O2 in the culture broth was examined to assess the radical reaction in lignin decomposition. As well, low molecular weight (LMW) organic acids were measured for analysis of its functional effect on LMEs. Furthermore, surface area and pore size distribution of chestnut shell were respectively determined to evaluate the modification of physical properties after biological processes. Consequently, the main objective of this work focused on entire understanding for the roles of LMEs, H2O2 and LMW organic acids in biodegradation of this substrate.
Materials and methods
2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), veratryl alcohol (VA), and standard organic acids like succinic, malic, tartaric, itaconic, oxalic, fumaric and citric acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Other chemicals were of analytical grade unless otherwise indicated.
Fungi and cultivation conditions
D. squalens (DSM 9615) and P. radiata (DSM 5111), obtained from the Deutsche Sammulung von Microorganismen und Zellkulturen (Braunschweig, Germany), were cultured on potato dextrose agar (PDA) plates at 28 °C for 5-7 d. These fungi were inoculated with eight plugs (1 cm Ã- 1 cm) in 250 mL Erlenmeyer flasks containing 40 mL medium under 150 rpm shaken speed and at 28 °C. The medium contained the following compositions (g L-1): 10.0 glucose, 0.5 NH4NO3 (ammonium nitrate), 0.8 KH2PO4, 0.2 Na2HPO4, 0.5 MgSO4â€¢7H2O, 0.01 FeSO4â€¢7H2O, 0.01 CuSO4â€¢5H2O, 0.005 MnSO4, 0.001 CaCl2â€¢2H2O, 0.001 ZnSO4â€¢7H2O, 0.0005 thiamine, adjusting initial pH to 6.0.
Commercial chestnut fruits (Castanea mollissima Blume) were obtained from a local market in Hangzhou (China) and peeled manually. Chestnut shell was washed thoroughly with distilled water to remove the dirt, and dried in an air oven at 60 °C for 48 h, then was ground and sieved through a 40 mesh sieve. 1 % (w/v) chestnut shell were added into the culture medium and sterilized at 115 °C for 30 min. After cooling to room temperature, each flask was then inoculated and incubated at 28 °C for 12 d under 150 rpm shaking speed at 28 °C in a dark chamber. The culturing flasks were plugged with sterile gauze, which allowed air exchange between the atmosphere and flask culture broth.
In this study, experimental designs were divided into five bioprocesses: (1) submerged fermentation grown with D. squalens for 12 d (DS process); (2) submerged fermentation grown with P. radiata for 12 d (PR process); (3) submerged fermentation grown with D. squalens and P. radiata for 12 d (DS+PR process). Experimental groups were supplied with chestnut shell, and the opposite groups without chestnut shell served as the control. Each group was performed in triplicate.
Protein concentration was determined using the Bradford protein quantitative kit (Tiangen, Beijing, China), which was similar to the well-documented Bradford protein assay . H2O2 quantitative assay kit (water compatible; Sangon Biotech, Shanghai, China) was used for analyses of hydrogen peroxide product , and the detailed microplate assay protocol followed the manufacturer's instructions. Reducing sugar in fermentation broths was determined using the 3, 5-dinitrosalicylic acid (DNS) colorimetric method .
The supernatant of fungal cultures was collected and assayed for LMEs activity at room temperature after centrifugation at 18,000 g for 5 min at 4°C. Laccase activity was determined by oxidation of 0.5 mM ABTS in 100 mM sodium acetate (pH 4.5) at 420 nm . Lignin peroxidase activity was measured by oxidation of 2.0 mM veratryl alcohol buffered with 100 mM sodium tartrate (pH 3.0) at 310 nm , and manganese peroxidase activity was assayed by following the formation of Mn3+-tartrate complex in 100 mM sodium tartrate (pH 4.5) in the presence of 0.5 mM MnSO4, at 238 nm . Peroxidase reactions were started by addition of 0.1 mM H2O2. One unit of enzyme activity was defined as 1 Î¼mol product formed per min, and the specific activity was expressed as units of enzyme activity per milligram of extracellular protein.
