Production Of Lipase In Solid State Fermentation Research Articles

Response surface optimization of lipase production by <i>Pseudomonas sp.</i> on a low-cost shea-nut cake using solid state fermentation

Shea nut cake (SNC) is the solid residue produced after the shea butter extraction process. The SNC could provide a low-cost substrate for  bacterial lipase production. Greater yield are among the benefits Solid State Fermentation (SSF). However, there have been less  instances of lipase production by bacteria than fungi in SSF. The current study aimed at improving bacterial lipase production on SNC in  SSF using Response Surface Methodology. Lipase-producing bacteria (LPB) were isolated from the shea butter mill effluent pond and screened for lipase production on a tributyrin agar plate. The influence of four independent parameters (temperature, pH, moisture  content and biosurfactant) on lipase production was studied using Box Behnken Design (BBD) of RSM. Results showed that isolate AO  (Pseudomonas sp.) displayed the highest zone of hydrolysis 2 (53 mm). A quadratic regression with R = 0.931 showed that the model is  best fitting, and predicted the optimum conditions of significant (P>0.05) growth parameters, including temperature at 40°C, moisture content at 50%, and biosurfactant at 2 mL, which produced 408.25 U/g of experimental lipase. The findings indicate that the model was useful for predicting optimal growth conditions for incresing lipase production in SSF and the low-cost agro- industrial SNC could be a promising substrate for industrial applications. 

Valorization of Macauba (Acromia aculeata) for Integrated Production of Lipase by Yarrowia lipolytica and Biodiesel Esters

Enzymatic biodiesel production is a potential eco-friendly alternative to the conventional chemical route which requires extensive study to reduce the costs associated with the application of commercial enzymes. Thus, this study aimed to develop a bioprocess using residues from macauba (Acrocomia aculeata) as raw material for lipase production in solid-state fermentation (SSF) by Yarrowia lipolytica. Then, the product obtained was used as a biocatalyst for the conversion (hydrolysis/esterification) of macauba acidic oil to biodiesel esters. Firstly, different SSF parameters (inoculum concentration, initial moisture content, and carbon and nitrogen levels) were investigated in a factorial design approach, using the cake from macauba fruit. Afterwards, moisture and urea concentration were shown to be statistically significant variables for lipase production. Lipase productnivities were 12.6 ± 0.6 U g−1 h−1 (at 24 h) for macauba fruit cake and 11.6 ± 1 U g−1 h−1 (at 20 h) for macauba pulp and peel cake. The solid enzymatic preparation (biocatalyst) showed optimized values at pH 6–7 at 37 °C, remaining stable (>70% retention) for 90 days at room temperature. Finally, enzymatic hydrolysis of the acidic oil from macauba reached 96% conversion (72 h) to fatty acids, and esterification of fatty acids reached 72% (biodiesel yield of 67%). The bioprocess described is a promising alternative for an integral and self-sufficient valorization of the macauba products.

A Temporal Evolution Perspective of Lipase Production by Yarrowia lipolytica in Solid-State Fermentation

Lipases are enzymes that, in aqueous or non-aqueous media, act on water-insoluble substrates, mainly catalyzing reactions on carboxyl ester bonds, such as hydrolysis, aminolysis, and (trans)esterification. Yarrowia lipolytica is a non-conventional yeast known for secreting lipases and other bioproducts; therefore, it is of great interest in various industrial fields. The production of lipases can be carried on solid-state fermentation (SSF) that utilizes solid substrates in the absence, or near absence, of free water and presents minimal problems with microbial contamination due to the low water contents in the medium. Moreover, SSF offers high volumetric productivity, targets concentrated compounds, high substrate concentration tolerance, and has less wastewater generation. In this sense, the present work provides a temporal evolution perspective regarding the main aspects of lipase production in SSF by Y. lipolytica, focusing on the most relevant aspects and presenting the potential of such an approach.

Statistical optimization of lipase production in solid-state fermentation by Aspergillus tamarii NDA03a and application of the fermented solid as a biocatalyst for biodiesel production

Background and objective Biodiesel, an attractive alternative fuel, is defined by the American Society for Testing and Materials (ASTM) as fatty acid methyl esters (FAME). Biodiesel is an ecofriendly fuel compared with many other transportation fuels. The aim of this study was to implement the statistical approaches for optimization of Aspergillus tamarii NDA03a mutant G lipase produced in solid-state fermentation (SSF), and then application of the dried fermented solid as a biocatalyst for biodiesel production from waste frying oil (WFO). Materials and methods A. tamarii NDA03a mutant (3G) was previously selected as a good lipase producer. Five oil residue meals were evaluated in the presence of wheat bran (WB) for their potential as enzyme inducers and substrates for the production of 3G lipase by SSF. The best oil residue meal was selected and used in subsequent experiments. The fermented solid thus obtained was collected, lyophilized, and used as a biocatalyst for waste frying oil transesterification to FAME. To optimize SSF conditions for lipase production using 3G, a Plackett–Burman design was used at first to screen the critical factors from several process variables, and finally, a central composite design was applied to further estimate the relationship between the variables and response as well as optimize the levels. Response was measured in terms of FAME yield. To verify the adequacy and accuracy of the model, validation experiments were also carried out. Results and conclusion The most favorable oil residue meal that enhances 3G lipase production by SSF was black cumin meal. Results of the Plackett–Burman design revealed that the factors contributing to the main effect were incubation temperature, incubation period, and moisture content. The optimal SSF conditions for lipase production were WB 10 g, black cumin meal 6% (w/w of WB), pH 8, temperature 28°C, moisture content 40%, molasses 1% (w/w of WB), and incubation period 3 days. Under these optimized conditions, produced FAME yield (65.55%) increased by 58% compared with the basal medium (41.46%). A good agreement between the experimental (65.55%) and predicted (65.03%) values was detected. The significance of this model was confirmed by its probability value and lack of fit (P<0.05) and clearly showed that the model was sufficient to describe the correlation between the FAME yield and the tested variables. The obtained results ascertained the success of response surface methodology as an efficient technique to optimize the lipase production in SSF and consequently the ability of application of the dried fermented solid as a biocatalyst for biodiesel production.

Artificial neural network hybridized with a genetic algorithm for optimization of lipase production from Penicillium roqueforti ATCC 10110 in solid-state fermentation

In the present work, an artificial neural network hybridized with a genetic algorithm (ANN-GA) has been applied to optimize Penicillium roqueforti ATCC 10110 lipase production in solid-state fermentation (SSF). For such a purpose, a feed-forward ANN with polynomial configuration 3-49-1 (i.e. 3 neurons in the input layer, 49 neurons in the hidden layer and 1 neuron in the output layer) was used to computationally model the experiment and a GA was used to optimize lipase production through the ANN model. The input variables optimized by the ANN-GA were fermentation time (1 day), incubation temperature (31.2 °C) and percentage moisture content (78%). Validation was performed by considering the optimal and central point conditions, thus obtaining a lipase activity value of 48.00 U g−1, which is three times greater than by using other methodologies. Furthermore, the ANN model was obtained using 28 essays (small dataset) with interpolation and generalization capability based on a significant and precise data choice and justified by mean square error and determination coefficient values. A total of 5.0 × 107 artificial tests were simulated from the small dataset of 28 experiments.

Optimization of Lipase Production in Solid-State Fermentation by Rhizopus Arrhizus in Nutrient Medium Containing Agroindustrial Wastes

Background: Rhizopus arrhizus is a potential microorganism for lipase production. Solid-state fermentation is used for microbial biosynthesis of enzymes, due to advantages, such as high productivity, utilization of abundant and low-cost raw materials, and production of enzymes with different catalytic properties. Objective: The objective of the research is optimization of the conditions for lipase production in solid-state fermentation by Rhizopus arrhizus in a nutrient medium, containing agroindustrial wastes. Method: Biosynthesis of lipase in solid-state fermentation by Rhizopus arrhizus was investigated. The effect of different solid substrates, additional carbon and nitrogen source, particles size and moisture content of the medium on enzyme production was studied. Response surface methodology was applied for determination of the optimal values of moisture content and tryptone concentration. A procedure for efficient lipase extraction from the fermented solids was developed. Results: Highest lipase activity was achieved when wheat bran was used as a solid substrate. The addition of 1% (w/w) glucose and 5% (w/w) tryptone to the solid medium significantly increased lipase activity. The structure of the solid medium including particles size and moisture content significantly influenced lipase production. A mathematical model for the effect of moisture content and tryptone concentration on lipase activity was developed. Highest enzyme activity was achieved at 66% moisture and 5% (w/w) tryptone. The addition of the non-ionic surfactant Disponyl NP 3070 in the eluent for enzyme extraction from the fermented solids increased lipase activity about three folds. Conclusion: After optimization of the solid-state fermentation the achieved 1021.80 U/g lipase activity from Rhizopus arrhizus was higher and comparable with the activity of lipases, produced by other fungal strains. The optimization of the conditions and the use of low cost components in solid-state fermentation makes the process economicaly effective for production of lipase from the investigated strain Rhizopus arrhizus.

Production of Lipases by a Newly Isolate of Aspergillus niger Using Agroindustrial Wastes by Solid State Fermentation

Background: Lipases are enzymes widely used in many industrial applications. However, its use can be increased by reducing costs of production, being important to this effort the screening of new microorganisms producing and the use of low cost culture mediums, such as agricultural residuals. Objective: To produce lipases using two new isolates of filamentous fungi in solid-state fermentation. Methods: The selection of significant variables on the lipase production was studied through a 27-3 Fractional Factorial Design. The studied variables were: the type of bran used as support in solid state fermentation (wheat or soy), nitrogen source (urea or nitrate), inducer (soy or olive oil), nitrogen source concentration (1 or 3%), inducer concentration (1 or 3%), pH (5 or 7) and filamentous fungal strain (Aspergillus O-4 or Aspergillus E-6). Also, a 24 Full Factorial Design was used in the sequence of optimization. Results: The first experimental design showed that the variables type of bran, nitrogen source, inducer, and pH were the most significant (p<0.05) on lipolytic activity, being the higher activity obtained of 45.95 U.g-1. These variables were studied in the second experimental design, in which high lipolytic activities were obtained (57 U) with soy bran, sodium nitrate as nitrogen source, olive oil as inducer and pH 5. Conclusion: The statistical methods of optimization enabled an increased lipase production in solidstate fermentation from Aspergillus niger (O-4). Keywords: Inducer, nitrogen source, pH, response surface methodology, valuation of residuals, fermentation.

Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor

Abstract A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g−1 at 18 h in the laboratory bioreactor to 113 U g−1 at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g−1 to 10.4 U g−1. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.

Extracellular Alkaline Lipase from a Novel Fungus
Curvularia sp. DHE 5: Optimisation of Physicochemical Parameters, Partial Purification and Characterisation.

Thirty isolated fungal strains were screened for lipase production using Phenol Red plates, containing tributyrin as lipidic substrate, and a novel fungus identified genetically as Curvularia sp. DHE 5 was found as the most prominent strain. Various agro-industrial substrates were evaluated as inert supports for lipase production in solid-state fermentation. The highest yield of lipase ((83.4±2.2) U/g on dry mass basis) was reported with wheat bran medium after seven days of fermentation at pH=7.0, temperature of 30 °C, 70% moisture content, inoculum size of 1.27·107 spore/mL and 2% olive oil as an inducer. Supplementation of the medium with 0.05% KCl as an ion source further increased lipase production to (88.9±1.2) U/g on dry mass basis. The enzyme was partially purified through ammonium sulphate fractionation (40%) followed by dialysis, and its optimum pH and temperature were reported at 8.0 and 50 °C, respectively, with remarkable pH and thermal stability.

Fungal Lipase Production by Solid-State Fermentation

Lipases are one of the most promising enzymes in the chemical and biopharmaceutical industries. Numerous applications have been reported including fine chemistry, detergents formulation and biodiesel synthesis. Lipases are commonly produced by a wide variety of yeasts and filamentous fungi in submerged fermentation or solid-state fermentation. Filamentous fungi and yeasts usually behave more efficiently in solid-state fermentation and show greater productivities when compared to submerged fermentation. Although filamentous fungi adequately growth in solid-state fermentation, there are some limitations for cultivating them by this process. Here, a review is made about lipase production in solid-state fermentation. This includes the analysis of solid-state fermentation as a promising technology, characterization of growth media and ambient factors such as moisture, pH and temperature.

Heterogeneity of buffalo haemoglobin ( Bos bubalis)

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g−1 at 18 h in the laboratory bioreactor to 113 U g−1 at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g−1 to 10.4 U g−1. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.