Utilization of Horticultural Waste for Laccase Production by <Emphasis Type="Italic">Trametes versicolor</Emphasis> Under Solid-State Fermentation

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.     

Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm³), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm³). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45–70 °C with the maximal activity at pH = 4.5.     

Bio-detoxification of Phorbol Esters and Other Anti-nutrients of Jatropha curcas Seed Cake by Fungal Cultures Using Solid-State Fermentation

Jatropha seed cake, a byproduct after biodiesel extraction, has several anti-nutrients and toxins. Solid-state fermentation was carried out for the detoxification of the Jatropha seed cake (JSC) using different fungal cultures. The reduction in the anti-nutritional components such as tannins, phytates, saponins, lectin and protease inhibitor, and phorbol esters on 6th, 9th, and 12th day of fermentation was analyzed. The phorbol ester content in the unfermented JSC was 0.83 mg/g, and the maximum degradation of phorbol esters to the extent of 75 % was observed in the case of JSC fermented with Cunninghamella echinulata CJS-90. The phytate degradation in the fermented JSC was in the range of 65–96 %. There was a gradual reduction of saponin content in the JSC from 6th to 12th day, and the reduction of saponin was in the range of 55–99 % after solid-state fermentation. The trypsin inhibitor activity and lectin were 1,680 trypsin inhibitor units (TIU) per gram and 0.32 hemagglutinating unit in the unfermented JSC, respectively. Trypsin inhibitor activity and lectin could not be detected in JSC after 12th day of solid-state fermentation. Tannins accounted for 0.53 % in unfermented JSC, and there was a marginal increase of tannins after solid-state fermentation. The results indicate that biological detoxification could be a promising method to reduce anti-nutritional compounds and toxins in the JSC.     

Solid-state Fermentation for Enhanced Production of Laccase using Indigenously Isolated <Emphasis Type="Italic">Ganoderma</Emphasis> sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.     

Screening and Production of Ligninolytic Enzyme by a Marine-Derived Fungal Pestalotiopsis sp. J63

Marine-derived fungi are prone to produce structurally unique secondary metabolites, a considerable number of which display the promising biological properties and/or industrial applications. Among those, ligninolytic enzymes have attracted great interest in recent years. In this work, about 20 strains were isolated from sea mud samples collected in the East China Sea and then screened for their capacity to produce lignin-degrading enzymes. The results showed that a strain, named J63, had a great potential to secrete a considerable amount of laccase. Using molecular method, it was identified as an endophytic fungus, Pestalotiopsis sp. which was rarely reported as ligninolytic enzyme producer in the literature. The production of laccase by Pestalotiopsis sp. J63 was investigated under submerged fermentation (SF) and solid state fermentation (SSF) with various lignocellulosic by-products as substrates. The SSF of rice straw powder accumulated the highest level of laccase activity (10,700 IU/g substrate), whereas the SF of untreated sugarcane bagasse provided the maximum amount of laccase activity (2,000 IU/ml). The value was far higher than those reported by other reports. In addition, it produced 0.11 U/ml cellulase when alkaline-pretreated sugarcane bagasse was used as growth substrate under SF. Meanwhile, the growth of fungi and laccase production under different salinity conditions were also studied. It appeared to be a moderately halo-tolerant organism.     

Phytosterols Elevation in Bamboo Shoot Residue through Laboratorial Scale Solid-State Fermentation Using Isolated Aspergillus niger CTBU

Aspergillus niger CTBU isolated from local decayed bamboo shoot residue was employed to solid-state fermentation (SSF) of bamboo shoot residue to elevate the content of phytosterols. Strain acclimatization was carried out under the fermentation condition using bamboo shoot as substrate for fermentation performance improvement. The optimal fermentation temperature and nitrogen level were investigated using acclimatized strain, and SSF was carried out in a 500-ml Erlenmeyer flask feeding 300-mg bamboo shoot residue chips under the optimal condition (33 °C and feeding 4 % urea), and 1,186 mg (100 g)^?1 of total phytosterol was attained after 5-day fermentation, in comparison, only 523 mg (100 g)^?1 of phytosterol was assayed in fresh shoots residue. HPLC analysis of the main composition of total phytosterols displays that the types of phytosterols and composition ratio of main sterols keep steady. This laboratorial scale SSF unit could be scaled up for raw phytosterols production from discarded bamboo shoot residue and could reduce its cost.     