Gradient ion chromatography measurements
Organic acids including succinic, malic, tartaric, itaconic, oxalic, fumaric and citric acid, were quantified by a Dionex ICS-3000 ion chromatography system (Dionex, Sunnyvale, CA, USA), equipped with an IonPac AS11-HC analytical column (4 Ã- 250 mm), an AG11-HC guard column (4 Ã- 50 mm), and a suppressed conductivity detector. The suppressor current was set to 50 mA, and the detector cell temperature was held at 30 °C. Sodium hydroxide (NaOH) solution was used as the eluent at a constant flow rate of 0.2 mL min-1. A multistep gradient was used for all separations with an initial injection volume of 10 Î¼L. The gradient procedures were as follows: 25 mM from 0 to 27 min; 25 to 50 mM from 27 to 30 min; 50 mM from 30 to 35 min; 50 to 25 mM from 35 to 42 min; and 25 mM from 42 to 50 min. Furthermore, inorganic anions, such as Cl-, NO3-, SO42-, H2PO4- were simultaneously detected to remove interferences on determination of organic anions. After centrifuged for 10 min at 18,000 g at 4 °C, the samples were injected through 0.22 µm millipore filter before detection by gradient ion chromatography.
Environmental scanning electron microscopic analysis
Centrifuged chestnut shell was treated by the critical point drying with liquid CO2 and coated with gold in a sputter coater before microscopic examination. The ESEM images of untreated and treated materials were done using a Philips XL-30 ESEM (Philips Company, Amsterdam, The Netherlands) operating at 20 kV.
Surface area and porosity examinations
The bioprocessed chestnut shell was collected by centrifugation, and washed thoroughly with distilled water. Then moisture and atmospheric gas were removed from the samples by application of vacuum and heat below 80 °C. The textural properties of the samples were obtained by performing nitrogen sorption measurements using Quantachrome Autosorb-1-C high performance surface area and pore size analyzer (Quantachrome Instruments, Florida, USA), and analyzed with Autosorb-1 program. Specific surface area was determined by the Brunauer-Emmett-Teller (BET) method, and pore size distribution was calculated using the Barret-Joyner-Halendar (BJH) method . In these experiments, chestnut shell processed in same procedure without the fungi served as the control.
All tests were conducted in triplicate; the results are given as mean value ± standard deviation (S.D.). Trends were considered as significant when mean values of compared sets were different at P < 0.05. Pearson's correlation test was used to determine the relations between each two variables among H2O2 concentration, oxalate concentration, laccase and MnP.
Results and Discussion
3.1. Comparison of different treatments on laccase production
D. squalens and P. radiata were selected as the fungi for laccase production using chestnut shell as the substrate, and three biological processes were compared in this study. As presented in Fig. 1a and 1b, laccase production was evaluated at different times in submerged fermentation with and without chestnut shell. Laccase activities in control groups decreased significantly from culture time 3 d to 12 d (Fig. 1a), while the production of laccase was dramatically improved in chestnut shell-containing groups cultivated with two white-rot fungi (Fig. 1b). In fact, different agricultural residues certainly determined the utilization of lignocellulosic materials and LMEs production . Chestnut shell was proven to be a potential substrate for laccase production by the white-rot fungi owing to its special characteristics. Clearly shown in Fig. 1a and 1b, the improved laccase production was greatly attributed to the presence of this substrate, especially for maintaining stable maximal production.
Mixed fungal cultivation appears to have advantages over monocultures in considering of the synergistic effect , but it depended on interactions between species, additional feedstock, and the micro-environmental or nutritional conditions. In this work, monocultures showed significantly more laccase formation than co-cultures of D. squalens and P. radiata containing chestnut shell. In Fig. 1b, the biological utilization with D. squalens in chestnut shell-containing culture led to the highest level of laccase production of 9.42 ± 0.73 U mg-1 at the 12th d, which was 6-15 times higher than other biological processes. It had a potential for improved laccase formation after 12 d. As well, monoculture of P. radiata gained the highest laccase specific activity of 3.03 ± 0.65 U mg-1 on day 9, which were still less than half of the corresponding activity of DS process with chestnut shell. In contrast, a high laccase activity was detected in co-cultivation of D. squalens and P. radiata at the third day when cultured without chestnut shell, but laccase activities of all inoculation approaches showed a huge decline from day 3 to day 12 (Fig. 1a).
