Tray Fermentation Research Articles

LACCASE PRODUCTION BY NEWLY ISOLATED GANODERMA LUCIDUM WITH SOLID STATE FERMENTATION CONDITIONS AND ITS USING FOR DYE DECOLORIZATION

Laccase production capacity of newly isolated fungus Ganoderma lucidum was studied during solid state fermentation on solid substrates. Because wheat bran was detected as the most effective solid substrate, effect of various culture parameters and some inducers on laccase production in wheat bran media was investigated. Wheat bran with moisture content of 75%, pH 5.0 and 30 °C was the most effective medium and 10 mM copper addition supported the highest amount of laccase. In glass tray fermenter 2973±220 U/L laccase activity was obtained. Crude laccase gave the highest activity at 70 °C. It was stable at 60°C for 6 hours and retained 37% activity at 70 °C for 2 hours. It decolorized 61% of Remazol Brilliant Blue R within 20 min. Results showed that this strain could produce high amount of laccase and suitable inducer such as copper could induce its laccase production. The crude laccase obtained could be used for textile dye decolorization applications.

Solid state fermentation of rice straw using Penicillium citrinum for chitosan production and application as nanobiosorbent

Chitosan production by Penicillium citrinum using rice straw was investigated by varying the moisture content (50–80%) and particle size of rice straw, and their optimum levels were found to be 70% and 4–5 mm, respectively. A lab scale tray fermenter was later employed to carry out the solid-state fermentation which yielded a maximum chitosan production of 9 g/kg of rice straw. Nanobiosorbents based on the fungal chitosan produced in this study was further prepared and evaluated for heavy metal removal. Magnetic iron (Fe 3 O 4 ) nanoparticles coated with chitosan and carboxymethyl chitosan (CMC) were used as the nano-biosorbents to remove Cr(VI) and Pb(II) from corresponding aqueous solutions. Maximum removal of Cr (VI) and Pb (II) were 42.43 and 178.2 mg/g with CMCNPs. Heavy metal removal kinetics by the nanobiosorbents followed the pseudo-second order kinetics. Furthermore, the nanobiosorbents were found suitable for reuse for up to three cycles of sorption-desorption. • Chitosan production by solid state fermentation of rice straw is reported. • Microwave-alkali pretreatment of rice straw is found to be effective. • Maximum chitosan titre of 9 g/kg is achieved using a tray fermenter. • Chitosan based nanobiosorbents showed very high removal of Cr(VI) and Pb(II). • The nanobiosorbents could be reused for heavy metal adsorption up to 3 cycles.

Potato pomace: An efficient resource for Monascus pigments production through solid-state fermentation

Monascus pigments are the important natural additives in food industrial production. To obtain more economic pigments production processes, the present study was performed to evaluate the feasibility of using pomace resource as substrate for pigments production. Petri dish fermentation was designed to seek the optimal process parameters, and the value of red, yellow and total pigments per dry fermented substrate could achieve 654.6, 1268.1 and 1922.7 OD units/g, respectively. Shallow tray fermentation experiments were used for investigating the potential industrial production of pigments using potato pomace as sole carbon. The final total pigments of 200g and 1000g shallow tray experiments could reach 1886.9 and 1737.4 OD units/g. The results in this work indicating that potato pomace could be an efficient and low cost substrate for the production of Monascus pigments, and will supply a valuable reference for the comprehensive utilization of potato resources and seeking the economical natural pigments process.

Adding value to agro-industrial waste for cellulase and xylanase production via solid-state bioconversion