Strategies for Enhancing Laccase Yield from <Emphasis Type="Italic">Streptomyces psammoticus</Emphasis> and Its Role in Mediator-based Decolorization of Azo Dyes

Enhanced production of laccases from Streptomyces psammoticus in solid-state fermentation was carried out using two different strategies: laccase inducers and scale-up process. Laccase yield was enhanced by a wide range of aromatic inducers. The best inducer was pyrogallol, which yielded 116 U/g as compared to the control (55.4 U/g). Scale-up studies in packed bed bioreactor was performed at different aeration rates. Aeration at 1.5 vvm was identified as the optimum condition for laccase production (75.4 U/g) in the column bioreactor. The enzyme yield was enhanced further by combining the best conditions from the first two experiments. Fermentation was carried out in bioreactors in the presence of 1 mM pyrogallol, which resulted in 3.9-fold increase in laccase yield (215.6 U/g). The role of laccase in azo dye decolorization was evaluated in the presence of four different laccase mediators, at different concentrations. 1-Hydroxybenzotriazole (HOBT) proved to be the best mediator for S. psammoticus laccase and decolorized the azo dyes efficiently. Acid orange, Methyl orange, and Bismarck brown were decolorized at the rates of 86%, 71%, and 75% respectively, by HOBT.     

Pythium irregulare Fermentation to Produce Arachidonic Acid (ARA) and Eicosapentaenoic Acid (EPA) Using Soybean Processing Co-products as Substrates

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.     

Simultaneous Production of Amyloglucosidase and Exo-Polygalacturonase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a Rotating Drum Reactor

Simultaneous production of amyloglucosidase (AMG) and exo-polygalacturonase (exo-PG) was carried out by Aspergillus niger in substrate of defatted rice bran in a rotating drum bioreactor (RDB) and studied by a 3¹ × 2² factorial experimental design. Variables under study were A. niger strains (A. niger NRRL 3122 and A. niger t0005/007-2), types of inoculum (spore suspension and fermented bran), and types of inducer (starch, pectin, and a mix of both). Solid-state fermentation process (SSF) was conducted at 30 °C under 60-vvm aeration for 96 h in a pilot scale. Production of AMG and exo-PG was significantly affected by the fungal strain and the type of inoculum, but inducers did not trigger any significant effect, an evidence of the fact that these enzymes are constitutive. The maximum activity of exo-PG was 84 U g_dm ^?1 whereas the maximum yield of AMG was 886.25 U g_dm ^?1.     

Decolorization kinetics of the azo dye drimaren blue X3LR by laccase

Summary An extracellular Drimaren Blue X3LR decolorizing enzymatic activity was found in the crude filtrate of Funalia trogii grown by solid-state fermentation using wheat bran and soya bean waste. Decolorization of the azo dye Drimaren Blue X3LR by the crude filtrate and partially purified enzyme of Funalia trogii were investigated and compared. In the absence of additional redox mediator, maximum decolorization ratios of 81.33 % and 77.4 % were observed for Drimaren Blue X3LR using crude filtrate and partially purified enzyme respectively. Decolorization yield was found to be higher with crude enzyme preparations. Na₂S₂O₅ inhibited laccase and dye decolorizing enzyme activities but a significant peroxidase activity inhibition was not observed. Since the reaction was catalyzed in the absence of H₂O₂ as co-substrate, it could be concluded that this enzyme is not a peroxidase but may be a laccase.. The kinetic parameters of decolorization were calculated according to Michaelis constant (Km of 1.700 x 10^-5 mol dm^-3 and W_max = 8.02 x10^-7 mol dm^-3 sec^-1).     

Effect of Synergistic Inducement on the Production of Laccase by a Novel Shiraia bambusicola Strain GZ11K2

In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as laccase secretion indicator. A novel laccase-producing strain was isolated and identified as Shiraia bambusicola Henn. strain GZ11K2 according to the morphological characteristics and the comparison of internal transcribed spacer ribosomal DNA gene sequences. In further investigation, the production of laccase by S. bambusicola GZ11K2 was greatly enhanced by the nontoxic inducers of copper sulfate and rhodamine B. Copper and rhodamine B were added into the cultivation medium at 24 and 12 h, respectively, and the maximum laccase production was obtained. Under the induction of 2.0 mM copper sulfate and 35 μM rhodamine B, an increment of about 80 times of laccase activity compared with that in the inducer-free medium and about 20 times compared with that in the single copper-supplemented medium was observed. Compared with other species, S. bambusicola GZ11K2 exhibits better laccase-producing characteristics with an activity of 16,400 U/L after 108 h, suggesting its potential ability for industrial application.     

