Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate

Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.     

Statistical Optimization of Alkaline Protease Production from Penicillium citrinum YL-1 Under Solid-State Fermentation

Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce. In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology. However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease. This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce. Furthermore, the culture conditions of alkaline protease production by P. citrinum YL-1 in solid-state fermentation were optimized by response surface methodology. First, three variables including peptone, initial pH, and moisture content were selected by Plackett–Burman design as the significant variables for alkaline protease production. The Box–Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine the optimal values of the variables. The maximal production (94.30 U/mL) of alkaline protease by P. citrinum YL-1 took place under the optimal conditions of peptone, initial pH, and moisture content (v/w) of 35.5 g/L, 7.73, and 136%, respectively.     

Pongamia pinnata seed cake: a promising and inexpensive substrate for production of protease and lipase from Bacillus pumilus SG2 on solid-state fermentation

The production of a protease and a lipase from Bacillus pumilus SG2 on solid-state fermentation using Pongamia pinnata seed cake as substrate was studied. The seed cake was proved to be a promising substrate for the bacterial growth and the enzyme production. The initial pH, incubation time and moisture content were optimized to achieve maximal enzyme production. Maximum protease production was observed at 72 h and that of the lipase at 96 h of incubation. The production of protease (9840 U/g DM) and lipase (1974 U/g DM) were maximum at pH 7.0 and at 60% moisture content. Triton X-100 (1%) was proved to be an effective extractant for the enzymes and their optimal activity was observed at alkaline pH and at 60 C. The molecular mass of the protease and lipase was 24 and 40 kDa, respectively. Both the enzymes were found to be stable detergent additives. The study demonstrated that inexpensive and easily available Pongamia seed cake could be used for production of industrially important enzymes, such as protease and lipase.     

Utilization of horticultural waste (Apple Pomace) for multiple carbohydrase production from Rhizopus delemar F2 under solid state fermentation

The brown rot fungus Rhizopus delemar F₂ was shown to produce extracellular thermostable and multiple carbohydrase enzymes. The potential of Rhizopus delemar F2 in utilizing apple pomace under solid state fermentation (SSF) is the purpose of the study. Solid state fermentation (SSF) is a very effective technique opposed to submerged fermentation in various aspects. Enhanced production of multiple carbohydrases 18.20?U?g^?1 of cellulose, 158.30?U?g^?1 of xylanase, 61.50?U?g^?1 of pectinase and amylase 21.03?U?g^?1 was released by microwave pretreatment of apple pomace at 450?W for 1?min and then by incubation the culture thus obtained at 30?°C for 6 days with moisture content of 1:4.5. Apple pomace can serve as a potential source of raw material for the production of multiple carbohydrases. Besides, it can find great commercial significance in production of bioethanol and various industries like textile, fruit juice, paper and pulp industry.     

Evaluation of waste chicken feathers as peptone source for bacterial growth

Aims: Peptones are one of the most expensive constituents of microbial media. This study was undertaken to prepare the peptone from waste chicken feathers through a new process. Methods and Results: The chemical analysis of chicken feather peptone (CFP) was performed. The ability of CFP to support the growth of the three test bacteria in liquid and agar media was comparable to those of three commercial peptones [tryptone peptone (TP), fish peptone and protease peptone (PP)]. Conclusions: CFP was found to be rich in ash (42·1 g 100 g^?1), protein (55·8 g 100 g^?1) and mineral contents. The maximum biomass yield (3·13 g l^?1) and colony number (83 × 10⁸ CFU ml^?1) for bacterium Bacillus subtilis were attained with CFP. The maximum biomass yields and colony numbers for Lactobacillus delbrueckii ssp. bulgaricus and Escherichia coli were reached in TP medium. Second high biomass yield (2·64 g l^?1) and colony number (75 × 10⁸ CFU ml^?1) for E. coli were achieved using CFP. Third high biomass yield (1·29 g l^?1) and colony number (90 × 10⁷ CFU ml^?1) for Lact. delbrueckii ssp. bulgaricus were obtained in CFP medium. Significance and Impact of the Study: Usability of waste chicken feathers as substrate for bacteria was investigated for the first time in the present study. The peptone may be used in industrial fermentations for production of antibiotics, organic acids, enzymes and biopolymer. It may be also used in clinical microbiology. A new chemical process was developed for peptone preparation. This process may be also employed for peptone preparation from other organic materials, especially fibrose protein‐containing materials.     

Cyanophycin production from feather hydrolysate using biotechnological methods

Abstract

Cyanophycin is a bacterial storage polymer for carbon, nitrogen and energy with emerging industrial applications. As efficient cyanophycin production is enhanced by peptone, but commercial peptones are very expensive, thereby increasing the overall production cost, an enzymatically produced feather hydrolysate (FH) is assessed as a cheap replacement of peptone to lower the costs and make cyanophycin production more economically feasible. Keratinase production using feather as the sole carbon/nitrogen source by S.pactum 40530 at 30-L fermentation scale was achieved within 93?h with degradation rate of 96.5%. A concentration of 60?g/L of FH, generated by keratinolytic activity (8?×?10³?U?g^?1L^?1d^?1) within 24?h, was used as the main carbon/peptone source to produce cyanophycin. The growth performances of E. coli DapE/L using FH was compared to that of casamino acids (CA) and up to 7.1?±?0.4 and 5.3?±?0.3?g/L of cell mass were obtained after 72?h from FH and CA, respectively. Cyanophycin production yielded 1.4?±?0.1g/L for FH with average molecular mass of 28.8 and 1.4?±?0.2 for CA with average molecular mass of 35.3, after 60?h. For the first time, FH generated by biotechnological methods from environmentally problematic, abundant and renewable feather bioresource was successfully used for cyanophycin biopolymer production.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

Protease production by thermo-alkaliphilic novel gut isolate Kitasatospora cheerisanensis GAP 12.4 from Gryllotalpa africana

In course of searching for proteolytic microbes from the gut of Gryllotalpa africana, a potent isolate GAP 12.4 was screened and identified as Kitasatospora cheerisanensis having protease activity 46.8?±?1.52?U/ml. Optimum conditions for the protease production (605.3?±?9.7?U/ml) were 7-d cultivation, 5% inoculum, pH 9.5, 55?°C, 150?rpm, and supplementation with 0.8% glucose and 0.6% ammonium sulfate. Surfactants such as SDS, EDTA, Tween 80 and Triton X-100 showed positive effect on enzyme production. Addition of biotin (50?μg/ml) promotes enzyme production maximally (674.15?±?4.13?U/ml). Further enhancement on addition of casein hydrolysate and molasses to the production medium was 709.20?±?7.53?U/ml and 744.26?±?9.71?U/ml, respectively. The isolate was also able to utilize agro-industries waste, green gram husk in solid-state fermentation for enzyme production (1675.02?±?21.58?U/ml). This thermo-alkaliphilic isolate may be a potent candidate for low cost protease production through management of agro-residues. It is the first report of protease production by a member of actinobacteria under the Kitasatospora genus.     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Molecular identification of a novel inulinolytic fungus isolated from and grown on tubers of Helianthus tuberosus and statistical screening of medium components

Of 24 fungi belonging to more than five genera isolated from tubers of rotten Helianthus tuberosus, 11-inulinolytic active isolates were able to develop halo zones around their fungal colonies, indicating inulinase activity. Alternaria, Aspergillus, Fusarium, Pencillium and Trichoderma were the most common inulinolytic genera, representing more than 90?% of the total positive inulinolytic fungi. Aspergillus tamarii and Pencillium citrinum quantitatively recorded better growth (5.5 and 4.7?mg?ml^?1) and inulinase production (21.53 and 20.15?U?ml^?1) in submerged culture. The enzyme preparation showed also invertase activity. Aspergillus tamarii, as the most potent producer of inulinase, was identified using the Inter Transcribed Spacer marker. The sequence comparisons showed that our molecularly identified strain (GU295949) is related more closely to A. tamarii strains of the gene bank. Statistical screening using the fractional factorial Plackett-Burman design with 12 run was applied for screening ten variables, the low levels of pH (4.8), inoculum size (10³ spore?g^?1), NH₄NO₃ (1.0?mg?g^?1) and MgSO₄ (0.12?mg?g^?1), were the most significant variables on A. tamarii inulinase production. The high inulinase/invertase ratio (1.841?C4.293) classified the enzyme preparation as inulinases, which can be used efficiently in production of fructose syrup from tubers of H. tuberosus.     

Supplementation of watermelon peels as an enhancer of lipase and esterase production by Yarrowia lipolytica in solid-state fermentation and their potential use as biocatalysts in poly(ethylene terephthalate) (PET) depolymerization reactions

Abstract

Yarrowia lipolytica is a yeast that presents high biotechnological potential due to its ability to produce many metabolites, among them lipases and esterases, which are important industrial biocatalysts. Since Brazil is an agroindustrial country, it generates an enormous diversity of residues or byproducts that can be used as a platform for biomolecules production. This work aims to evaluate lipase and esterase production by Y. lipolytica via solid-state fermentation using soybean bran and soybean bran supplemented with watermelon peels in different contents, and subsequent use of the enzyme extracts for poly(ethylene terephthalate) (PET) hydrolysis. Supplementation of watermelon peels in the lowest content led to an improvement of lipase activity in almost 31%, reaching 75.22?U g^?1. Esterase productivity was 1.5-fold higher when 20?wt% of watermelon peels were added to the media culture. Timecourse evaluation of enzymes production showed a maximum lipase activity in 14?h and similar esterase activity in 14 and 20?h of fermentation. Proteases production were also intensified in supplemented samples. Enzymes produced with 5?wt% watermelon peels supplementation led to higher terephthalic acid concentration (up to 42.02?µmol L^?1) during PET depolymerization. Results suggest a great potential of enzyme production in low cost fermentative media to act as biocatalysts in PET hydrolysis reactions.     