The lignocellulosic biomass is presenting an enormous potential in biofuel production, which requires a hydrolyzing step in the bioconversion process. The biomass is hydrolyzed using costly enzymes like cellulase and xylanase. These enzymes can be produced by using lignocellulosic biomass as feedstock, and on-site produced crude enzyme can saccharify biomass. The integration of both approaches can lower down the cost of biofuel production. Keeping this in view, the present study was conducted to explore various agro-industrial waste as carbon sources and best-suited conditions for enhanced cellulase and xylanase production by Aspergillus flavus under solid-state fermentation. Among all studied biomass, microwave alkali-pretreated rice straw was found most suitable as a carbon source for enzyme production. Maximum enzyme activities FPase 12.5 IU/gds, CMCase 235 IU/gds, β-glucosidase 190 IU/gds, and xylanase 180 IU/gds were obtained under optimum conditions of initial pH 5.5, temperature 30 °C, substrate to liquid ratio 1:3.5 (w/v), particle size 500–1000 μm, and NaNO3 on the 5th day of the incubation period. Enzyme activities FPase, CMCase, β-glucosidase, and xylanase were enhanced by approximately 1.27-, 1.18-, 1.06-, and 1.09-fold during tray fermentation. The adopted approach utilized low-cost lignocellulosic biomass for enzyme production as an additional avenue to biomass valorization along with solid waste management and its reduction which would otherwise cause environmental pollution problems.

The Effects of Operational Conditions in Scaling Up of Xylanase Enzyme Production for Xylitol Production

The biological route to produce xylitol from Oil Palm Empty Fruit Bunches (EFBs) comprises of EFBs pretreatment, enzymatic hydrolysis, fermentation, and downstream separation of the produced xylitol. Due to the specificity in the hemicellulose composition of EFBs, a xylanase enzyme that has a high affinity to EFBs is required to hydrolyze the EFBs into xylose. In this research, the influences of aeration, humidity, and mixing in xylanase production were mapped. The xylanase production was performed by Aspergillus fumigatus ITBCCL170 in a solid-state fermentation using a tray fermenter with EFBs as the substrate. The optimal configuration was further scaled up into xylanase production using 1000 g of EFBs as the substrate. The results showed that the highest enzyme activity was 236.3 U/g EFB, obtained from the use of humid air airflow of 0.1 LPM, and mixing was performed once a day. The scaling up resulted in a lower xylanase activity and call for a better design of the fermenter.Keywords: aeration, humidity, mixing, OPEFBs, tray fermenter, xylanase, xylitol

Production and optimization of lipase using Aspergillus niger MTCC 872 by solid-state fermentation

BackgroundLipases are serine hydrolases that degrade triglycerides, an attribute that treasures wide applications in biodiesel production, detergent, chemical industries, etc. The most sought after the application is in the high quality and economical production of biodiesel under mild reaction conditions and simplified product separation. For the said application, fungal lipases are ideal catalysts that could effectively catalyze esterification and transesterification reactions with their specific ability to release fatty acids from 1, 3 positions of acylglycerols.ResultsIn the present work, to facilitate bulk synthesis, lipase production using Aspergillus niger MTCC 872 was studied by solid-state fermentation (SSF). The chosen fungal strain was evaluated for lipase production using a mixture of agro-industrial substrates viz. rice husk, cottonseed cake, and red gram husk in various combinations at flask level. Tri-substrate mixture (rice husk, cottonseed cake, and red gram husk) combined in the ratio of 2:1:1 has shown the maximum lipase activity 28.19 U/gds at optimum cultivation conditions of temperature 40 °C, moisture content 75% (v/w), pH 6.0 and initial spore concentration of 5.4 million spores per mL. Further studies were performed for scale-up of lipase from flask level to lab scale using tray fermenter. Lipase activity was found to be 24.38 U/gds and 21.62 U/gds for 100 g and 1000 g substrate respectively.ConclusionThis is the first report on the production of lipase from Aspergillus niger MTCC 872 using tri-substrate mixture of rice husk (RH), cottonseed cake (CSC), and red gram husk (RGH). Moreover, comparison between individual, binary, and tri-substrate mixture was carried out for which the highest lipase activity was observed for tri-substrate mixture. In addition, comparable results were found when scale-up was performed using tray fermenter. Thus, the current work signifies usage of agro-industrial residues as substrates for enzyme production by solid-state fermentation process as an effective alternative to submerged fermentation for industrial applications.

Studies on a better laccase-producing mutant of Fusarium incarnatum LD-3 under solid substrate tray fermentation.