Fed-Batch Enzymatic Saccharification of High Solids Pretreated Lignocellulose for Obtaining High Titers and High Yields of Glucose

1-Deoxynojirimycin (DNJ) is an efficient α-glucosidase inhibitor (α-GI) with potential applications in the prevention and treatment of diabetes. In this study, 16 Bacillus strains were screened for α-GI rate, and the strain HZ-12 with the highest α-GI rate was identified as Bacillus amyloliquefaciens through the analysis of physiological biochemical characteristics and 16S rDNA sequence. By LC-MS/Q-TOF analysis, the α-GI component produced by B. amyloliquefaciens HZ-12 was identified as DNJ. Soybean was used as the substrate for the solid-state fermentation; 870 mg/kg DNJ was produced by B. amyloliquefaciens HZ-12 after optimizing the fermentation conditions and media, which was 3.83-fold higher than the initial yield. Also, evaluations of nutraceutical enrichment in the form of anticoagulant activity, antioxidant activity, total nitrogen (TN), and total reducing sugars (TRS) of the B. amyloliquefaciens HZ-12 fermented soybeans were substantially higher than unfermented soybeans. This study provided a promising strain for high-level production of DNJ and produced nutraceutical enriched soybeans by fermentation.     

Solid-State Fermentation of Rapeseed Meal with the White-Rot Fungi Trametes versicolor and Pleurotus ostreatus

Rapeseed meal is valuable high-protein forage, but its nutritional value is significantly reduced by the presence of a number of antinutrients, including phenolic compounds. Solid-state fermentation with white-rot fungi was used to decrease the sinapic acid concentration of rapeseed meal. After 7 days of growth of Trametes versicolor and Pleurotus ostreatus, the sinapic acid content of rapeseed meal was reduced by 59.9 and 74.5 %, respectively. At the end of the experiment, sinapic acid concentration of T. versicolor cultures decreased by 93 % of the initial value; in the case of cultures of P. ostreatus, 93.2 % reduction was observed. Moreover, cultivation of white-rot fungi on rapeseed meal resulted in the intensive production of extracellular laccase, particularly strong during the late phases of growth of T. versicolor. The obtained results confirm that both fungal species may effectively be used to decompose antinutritional phenolics of rapeseed meal. Rapeseed meal may also find use as an inexpensive and efficient substrate for a biotechnological production of laccase by white-rot fungi.     

Functional Magnetic Mesoporous Nanoparticles for Efficient Purification of Laccase from Fermentation Broth in Magnetically Stabilized Fluidized Bed

A magnetically stabilized fluidized bed (MSFB) with the Cu²⁺-chelated magnetic mesoporous silica nanoparticles (MMSNPs-Cu²⁺) was established to purify laccase directly from the fermentation broth of Trametes versicolor. The MMSNPs-Cu²⁺ particles in the MSFB maintained a stable bed expansion of two to threefold at a flow rate of 120–180 cm/h. At the optimal magnetic field intensity of 120 Gs, both the maximal Bodenstein number and the smallest axial dispersion coefficient were achieved, which resulted in a stable fluidization stage. The dynamic binding capacity of laccase in the MSFB decreased from 192.5 to144.3 mg/g when the flow velocity through the bed increased from 44.2 to 69.8 cm/h. The MSFB with MMSNPs-Cu²⁺ achieved efficient laccase purification from the fermentation broth with 62.4-fold purification of laccase and 108.9 % activity yield. These results provided an excellent platform for the application of these magnetic mesoporous nanoparticles integrated with the MSFB in developing novel protein purification process.     

Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by β-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 °C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0–5.5. They were stable in the pH range 5.0–10.0 and 5.5–8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55C and 60 °C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 °C.     

Production of ligninolytic enzymes for dye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus and Phanerochaete chrysosporium under solid-state fermentation

Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 U/g and manganese peroxidase of 982 U/g was obtained on d 20 and lignin peroxidase of 656 U/g on d 25 at 28±1 °C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.     

Effect of Biosurfactants on Laccase Production and Phenol Biodegradation in Solid-State Fermentation

The effects of two biosurfactants, tea saponin (TS) and rhamnolipid (RL), on the production of laccase and the degradation of phenol by P. simplicissimum were investigated in solid-state fermentation consisting of rice straw, rice bran, and sawdust. Firstly, the effects of phenol on the fermentation process were studied in the absence of surfactants. Then, a phenol concentration of 3 mg/g in the fermentation was selected for detailed research with the addition of biosurfactants. The results showed that TS and RL at different concentrations had stimulative effects on the enzyme activity of laccase. The highest laccase activities during the fermentation were enhanced by 163.7%, 68.2%, and 23.3% by TS at concentrations of 0.02%, 0.06%, and 0.10%, respectively. As a result of the enhanced laccase activity, the efficiency of phenol degradation was also improved by both biosurfactants. RL caused a significant increase of fungal biomass in the early stage of the fermentation, while TS had an inhibitory effect in the whole process. These results indicated that RL could mitigate the negative effects of phenol on fungal growth and consequently improve laccase production and phenol degradation. TS was potentially applicable to phenol-polluted solid-state fermentation.     