In chesnut shell-containing cultures, P. radiata caused to a negative effect on co-cultivation with D. squalens for laccase production, though its monoculture exhibited higher enzyme activity than mixed fungal cultures. By contrast, D. squalens was proven to be a better fungus than P. radiata for the utilization of chestnut shell. Considering the significant activity change of laccase in DS+PR group between submerged broths with and without chestnut shell, it indicated that the synergistic effect between D. squalens and P. radiata was inhibited in the culture containing chestnut shell as the substrate. However, the exact mechanism underlies required future elucidation.
Comparison of different treatments on peroxidase production
MnP and LiP are two important fungal class II peroxidases in lignin biodegradation . MnP is secreted by most of the white-rot fungi , including D. squalens and P. radiata . In Fig. 2a-b, MnP activity was reduced in control groups from day 3 to day 12, but enhanced in the fermentation cultures containing chestnut shell mainly after 3 d cultivation, which was similar to laccase production. MnP reached the highest level at the 9th days in experimental groups, after which MnP started to decrease. Obviously, MnP activities in chestnut shell groups were far below all groups without this substrate at the third day (Fig. 2b). So chestnut shell was not a desirable substrate for MnP production under this condition, and the addition of chestnut shell only resulted in a limited improvement of MnP.
Although three LiP encoding genes found were also presented in white-rot fungus P. radiata , and LiP produced by P. radiata was found in batch and semi-continuous cultivations . Surprisingly, no LiP activity was measured in the extracellular broths of three processes by D. squalens and P. radiata in this investigation, which in accordance with the previous finding when cultured with other agricultural biomass such as corn stalk, corncob, wheat straw and rice stalk . Based on the whole genome sequence of D. squalens , it verified that this fungus does not possess any LiP encoding genes. The present work inferred that LiP production by P. radiata was possibly inhibited in the submerged fermentation.
Change of residual reduced sugar, hydrogen peroxide and oxalate involved in degradation of chestnut shell
In the cultures containing chestnut shell, D. squalens and P. radiata produced laccase and MnP throughout the cultivation process. Clearly, biosynthesis of abundant laccase primarily occurred at the latter stage (from day 9 to day 12). LMEs were affected by nutrition condition of carbon sources , meanwhile, reducing sugar consumption was related to fungal biomass . To trace the change of available carbon sources consumed by the white-rot fungi, we examined the concentration of residual reduced sugar in fermentation broths. As shown in Fig. 3a-b, the consumption rate of reduced sugar in control groups was faster than the groups cultured with chestnut shell, especially in the groups inoculated early with D. squalens (such as DS, DS+PR without substrate). D. squalens gave rise to a steeper consumption of carbon source in cultures without chestnut shell. Possibly, sugar formation derived from chestnut shell in the culture medium owing to the degradation of white-rot fungus was decelerated (Fig. 3b). An accurate correlation between microbial growth, substrate consumption and laccase synthesis was provided in the submerged non-homogeneous broths . The reduced production of laccase and MnP in control cultures after 3 d was probably due to the decreasing available carbon nutrients. Decomposition of chestnut shell compensated for consumable carbon source, which favored to the sustainable production of LMEs.