Forty-seven (47) mutant strains were generated from the wild-type fungus, Fusarium incarnatum strain LD-3 after exposure to ultraviolet radiation (UV) and a further seventeen (17) mutants were generated after exposure to ethyl methane sulfonate (EMS). Amongst these, the mutant strain, identified as UC-14, was the most promising laccase producer and produced threefold more laccase than the wild strain LD-3. Solid substrate tray fermentation using wheat straw and rice bran showed a twofold increase in laccase productivity and a fivefold loss of total organic matter (TOM) by mutant UC-14 over the wild strain LD-3. The mutant strain UC-14 also showed 25% and 54% weight loss of TOM after 36 days of fermentation which was 10% higher than the wild-type LD-3. Scanning electron microscopy suggested that the delayed condidiation in mutant strain UC-14 may be responsible for better laccase production.

RETRACTED: Cellulase production through solid-state tray fermentation, and its use for bioethanol from sorghum stover

The production of cellulase by Trichoderma reesei RUT C-30 under solid-state fermentation (SSF) on wheat bran and cellulose was optimized employing a two stage statistical design of experiments. Optimization of process parameters resulted in a 3.2-fold increase in CMCase production to 959.53IU/gDS. The process was evaluated at pilot scale in tray fermenters and yielded 457IU/gDS using the lab conditions and indicating possibility for further improvement. The cellulase could effectively hydrolyze alkali pretreated sorghum stover and addition of Aspergillus niger β-glucosidase improved the hydrolytic efficiency 174%, indicating the potential to use this blend for effective saccharification of sorghum stover biomass. The enzymatic hydrolysate of sorghum stover was fermented to ethanol with ∼80% efficiency.

Biodegradation of waste grease by Penicillium chrysogenum for production of fatty acid

The aim of present work was to effectively remediate grease waste by Penicillium chrysogenum. For efficient degradation, grease waste was pre-treated using various lipases, among them lipolase was the best. The pretreated grease was used as a substrate by P. chrysogenum resulting into the production of fatty acids. Process was optimized by response surface methodology (RSM) using four variables viz; FeCl2 (mM), spore concentration (spores/ml), time period (days) and amount of grease (g). The optimized conditions viz; FeCl2 1.25mM, culture amount 5×1011spores/ml and time period 16days led to the production of 6.6mg/g fatty acid from 10.0g of pre-treated grease mixed with 5.0g wheat bran in 10.0ml czapek-dox medium under solid state fermentation. The fermented media was extracted with hexane and subjected to GCMS analysis, which showed the presence of higher amount of palmitic acid. It was purified by crystallization method and 2.8g of palmitic acid was recovered from 1.0kg grease waste in tray fermentation.

Enhanced enzymatic hydrolysis of mild alkali pre-treated rice straw at high-solid loadings using in-house cellulases in a bench scale system

In the present study, scale-up systems for cellulase production and enzymatic hydrolysis of pre-treated rice straw at high-solid loadings were designed, fabricated and tested in the laboratory. Cellulase production was carried out using tray fermentation at 45°C by Aspergillus terreus in a temperature-controlled humidity chamber. Enzymatic hydrolysis studies were performed in a horizontal rotary drum reactor at 50°C with 25% (w/v) solid loading and 9 FPUg(-1) substrate enzyme load using in-house as well commercial cellulases. Highly concentrated fermentable sugars up to 20% were obtained at 40h with an increased saccharification efficiency of 76% compared to laboratory findings (69.2%). These findings demonstrate that we developed a simple and less energy intensive bench scale system for efficient high-solid saccharification. External supplementation of commercial β-glucosidase and hemicellulase ensured better hydrolysis and further increased the saccharification efficiency by 14.5 and 20%, respectively. An attempt was also made to recover cellulolytic enzymes using ultrafiltration module and nearly 79-84% of the cellulases and more than 90% of the sugars were recovered from the saccharification mixture.