In Silico and in Vitro Physicochemical Screening of Rigidoporus sp. Crude Laccase-assisted Decolorization of Synthetic Dyes—Approaches for a Cost-effective Enzyme-based Remediation Methodology

The objective of this paper is to compare in silico data with wet lab physicochemical properties of crude laccase enzyme isolated from Rigidoporus sp. using wheat bran as solid substrate support towards dye decolorization. Molecular docking analysis of selected nine textile and non-textile dyes were performed using laccase from Rigidoporus lignosus as reference protein. Enzyme-based remediation methodology using crude enzyme enriched from solid state fermentation was applied to screen the effect of four influencing variables such as pH, temperature, dye concentration, and incubation time toward dye decolorization. The extracellular crude enzyme decolorized 69.8 % Acid Blue 113, 45.07 % Reactive Blue 19, 36.61 % Reactive Orange 122, 30.55 % Acid Red 88, 24.59 % Direct Blue 14, 18.48 % Reactive Black B, 16.49 % Reactive Blue RGB, and 11.66 % Acid Blue 9 at 100 mg/l dye concentration at their optimal pH at room temperature under static and dark conditions after 1 h of incubation without addition of any externally added mediators. Our wet lab studies approach, barring other factors, validate in silico for screening and ranking textile dyes based on their proximity to the T1 site. We are reporting for the first time a combinatorial approach involving in silico methods and wet lab-based crude laccase-mediated dye decolorization without any external mediators.     

Gibberellic acid production by solid-state fermentation in coffee husk

Five strains of Gibberella fujikuroi and one of Fusarium moniliforme were screened for the production of gibberellic acid (GA₃) in coffee husk, and based on the results, one strain, G. fujikuroi LPB-06, was selected. The comparative production of GA₃ by solid-state fermentation and submerged fermentation indicated better productivity with the former technique, mainly with pretreated substrate. The GA₃ accumulation was 6.1 times higher in the case of solid-state fermentation. Considering the C:N relation, higher yields of GA₃ were achieved using a mixed substrate comprising coffee husk and cassava bagasse (7:3, dry wt), increasing the results twice. Supplementation of an optimized saline solution containing 0.03% FeSO₄ and 0.01% (NH₄)₂SO₄ enhanced the accumulation of GA₃ 1.7 times in the fermented substrate. Under the finally optimized condition, the culture gave a maximum of 492.5 mg of GA₃/kg of dry substrate, with a pH of 5.3, moisture of 75%, and incubation temperature of 29°C. GA₃ yield was almost 13 times more than the initial results.     

Valorization of By-Products from Palm Oil Mills for the Production of Generic Fermentation Media for Microbial Oil Synthesis

This study demonstrates the production of a generic nutrient-rich feedstock using by-product streams from palm oil production that could be used as a substitute for commercial fermentation supplements. Solid-state fermentations of palm kernel cake (PKC) and palm-pressed fiber (PPF) were conducted in tray bioreactors and a rotating drum bioreactor by the fungal strain Aspergillus oryzae for the production of crude enzymes. The production of protease was optimized (319.3 U/g) at an initial moisture content of 55 %, when PKC was used as the sole substrate. The highest free amino nitrogen (FAN) production (5.6 mg/g) obtained via PKC hydrolysis using the crude enzymes produced via solid-state fermentation was achieved at 50 °C. Three initial PKC concentrations (48.7, 73.7, and 98.7 g/L) were tested in hydrolysis experiments, leading to total Kjeldahl nitrogen to FAN conversion yields up to 27.9 %. Sequential solid-state fermentation followed by hydrolysis was carried out in the same rotating drum bioreactor, leading to the production of 136.7 U/g of protease activity during fermentation and 196.5 mg/L of FAN during hydrolysis. Microbial oil production was successfully achieved with the oleaginous yeast strain Lipomyces starkeyi DSM 70296 cultivated on the produced PKC hydrolysate mixed with commercial carbon sources, including glucose, xylose, mannose, galactose, and arabinose.