In terms of antioxidant effect of chestnut shell extract and H2O2-involved in MnP activity , hereby to examine the content of H2O2 is helpful to reveal the inhibitory effect on MnP production by D. squalens and P. radiata in the submerged culture containing chestnut shell. Moreover, hydrogen peroxide (H2O2) is well-knownly participated in the lignin-containing biomass degradation by the white-rot fungi. Thus, in this study H2O2 was also comparatively examined. Results presented in Fig. 4a-b revealed that H2O2 concentration in chestnut shell-containing submerged cultures with three bioprocesses increased quickly at earlier stage, and reached to the peak value on day 9, then started to decrease after 9th d. Interestingly, the specific activity of MnP reached to the peak point just on that culture time (Fig. 4b). Particularly, the concentration of H2O2 in the culture medium pretreated by D. squalens alone was always higher than other experimental groups from day 3 to day 12 (Fig. 4a A and 4b A). As a primary donor, the content of H2O2 partly reflects the situation of free radical generation in broths. In submerged fermentation, LMEs were generated and secreted into the broths; free radical reaction was strengthened quickly. H2O2 production strongly correlated with laccase production. Likely, the free radical attacks triggered by laccase provided a possible source of H2O2. This synergetic effect was used in utilization of hemicellulase for improvement of teak veneer surface color . Simultaneously, H2O2 can initiate enzyme activity of MnP and accelerate the biodelignification of chestnut shell. Based on the time-varying correlations data in Table 1, H2O2 was strongly correlative with laccase and MnP activity in the broths containing chestnut shell except for cultivation with D. squalens, which still had a weak time-dependent correlation with H2O2. The data inferred that D. squalens and P. radiata showed completely different in regulating LMEs production under the chestnut shell-presented culture medium. Lignin biodegradation proceeds via a free radical process in the presence of molecular oxygen and transition metals. Reductive radical intermediates such as the semiquinone radical reduce molecular oxygen to produce superoxide, which in turn reduces Fe3+ or disproportionates into H2O2 and molecular oxygen.
Lignin biodegradation is a complex oxidative-reductive process that involves the action of a multienzyme system and the mediation of some small molecules. As been reported, the white-rot fungi also secrete a number of low-molecular mass compounds including oxalic and glyoxylic acids in submerged cultures, oxalic acid during solid-state fermentation of wheat straw and several fatty acids during solid-state fermentation of wood . Therefore, we conducted the determination of low-molecular weight acids among three bioprocesses (Fig. 4a-b) in this work. Among the examined organic and inorganic acids, only oxalate and inorganic anions (Cl-, NO3-, SO42- and H2PO4-) were detected by gradient ion chromatography. Other organic acids were observed without detectable quantity, which including succinic, malic, tartaric, itaconic, fumaric and citric acid. Shown in Fig. 4a-b, mass concentration of oxalate rapidly declined at an initial stage of fermentation time from day 3 to day 6, and then fell slowly, even slightly rebounded. Oxalic acid is produced by a wide variety of fungi, including brown-rots, white-rots, mycorrhizae, plant pathogens, and Aspergillus niger. The regulating role played in lignin-containing substrate degradation were specifies-dependent.
Until present, low molecular weight organic acids were known to involve in lignin degradation . As a major organic acid produced by D. squalens and P. radiata, oxalate was synthesized and secreted to extracellular medium in early stage. It was not accumulated but rather decomposed into carbon dioxide and formate anion radical (CO2 â€¢-), and further oxidized to CO2 and superoxide anion radicals (O2â€¢- or HOOâ€¢) under aerobic conditions . Furthermore, the synthesis and secretion of oxalate at early stage provided glucose metabolism in the submerged fermentation with NADH . Simultaneously, it was an H2O2-dependent reaction to oxidize Mn2+ to Mn3+ by MnP, thus an organic chelator was needed to stabilize the formation of Mn3+ . The cleavage of oxalate resulted in formation of superoxide, and led to the increase of H2O2, and more oxalate was oxidized to form H2O2 along with the production of MnP, thereby MnP could be independent of H2O2 in the presence of oxalate. These findings also confirmed the viewpoints on lignin conversion by MnP overviewed by Hofrichter . In general, the strong negative correlation with concentration of H2O2, laccase and MnP activity demonstrated that oxalate probably plays a key role in LMEs production and biological degradation of chestnut shell.
Surface areas and pore size distribution of submerged treated chestnut shell
The activated carbon prepared from chestnut shell and grapeseed was capable of an effective adsorbent for the removal of copper ions from aqueous solutions . The physical structure of chestnut shell should be paid more attention when endured to biological treatments. In this work, we conducted the biomass textural examination and comparison of chestnut shell with and without biotreatments by the selected white-rot fungi. The high magnification images of biomass obtained through ESEM were shown in Fig. 5, which helps to visualize the clear surface morphological differences. The compact structures were compared in Fig. 5A-D and 5A'-D' at two levels of magnification, and showing the exposure of cell lumen with approximate 10-30 Î¼m diameter and cell wall pits with approximate 1-3 Î¼m diameter (noted by arrows) in the treated substrate belonging to DS group more clearly compared to other groups (Fig. 5A'-D'). Zeng, et al. (2011) also discovered that biological pretreatment by Phanerochaete chrysosporium gave rise to exposure of cellulose microfibrils using scanning electron microscopic analysis, and the apparent structure of wheat straw was visibly changed by the fungus. The microscopic analysis in this study revealed that the weak part of solid tissue was easily attacked by extracellular lignin-modifying enzymes (specially MnP or Laccase). However, long pretreatment time was generally needed to achieve substantial lignin degradation . Consequently limited biodegradation was observed in the compact structure of chestnut shell after 12 d treatment, that likely due to sclerenchyma tissue of cutin and much more recalcitrant epidermis tissue making this structure resistant to enzymes. In this study, D. squalens was found to demonstrate a better biodegradability on the weak part of chestnut shell.
Though the data from ESEM examination showed the change of outer structure of chestnut shell, it is still under investigation whether the inner structure or physical property of chestnut shell changed or not. As a consequence, specific surface areas of chestnut shell under the treatment of three processes at 12 d were compared by multipoint BET analysis (Table 2). Results revealed that specific surface area in the microbial treatments was far higher than the control group without any treatments. The highest multipoint BET surface area was found to be 1.216 m2 g-1 samples in DS group, which was more than twice the untreated sample. Physical properties of the untreated and the processed samples by D. squalens were shown in Table 3, revealing that specific surface area and total pore volume were efficiently improved by means of biological treatment with D. squalens. Besides, the average pore diameter of DS group was smaller than the control. In addition to the change of surface area, the absorption property should be considered as well. The nitrogen adsorption-desorption isotherms of DS group were provided in Fig. 6. The pore size distribution calculated by BJH method using desorption branch showed two peaks at 17.1 nm and 1.6 nm in diameter, respectively. Many mesopores below 150 nm in diameter were clearly observed. The pore size distribution was indicative of plenty of small mesopores in diameter below 150 nm formed, which means that biologic treatment by fungus D. squalens actually results in the destruction on the complete structure of chestnut shell.
The ESMs data indicated that biological treatment by the selected white-rot fungi actually led to the destruction of chestnut shell. The improvement of BET surface area greatly attributed to lignin decomposition of chestnut shell by white-rot fungi. Considering that higher laccase activity produced by D. squalens alone at 12 d, it was more aggressive for the removal and/or modification of lignin from this biomass making the accessible surface for hemicellulose and cellulose. The physical characteristics of chestnut shell, especially processed with D. squalens, were vastly modified, which replying on the radical attacks stemmed from laccase and MnP. In recent years, agricultural waste products like coconut shell and chestnut shell, were often used as adsorbent to remove pesticides and metal ion in waste water treatment . Herein bioprocess with D. squalens provides an auxiliary method for physicochemical treatment of chestnut shell, also a sustainable strategy on biological process of this biomass for laccase production, which is an eco-friendly enzyme with a great potential in industrial application . Further researches are needed in achieving higher yield of laccase, and adsorption material development of pesticides and heavy metal cations.
In summary, biological treatment with D. squalens gave the highest laccase activity with a maximum value of 9.42 ± 0.73 U mg-1 in submerged fermentation. This bioprocess was proven to be more efficient in degradation of chestnut shell, and the surface microstructure of this biomass was obviously modified by the fungus compared with the control. Laccase, MnP, H2O2 and oxalate were all confirmed to participate in this process. These findings provided us with more understanding for limited knowledge on biodegradation of chestnut shell, and proposed a potential approach for commercial exploitation of this feedstock.