Fermented Apple Pomace as a Feed Additive to Enhance Growth Performance of Growing Pigs and Its Effects on Emissions

Apple pomace is a by-product from the apple processing industry and can be used for the production of many value-added compounds such as enzymes, proteins, and nutraceuticals, among others. An investigation was carried out to study the improvement in the protein content in apple pomace by solid-state fermentation using the fungus Phanerochaete chrysosporium by tray fermentation method. The effect of this protein in terms of how it enriched apple pomace as animal feed for pigs has also been studied. There was a 36% increase in protein content in the experimental diet with 5% w/w fermented apple pomace. The efficiency of conversion of ingested food was increased from 43.5 ± 2.5 to 83.1 ± 4.4 in the control group and the efficiency of conversion of feed increased from 55.4 ± 4.5 to 92.1 ± 3.6 in the experimental group during the animal feed experiment. Similarly, the effect of a protein enriched diet on odor emission and greenhouse gas emission has also been studied. The results demonstrated that the protein enrichment of apple pomace by solid state cultivation of the fungus P. chrysosporium makes it possible to use it as a dietary supplement for pigs.

Production of Proteases in a Novel Trickling Tray Bioreactor

A novel trickling tray bioreactor was constructed for the production of an alkaline protease by Aspergillus oryzae PTCC 5164. A mixture of rice bran, wheat bran, soybean meal, and wheat flour with the respective mass ratio of 3, 1, 0.6 and 0.4 was used as the substrate. The substrate was spread on the trays and inoculated with spores of the fungus. After the initial growth, a solution was trickled on the trays to wash the produced protease out of the bioreactor during the fermentation. The process continued for 5 days and produced the maximum value of 748 U per gram of dry substrate (748 U/g ds ± 38.72) of the alkaline protease in the trickling medium on the third day, with a gradual decrease in enzyme amount thereafter. The performance of the trickling tray bioreactor was compared with the performance of conventional solid state fermentation conducted in flasks. In the conventional solid state fermentation the maximum value of 530 U/g ds ± 21.22 of the alkaline protease was harvested on the third day. Similar to the trickling tray fermentation a gradual decrease in the enzyme amount was observed after the third day. The results proved that the extraction of the enzyme from the substrate during fermentation could stimulate further enzyme production.

Multi-component thermostable cellulolytic enzyme production by Aspergillus niger HN-1 using pea pod waste: Appraisal of hydrolytic potential with lignocellulosic biomass

Solid state fermentation with pea pod waste and Aspergillus niger HN-1 resulted in filter paper cellulase (FP) and β-glucosidase (BGL) activity of 30 FPU/gds and 270 U/gds, respectively. Validation with the response surface optimized parameters (moisture content: 65%, pH 6.0, temperature: 33 °C, time: 84 h) in a solid-state tray fermentation enhanced FP and BGL activity by about 40 and 28%, respectively. Multi-component enzyme from A. niger HN-1 produced FP, BGL, endoglucanase (EG), cellobiohydrolase (CBHI), xylanase, α-l-arabinofuranosidase, β-xylosidase and xylan esterase with activities of 41.07 ± 2.11 FPU/gds, 345.69 ± 17.1, 480.3 ± 21.5, 52.1 ± 1.5, 2800.5 ± 88.4, 88.1 ± 9.3, 280.8 ± 11.4 and 3321.7 ± 14.8 U/gds, respectively. Enzyme was optimally active at temperature and pH of 55 °C and 5.0, respectively and demonstrated thermostability by retaining >95% activity for 6 h at 55 °C. SDS-PAGE showed the presence of 11 protein bands with molecular mass ranging between 20 and 200 kDa, while zymogram revealed the presence of multiple forms of EG, CBH and BGL with varying molecular mass. Hydrolysis of sweet sorghum bagasse at relatively high substrate loading (15%, w/v) with crude enzyme at 20 FPU/gds in thermostatically controlled glass reactor led to conversion of 82–91% of holocellulose to fermentable sugars in just 24 h as evident from HPLC analysis, showing promise for the reported enzyme in bioprocessing applications.