Production of recombinant beta-amylase of Bacillus aryabhattai

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai β-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for β-amylase production, containing glucose (7.5?g·L^?1), pig bone peptone (40.0?g·L^?1), Mg²⁺ (0.05?mol·L^?1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the β-amylase yield reached 925.4?U mL^?1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4?g L^?1. Through this cultivation approached 30?°C with 0?g·L^?1 initial glucose concentration, the maximum β-amylase activity reached 5371.8?U mL^?1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.     

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam gave maximum activities of 220 U lignin peroxidase ml^–1, 150 U manganese peroxidase ml^–1, 50 U laccase ml^–1 and 6.2 U protease ml^–1 at the retention time of 24 h for 60 days. Protease secretion destabilized the produced lignin peroxidase, manganese peroxidase and laccase.     

Solid-state protease production using anchovy waste meal by moderate halophile <Emphasis Type="Italic">Serratia proteamaculans</Emphasis> AP-CMST isolated from fish intestine

The possibility of utilising anchovy waste meal as a substrate for protease production by the fish gut isolate Serratia proteamaculans AP-CMST was assessed through solid-state fermentation. A time course for protease production revealed 72 h to be the optimum duration for higher production (146.24 U/g). The most suitable pH, temperature and moisture level observed for higher protease production were pH 7 (123.5 U/g), 30°C (97.22 U/g) and 75% (126.7 U/g), respectively. Protease production by S. proteamaculans AP-CMST was high in medium with added xylose (198.21 U/g), peptone (118.42 U/g), Triton X-100 (152.56 U/g) and manganese sulphate (178.33 U/g) when compared to other tested medium components. The halotolerancy of S. proteamaculans AP-CMST for protease production was 4% sodium chloride (155.65 U/g). Enzyme recovery from fermented anchovy waste meal was greatest (130.52 U/g) when 10% ethyl acetate was used as the extractant, and the optimum time range for extraction was 90–120 min.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Effect of the different parts of the corn cob employed as a carrier on ligninolytic activity in solid state cultures by P. chrysosporium

Outside and inside corn cob were used to study ligninolytic enzymes produced by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during solid state fermentation conditions. In a previous work by employing a mixture of outside and inside corn cob, we achieved a maximum MnP activity of 96?U l^?1 but LiP activities were low. In the present work we determined which part of the corn cob is more suitable in order to obtain high ligninolytic activities. We could find MnP activities about 300?U l^?1 by employing inside corn cob as a carrier and a maximum LiP activity of 24?U l^?1. ?In a subsequent experiment, using inside corn cob as a carrier, we could considerably improve ligninolytic enzymes production, by supplementing the medium with Tween 80 (0.5% v/v). We obtained a maximum MnP activity of 384?U l^?1and a maximum LiP activity of 155?U?l^?1.     

A Low-Cost Fermentation Medium for Thermophilic Protease Production by Streptomyces sp. 594 Using Feather Meal and Corn Steep Liquor

Protease production by Streptomyces sp. 594 was obtained after submerged fermentation (SF) and solid-state fermentation (SSF) using feather meal (FM) and corn steep liquor (CSL) as sole sources of carbon and nitrogen. Enzyme productions were 13.4 U ml⁻¹ in SF and 21.5 U g⁻¹ in SSF; these values were approximately 86% and 39% higher, respectively, than those obtained previously when yeast extract was used in place of CSL. The proteases, which belong to the serine and metalloproteinase classes, were active at high temperatures (55°C to 90°C) and over a wide range of pH values (5.0 to 10.0). Thus, these thermophilic proteases have shown interesting properties for industrial purposes. As far as we are concerned, this is the first contribution toward the microbial production of thermophilic proteases by a streptomycete using a low-cost medium composed of industrial poultry (FM) and corn processing by-products (CSL).     

Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min^?1 ml^?1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X₁); peptone (X₂) and soy bean flour (X₃) were examined, each at three different levels coded ?1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min^?1 ml^?1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl₂ 0.02, Y.E. 2.5, K₂HPO₄ 1.0, MgSO₄.7H₂O 0.2 and Fe₂ (SO₄)₃ 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min^?1 ml^?1.     

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8?U mL^?1 under the conditions of 180?rpm, an aeration rate of 1.5 vvm, 30?°C, pH_initial of 5.5, 5?×?10⁶ spores mL^?1, 32?g L^?1 pectin, 10?g L^?1 of yeast extract and 0.5?g L^?1 magnesium sulfate and bioproduction for 36?h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180?rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8?U mL^?1), also contained activity pectin methylesterase (6.0?U mL^?1) and pectin lyase (6.61?U mL^?1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.