Mixed-solid substrate fermentation. A novel process for enhanced lipase production by Candida rugosa

Candida rugosa was cultivated in a mixed-solid substrate containing coconut oil cake (COC) and fine and coarse wheat bran (1:1:1) with an initial water activity (a_w) of 0.92. The substrate was modified by adding a mineral solution (5%), corn steep liquor (6%), maltose (2%), peptone (3%), olive oil (10%), gum arabic (0.4%), different fatty acids (0.3%) and Tweens (0.5%). Fermentation in a column fermenter significantly improved the lipase yield to 118.2 Units per gram of dry fermented substrate [U/gds] at 72 h. This result was obtained 24 hours earlier than in our former studies (87.76 U/gds at 96 h) in COC, and the yield showed a 38% increase. Growth was measured indirectly by determining the glucosamine content in the cell wall of the yeast contained in the fermented matter, after its hydrolysis.     

Coconut oil cake--a potential raw material for the production of alpha-amylase

Solid-state fermentation (SSF) was carried out using coconut oil cake (COC) as substrate for the production of alpha-amylase using a fungal culture of Aspergillus oryzae. Raw COC supported the growth of the culture, resulting in the production of 1372 U/gds alpha-amylase in 24 h. Process optimization using a single parameter mode showed enhanced enzyme titre, which was maximum (1827 U/gds) when SSF was carried out at 30 degrees C for 72 h using a substrate with 68% initial moisture. Supplementation with glucose and starch further enhanced enzyme titre, which was maximum (1911 U/gds) with 0.5% starch. However, maltose inhibited the enzyme production. Studies on the effect of addition of external organic and inorganic nitrogenous compounds further showed a positive impact on enzyme synthesis by the culture. Increase of 1.7-fold in the enzyme activity (3388 U/gds) was obtained when peptone at 1% concentration was added to the fermentation medium. The enzyme production was growth-related, the activity being the maximum when the fungal biomass was at its peak at 72 h. Use of COC as raw material for enzyme synthesis could be of great commercial significance. To the best of our knowledge this is the first report on alpha-amylase production using COC in SSF.     

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.     

Comparison of phytase production on wheat bran and oilcakes in solid-state fermentation by Mucor racemosus

Comparisons were made for phytase production using wheat bran (WB) and oilcakes as substrates in solid-state fermentation (SSF) by Mucor racemosus NRRL 1994. WB was also used as mixed substrate with oil cakes. Sesame oil cake (SOC) served as the best carbon source for phytase synthesis by the fungal strain as it gave the highest enzyme titres (30.6 U/gds). Groundnut oil cake (GOC) also produced a reasonably good quantity of enzyme (24.3 U/gds). Enzyme production on WB was surprisingly much less (almost 3.5 times less in comparison to SOC). Mixing WB with SOC (1:1 ratio) resulted in better phytase activity (32.2 U/gds). Optimization of various process parameters such as incubation time, initial moisture content and inoculum concentration was carried out using the single variable mode optimization technique. Under optimized conditions, the production of phytase reached 44.5 U/gds, which was almost 1.5-fold higher than the highest yield obtained with any individual substrate used in this study and was more than 4-fold higher than that obtained from WB.     

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.     

Sequential optimization and large scale production of lipase using tri-substrate mixture from Aspergillus niger MTCC 872 by solid state fermentation

Tri-substrate mixture of Prosopis juliflora (PJ), red gram husk (RGH) and cotton seed cake (CSC) has been studied for the production of lipase (E.C. 3.1.1.3) using Aspergillus niger MTCC 872 in solid state fermentation. Simplex centroid mixture design (SCMD) was implemented to optimize the tri-substrate mixture composition consisting of PJ, RGH and CSC. Mixture taken in the ratio of 6.66:1.66:1.66 for PJ:RGH:CSC has shown highest lipase activity of 212.20 ± 6.36 U/gds at 30 °C, 7 pH and 70 % initial moisture content (v/w). Sequential optimization of physical parameters was done using the central composite face-centered design (CCFD). The optimum mixture composition has shown the highest lipase activity of 269.87 ± 8.09 U/gds at 35 °C, 7 pH and 70 % initial moisture content (v/w). ANOVA analysis for SCMD and CCFD confirms the model’s significance with R² values of 0.9989 and 0.968. A 1.27 fold increased lipase activity was obtained after physical parameters optimization. Large scale production using 1 kg substrate was carried out in tray bioreactor with different bed heights and the highest lipase activity of 208.79 ± 6.26 U/gds was obtained. This study signifies the enhancement of lipase production using substrate PJ for lipase production along with the other agricultural residues.     

Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

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.     

L-glutaminase production by <Emphasis Type="Italic">Trichoderma koningii</Emphasis> under solid-state fermentation

Solid state fermentation was conducted for the production of L-glutaminase by Trichoderma koningii Oud.aggr. using different agro-industrial byproducts inlcuding wheat bran, groundnut residues, rice hulls, soya bean meal, corn steep, sesamum oil cake, cotton seed residues and lentil industrial residues as solid substrates. Wheat bran was the best substrate for induction of L-glutaminase (12.1 U/mg protein) by T. koningii. The maximum productivity (23.2 U/mg protein) and yield (45.0 U/gds) of L-glutaminase by T. koningii occurred using wheat bran of 70% initial moisture content, initial pH 7.0, supplemented with D-glucose (1.0%) and L-glutamine (2.0% w/v), inoculated with 3 ml of 6 day old fungal culture and incubated at 30°C for 7 days. After optimization, the productivity of L-glutaminase by the solid cultures of T. koningii was increased by 2.2 fold regarding to the submerged culture.     

响应面法优化洋葱假单胞菌产脂肪酶液体发酵工艺

用响应面法对洋葱假单胞菌G-63液体发酵产脂肪酶条件进行了优化。首先运用单因子试验筛选出麦芽糖和豆粉水解液为最适碳源和氮源。在此基础上,通过Plackett-Burman设计试验,对影响产酶条件的11个相关因子进行评估并筛选出具有显著效应的3个因子:橄榄油、豆饼粉水解液以及初始pH值。在用最陡爬坡实验逼近以上3个因子的最大响应区域后,采用响应面分析法,确定出橄榄油、豆粉水解液的最佳浓度和最佳初始pH值分别为4.337%,1.956%和8.38。优化后液体发酵培养基中脂肪酶活力提高到44.39 U/mL,比初始酶活13.45 U/mL提高了3.3倍。     

Production and Regulation of Lipase Activity from <Emphasis Type="Italic">Penicillium restrictum</Emphasis> in Submerged and Solid-State Fermentations

Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2–14.1 mg/mL) and SSF (7.0–8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression.     

Microbial production of extra-cellular phytase using polystyrene as inert solid support

Aspergillus ficuum TUB F-1165 and Rhizopus oligosporus TUB F-1166 produced extra-cellular phytase during solid-state fermentation (SSF) using polystyrene as inert support. Maximal enzyme production (10.07 U/g dry substrate (U/gds) for A. ficuum and 4.52 U/gds for R. oligosporus) was observed when SSF was carried out with substrate pH 6.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1.3 x 10(7) spores/5 g substrate, for 72 h for A. ficuum and with substrate pH 7.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1 x 10(6) spores/5 g substrate for 96 h for R. oligosporus. Results indicated scope for production of phytase using polystyrene as inert support.     

Alkaline lipase production by Citrobacter freundii IIT-BT L139

Around 150 lipase producing bacterial isolates were screened from the local soils enriched with oil. Citrobacrer freundii IIT-BT L139, an isolated microbial strain, produced lipase that had high activity (8.8 U/ml) at pH 9.0 and 40 degrees C. The 16S rDNA phylogenetic studies showed that Citrobacter freundii belongs to the family Enterobacteriaceae and later confirmed by the microbial identification. Suitable C and N sources for lipase production were deduced to be starch and peptone-urea, respectively. In a controlled fermenter (1 L), the lipase activity was found to increase by 36% (12 Uml(-1)). The variation of lipase activity, pH and dissolved oxygen (DO) during growth of the organism in the controlled batch fermenter were monitored. The rheological characteristics of the fermentation broth indicated that it behaved like a Newtonian fluid throughout the fermentation. The fermentation time was comparatively short (60 h). The lipase was also found to be substantially resistant to common detergents. This lipase was, thus, characterized as alkaline, thermostable and solvent stable, which was essentially desirable in pharmaceutical, detergent and other industrial applications or production.     