Development of Panax quinquefolius Yaoqu and Panax quinquefolius Sake and determination of ginsenosides Rg1, Rb1, and Re in both samples by HPLC–MS/MS

The mixed starter composed of high-quality Panax quinquefolius Yaoqu has been developed in our laboratory. Compared to traditional rice-koji, Panax quinquefolius Yaoqu demonstrated apparently higher saccharifying and liquefying power in shallow tray fermentation, and the Panax quinquefolius Sake’s indexes were better than rice-koji Sake’s. A sensitive determination method of ginsenosides Rg1, Rb1, Re in Panax quinquefolius Yaoqu and Panax quinquefolius Sake using high-performance liquid chromatography–tandem mass spectrometry was developed and validated with electrospray ionization in positive multiple reactions monitoring mode. The physical and chemical indexes of Panax quinquefolius Yaoqu and Panax quinquefolius Sake were better than rice-koji’s and rice-koji Sake’s. Specifically, saccharifying power, liquefaction power, amino acid nitrogen, total acid, and total sugar were improved to 1,764.95 U/g dry Qu, 56.61 U/g dry Qu, 0.56 g/L, 3.23 g/L, and 85 g/L, respectively, which were 31.52, 48.70, 211.10, 18.75, and 18.06 % higher than the rice-koji’s and rice-koji Sake’s.

Response surface optimization for enhanced production of cellulases with improved functional characteristics by newly isolated Aspergillus niger HN-2

Fungi isolated from partially decayed wood log samples showing characteristic diversity for spore colour, colony morphology and arrangement of spores were assessed for cellulolytic enzyme production. Isolates showing a cellulolytic index of ≥2.0 were assayed for filter paper (FP) cellulase and β-glucosidase (BGL) production. Molecular characterization confirmed the identity of the selected cellulolytic isolate as a strain of Aspergillus niger (A. niger HN-2). Addition of 2% (w/v) urea enhanced FP and BGL activity by about 20 and 60%, respectively. Validation studies conducted at parameters (29°C, pH 5.4, moisture content 72% and 66h) optimized through response surface methodology in a solid-state static tray fermentation resulted in FP, BGL, cellobiohydrolase I (CBHI), endoglucanase (EG), xylanase activity and protein content of 25.3 FPU/gds, 750IU/gds, 13.2IU/gds, 190IU/gds, 2890IU/gds and 0.9mg/ml, respectively. In comparison, A. niger N402 which is a model organism for growth and development studies, produced significantly lower FP, BGL, CBHI, EG, xylanase activity and protein content of 10.0 FPU/gds, 100IU/gds, 2.3IU/gds, 50IU/gds, 500IU/gds and 0.75mg/ml, respectively under the same process conditions as were used for A. niger HN-2. Process optimization led to nearly 1.8- and 2.2-fold increase in FP and BGL activity, respectively showing promise for cellulase production by A. niger HN-2 at a higher scale of operation. Zymogram analysis revealed two isoforms each for EG and cellobiohydrolase and three isoforms for BGL. Crude cellulase complex produced by A. niger HN-2 exhibited thermostability under acidic conditions showing potential for use in biofuel industry.

STATISTICAL OPTIMIZATION OF BIOPROCESS PARAMETERS FOR ENHANCED GALLIC ACID PRODUCTION FROM COFFEE PULP TANNINS BY Penicillium verrucosum

Gallic acid (3,4,5-trihydroxybenzoic acid) was produced by microbial biotransformation of coffee pulp tannins by Penicillium verrucosum. Gallic acid production was optimized using response surface methodology (RSM) based on central composite rotatable design. Process parameters such as pH, moisture, and fermentation period were considered for optimization. Among the various fungi isolated from coffee by-products, Penicillium verrucosum produced 35.23 µg/g of gallic acid on coffee pulp as sole carbon source in solid-state fermentation. The optimum values of the parameters obtained from the RSM were pH 3.32, moisture 58.40%, and fermentation period of 96 hr. Gallic acid production with an increase of 4.6-fold was achieved upon optimization of the process parameters. The results optimized could be translated to 1-kg tray fermentation. High-performance liquid chromatography (HPLC) analysis and spectral studies such as mass spectroscopy (MS) and 1H-nuclear magnetic resonance (NMR) confirmed that the bioactive compound isolated was gallic acid. Thus, coffee pulp, which is available in enormous quantity, could be used for the production of value-added products that can find avenues in food, pharmaceutical, and chemical industries.

Production and optimization of l-asparaginase from Cladosporium sp. using agricultural residues in solid state fermentation

Abstract The present communication deals with the production of l -asparaginase from Cladosporium sp. in solid state fermentation (SSF) using agro-industrial residues. When used as sole source for growth in SSF, wheat bran supported maximum enzyme production by Cladosporium sp. followed by rice bran and bagasse. Central composite rotatable designs (CCRDs) with five process variables were studied each at levels −2, −1, 0, +1 and +2. A 120 h fermentation time under aerobic condition optimized using response surface methodology (RSM) indicated that with moisture content of 58%, pH of 5.8, incubation temperature of 30 °C appeared optimal for enzyme production by SSF. Enzyme yield (3.74 U) was maximum at these optimized conditions. Presence of Tween 20 enhanced enzyme production by 1.3 folds. A bed height of 3 and 5 cm was appropriate to obtain high enzyme production. The results optimized could be translated to 1 kg tray fermentation. The study suggested that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an l -asparaginase production process based on wheat bran as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries. This result is of significant interest due to the low cost and abundant availability of residues.

Modeling Menaquinone 7 production in tray type solid state fermenter

Modeling Menaquinone 7 production in tray type solid state fermenter

Improved xylanase production using apple pomace waste by Aspergillus niger in koji fermentation

Xylanase production by Aspergillus niger NRRL‐567 in solid‐state fermentation (koji fermentation) was optimized using 24 factorial design and response surface methodology. The evaluated variables were the initial moisture level and concentration of inducers [veratryl alcohol (VA), copper sulphate (CS), and lactose (LAC)], leading to the response of xylanase production. Initial moisture level and LAC were found to be the most significant variable for xylanase production (p<0.05). The highest xylanase production was observed with 3578.8 ± 65.3 IU/gds (gram dry substrate) under optimal conditions using initial moisture of 85% (v/w), pH 5.0 and inducers VA (2 mM/kg), LAC 2% (w/w), and CS (1.5 mM/kg) after 48 h of incubation time. Higher xylanase activity of 3952 ± 78.3 IU/gds was attained during scale‐up of the process in solid‐state tray fermentation under optimum conditions after 72 h of incubation time. The present study demonstrates that A. niger NRRL‐567 can efficiently be used to achieve xylanase production with an economical and environmental benefit in solid‐state tray fermentation. The developed process can be used to develop an effective process for commercially feasible bioproduction of xylanases for speciality applications, such as conversion of lignocellulosic biomass to biofuels and other value‐added products.

Lactoserum as a moistening medium and crude inducer for fungal cellulase and hemicellulase induction through solid-state fermentation of apple pomace

Abstract Objectives The potential of lactoserum as a crude inducer and moistening medium for fungal cellulase and hemicellulase induction was tested through solid-state fermentation. Methodology Solid-state fermentation was conducted in Erlenmeyer flasks and plastic trays using apple pomace (AP) as solid substrate by Aspergillus niger NRRL 567 and A. niger NRRL 2001. Results Solid-state tray fermentation resulted in cellulase activities (IU g−1 dry weight basis) of 383.7 ± 17.9, 425.3 ± 21.3, 336.1 ± 16.2 and 4868 ± 39.8, respectively for FPase (filter paper cellulase), CMCase (carboxymethyl cellulase), BGL (β-glucosidase) and xylanase using A. niger NRRL 567. Similarly, A. niger NRRL 2001 resulted in enzyme activities (IU g−1) of 401 ± 23.8, 544.7 ± 24.5, 285.4 ± 11.7 and 4580.7 ± 34.5, respectively for FPase, CMCase, BGL and xylanase. The enzyme loading of FPase 50 IU g−1 substrate in the hydrolysis of AP resulted in (g kg−1): 509.3 ± 22.9 total sugar (48 h), 109.3 ± 5.7 reducing sugar (48 h), 16.1 ± 0.8 glucose (60 h) and 10.4 ± 2.0 xylose, respectively whereas hydrolysis of BSG resulted in (g kg−1): 375.7 ± 16.2 total sugars (36 h), 114.6 ± 4.0 reducing sugar (60 h), 18.1 ± 0.9 glucose (60 h) and 19.8 ± 2.0 xylose (48 h), respectively. Conclusions This study demonstrated the potential of SSF as a simple and cheap technology for higher cellulase and hemicellulase production using negative cost AP waste which could eventually help to utilize abundant lignocellulosic biomass for production of biofuels and other value-added products.