Hydrolysis of olive oil catalyzed by surfactant-coated<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase in a hollow fiber membrane reactor

In this study, we invetigated the hydrolysis of olive oil catalyzed by a surfactant-coatedCandida rugosa lipase in a hydrophilic polyacrylonitrile hollow fiber membrane reactor and then compared the results to those using the native lipase. The organic phase was passed through the hollow inner fibers of the reactor and consisted of either the coated lipase and olive oil dissolved in isooctane or the coated lipase dissolved in pure olive oil. The aqueous phase was pumped through the outer space. After 12 h and with conditions of 30°C, 0.12 mg enzyme/mL and 0.62 M olive oil, the substrate conversion of the coated lipase reached 60%. This was twice the conversion for the same amount of native lipase that was pre-immobilized on the membrane surface. When using pure olive oil, after 12 h the substrate conversion of the coated lipase was 50%. which was 1.4 times higher than that of the native lipase.     

Mixed substrate solid state fermentation for production and extraction of lipase from Aspergillus niger MTCC 2594

A novel mixed substrate solid-state fermentation (SSF) process has been developed for Aspergillus niger MTCC 2594 using wheat bran (WB) and gingelly oil cake (GOC) and the results showed that addition of GOC to WB (WB : GOC, 3 : 1, w/w) increased the lipase activity by 36.0% and the activity was 384.3+/-4.5 U/g dry substrate at 30 degrees C and 72 h. Scale up of lipase production to 100 g and 1 kg tray-level batch fermentation resulted in 95.0% and 84.0% of enzyme activities respectively at 72 h. A three-stage multiple contact counter-current extraction yielded 97% enzyme recovery with a contact time of 60 min. However, extraction by simple percolation and plug-flow methods resulted in decreased enzyme recoveries. The mixed substrate SSF process has resulted in a significant increase in specific activity (58.9%) when compared to a submerged fermentation (SmF) system. Furthermore, an efficient process of extraction has been standardized with this process. Use of GOC along with WB as potential raw materials for enzyme production could be of great commercial significance. This is the first report on the production and extraction of lipase from Aspergillus niger using mixed solid substrates, WB and GOC, which are potential raw materials for the production of enzymes and other value-added products.     

Evolutionary and swarm intelligence‐based approaches for optimization of lipase extraction from fermented broth

Evolutionary and swarm intelligence‐based optimization approaches, namely genetic algorithm (GA) and particle swarm optimization (PSO), were utilized to determine the optimal conditions for the lipase extraction process. The input space of the nonlinear response surface model of lipase extraction served as the objective function for both approaches. The optimization results indicate that the lipase activity was significantly improved, more than 20 U/g of dry substrate (U/gds), in both approaches. PSO (133.57 U/gds in the 27th generation) outperforms GA (132.24 U/gds in the 320th generation), slightly in terms of optimized lipase activity and highly in terms of convergence rate. The simple structure associated with the effective memory capability of PSO renders it superior over GA. The proposed GA and PSO approaches, based on a biological phenomenon, are considered as natural and thus may replace the traditional gradient‐based optimization approaches in the field of downstream processing of enzymes.     

A new process for simultaneous production of tannase and phytase by <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> in solid-state fermentation of orange pomace

The production of enzymes such as tannases and phytases by solid-state fermentation and their use in animal feed have become a subject of great interest. In the present work, Paecilomyces variotii was used to produce tannase and phytase simultaneously. Solid-state fermentation, a process initially designed for tannase production, was implemented here using orange pomace as substrate. Orange pomace is the waste product of the large orange juice industry in Brazil, and it has also been used as an ingredient in animal feed. In addition to enzymatic production, biotransformation of the phenolic content and antioxidant capacity of the orange pomace were analyzed after fermentation. Fermentation conditions, namely moisture level and tannic acid concentration rate, were studied using CCD methodology. The response surface obtained indicated that the highest tannase activity was 5,000 U/gds after 96 h at 59% (v/w) and 3% (w/w) and that of phytase was 350 U/gds after 72 h at 66% (v/w) and 5.8% (w/w) of moisture level and tannic acid concentration, respectively. The amount of tannase production was similar to the levels achieved in previous studies, but this was accomplished with a 7% (w/w) reduction in the amount of supplemental tannic acid required. These results are the first to show that P. variotii is capable of producing phytase at significant levels. Moreover, the antioxidant capacity of orange pomace when tested against the free radical ABTS was increased by approximately tenfold as a result of the fermentation process.     

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> using surface response methodology and inulin hydrolysis

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl₂. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.     

Phytase production by a thermophilic mould Sporotrichum thermophile in solid state fermentation and its potential applications

Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett-Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120 h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76 U/g DMR) was attained at a substrate bed depth of 1.5 cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate.