Influence of carbon and nitrogen sources on lipase production by a newly isolated Candida viswanathii strain

Microorganisms can produce lipases with different biochemical characteristics making necessary the screening of new lipase-producing strains for different industrial applications. In this study, 90 microbial strains were screened as potential lipase producers using a sensitive agar plate method with a suitable medium supplemented with Tween 20 and also a liquid culture supplemented with olive oil. The highest cell growth and lipase production for Candida viswanathii were observed in triolein and oleic acid when used as the only pure carbon source. Renewable low-cost triacylglycerols supported the best cell growth, and olive oil was found to be the best inducer for lipase production (19.50 g/L and 58.50 U). The selected conditions for enzyme production were found with yeast extract as nitrogen source and 1.5 % (w/v) olive oil (85.70 U) that resulted in a good cell growth yield (Y_X/S?=?1.234 g/g) and lipase productivity (1.204 U/h) after 72 h of shake-flask cultivation. C. viswanathii lipase presented high hydrolytic activity on esters bonds of triacylglycerols of long-chain, and this strain can be considered an important candidate for future applications in chemical industries.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast

From the soil samples of various locations, 245 strains of microorganisms were isolated by the enrichment culture method using olive oil as a carbon source. Of these microorganisms one deuteromycotinous yeast was the best producer of extracellular lipase, and the strain WU-C12 was identified as Trichosporon fermentans from the morphological and taxonomical properties. When cultivated at 30°C for 4 d in the medium containing 8% (w/v) corn steep and 3% (v/v) olive oil as sources of nitrogen and carbon, T. fermentans WU-C12 produced 126 U/ml of extracellular lipase. When 3% (v/v) tung oil was used instead of 3% (v/v) olive oil, 146 U/ml of the lipase was produced. Although lipase production decreased to 40 U/ml by the addition of 2% (w/v) glucose to the corn steep-olive oil medium, the strain WU-C12 produced 34 U/ml of lipase in the medium containing 2% (w/v) glucose instead of 3% (v/v) olive oil. On the other hand, T. fermentans WU-C12 could grow and produce lipase in the medium containing n-paraffin as a carbon source.     

固液态发酵中橄榄油对Rhizopus chinensis全细胞脂肪酶的影响

主要对华根霉全细胞脂肪酶固态和液态两种发酵过程进行比较,并着重探讨不同培养方式下橄榄油对其合成活力和水解活力的影响。结果表明：液态培养较有利于菌体生长,对脂肪酶的生产也有一定的促进作用。橄榄油的加入不仅有利于菌体生长、提高脂肪酶水解活力,更可使脂肪酶的合成活力显著增加,液态发酵下的效果更为明显。橄榄油在整个发酵过程中可能既作为碳源又是脂肪酶的诱导物。另外,全细胞脂肪酶的水解活力和合成活力在固液态发酵条件下均存在不对应性,表明华根霉可能产性质不同的脂肪酶同功酶。     

Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method

Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions.     

Production and partial purification of cellulase from a novel fungus,Aspergillus flavus BS1

This study describes the isolation and characterization of a novel fungus, Aspergillus flavus BS1 and its cellulolytic activities with special emphasis on endoglucanase production. Preliminary screening studies showed that A. flavus BS1 was a potent strain for the production of cellulase. To study the cellulolytic activities in detail by submerged fermentation (SmF), productions of endoglucanase, exoglucanase, and β-glucosidase were estimated from the basal salt medium (BSM) supplemented with 1 % carboxy methyl cellulose (CMC). CMC medium supported the maximum yield of endoglucanase (2,793 U/ml) on day 5 of incubation at 28 °C and 150 rpm, which was higher than that obtained with naturally available supplements (flour) from banana, tapioca, potato, or banana peel. During cellulase production by solid-state fermentation, 10 % (w/w) tapioca flour in sawdust (teak wood) moisturized with BSM (1:2, w/v) supported maximum cellulase yield (5,408 U/g dry substrate) on day 3 at 28 °C, which was 2-fold higher than that obtained during SmF. The active cellulase was qualitatively estimated by polyacrylamide gel electrophoresis (PAGE). Native-PAGE (0.25 % CMC impregnated on the 10 % gel) activity staining with congo-red showed a clear zone for CMCase activity, whereas SDS-PAGE showed a distinct band. In conclusion, this study showed that A. flavus strain BS1 is a potent strain for the production of cellulase on lignocellulosic media, the hot enzyme for bioethanol production from the lignocellulosic biomass by SSF.     

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.     

Lipase production by Serratia marcescens strain SN5gR isolated from the scat of lion-tailed macaque (Macaca silenus) in Silent Valley National Park, a biodiversity hotspot in India

An extracellular lipase-producing bacterium was isolated from a fecal sample of lion-tailed macaque (Macaca silenus), an endangered Old World monkey that is endemic to the Western Ghats of South India. Morphological, biochemical and molecular analyses identified the bacterium as Serratia marcescens. Production of lipase was investigated in shake-flask culture. Optimum tributyrin concentration of 1.5 % was found to be the most suitable triglyceride to increase lipase production (13.3 U ml^?1). The next best lipid source observed was olive oil (11.94 U ml^?1), followed by castor oil, coconut oil and palm oil. Analyzing the effect of different carbon sources on lipase production revealed that 2 % glucose yielded higher lipase production than the other tested carbon sources. Investigations on suitable nitrogen source for lipase production revealed that 2 % meat extract yielded higher lipase production. The most suitable trace element for maximum lipase production was zinc sulfate, followed by magnesium sulfate and copper sulfate. Partial characterization of the crude lipase revealed that pH 7.0 and a temperature of 40 °C gave optimal lipase activity. Enzymatic activity of the crude sample was retained over a wide temperature range (20–75 °C), and 70 % of enzyme activity was retained at 60 °C. Testing the effect of various organic solvents on lipase activity revealed that hexadecane increased lipase activity by 85 % over the control.     

Use of evolutionary operation (EVOP) factorial design technique to develop a bioprocess using grease waste as a substrate for lipase production

The aim of the present work was to develop a bioprocess using EVOP-factorial design technique employing grease waste as a substrate for the production of lipase. A newly isolated fungal strain of Penicillium chrysogenum was explored for the fermentation process. Solid-state fermentation (SSF) was carried out using grease waste and Czapek-dox medium, supplemented with wheat bran. The yield of lipase was 38 U/ml when SSF was carried out at 32 °C for 8 days and grease:wheat bran:Czapek-dox media in 1:1:2 (w/w/v). Different physicochemical parameters affecting the production of lipase were optimized through evolutionary operation (EVOP) factorial design technique and after optimization yield was enhanced up to 46 U/ml at 30 °C, pH 7.0 with 1:1:2 (w/w/v) grease waste:wheat bran:Czapek-dox media. Industrial grease waste has never been reported before for the production of industrially important lipase enzyme.     

Coconut cake – a potent substrate for the production of lipase by Candida rugosa in solid-state fermentation

Investigations were conducted with the aim of producing extracellular lipase from Candida rugosa by solid-state fermentation (SSF), using coconut oil cake (COC) as a solid substrate. To optimize production, various modifications were made to enrich the substrate by supplementing it with mineral solution, different carbon sources and several inorganic as well as organic nitrogen sources. Among them, urea (1%), peptone (3%) and maltose (5%) were found to be most suitable. Addition of olive oil (10%) encouraged lipase synthesis. The maximum lipase activity in the enriched substrate was 87.76 units per gram of dry fermented substrate [U/gds] compared to 25.81 U/gds in the raw cake at 96 h of fermentation, and growth was as high as 14.44 mg/gds of glucosamine. This was reached at 72 h in the enriched substrate. C. rugosa growth was calculated indirectly by estimating the glucosamine content in the cell wall after its hydrolysis. The enzyme yield was far better than any values reported as yet.     

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.     

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.     

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.     

Alkaline lipase activity from the marine protists,thraustochytrids

Two thraustochytrid protists of the genus Thraustochytrium isolated from coastal and mangrove habitats of Goa, India were studied for extracellular alkaline lipase production. Maximum lipase production was supported by a combination of peptone and yeast extract in the growth medium while strong inhibition of enzyme production was observed in presence of glucose. The inducible nature of the enzyme production was evidenced by the requirement of olive oil in the medium. Lipase production was salt-dependent and optimum production required 3.4% (w/v) crude sea salt. Ideal conditions for maximum production of lipases were therefore adopted as incubation at 30 ± 2°C for 168 h at an initial pH of 6.0 in a medium consisting of 0.5% peptone, 0.01% yeast extract, 0.5% olive oil and 3.4% crude salt. Extracellular lipase production by the two thraustochytrid isolates [designated TZ (ATCC #PRA-295) and AH-2 (ATCC #PRA-296)] was increased threefold under these optimized culture conditions. This appears to be the first report on optimization of cultivation conditions for the production of alkaline lipases by thraustochytrids.     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Optimization of Cultivating Conditions for Triterpenoids Production from Antrodia cinnmomea

The submerged cultivating conditions for triterpenoids production from Antrodia cinnamomea were optimized using uniform design method and the one-factor-at-a-time method was adopted to investigate the effect of plants oils and glucose supply on triterpenoids production and mycelia growth. Corn starch and culturing time were identified as more significant variables for triterpenoids production. The optimal conditions for triterpenoids production was 20.0 g/L corn starch, 20.0 g/L wheat bran, 1.85 g/L MgSO4, initial pH 3 and 16 days of cultivation. In addition, investigation of plant oils and glucose supply showed that 0.3 % (v/v) olive oil supply at the beginning of fermentation stimulated mycelia growth and significantly increased triterpenoids production; 0.2 % (w/v) glucose supplement at 10th day enhanced production of triterpenoids with slight effect on biomass, which is reported for the first time. The triterpenoids production experimentally obtained under the optimal conditions was 7.23 % (w/w). The uniform design method may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoids production from A. cinnamomea.     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

Optimization of extracellular lipase production from the biocontrol fungus Nomuraea rileyi

Lipases are important cuticle-degrading enzymes that hydrolyze the ester bonds of waxes, fats and lipoproteins during the infection of insects by the fungus Nomuraea rileyi. Lipase production by the N. rileyi strain MJ was optimized by varying environmental and nutritional conditions in culture medium containing different vegetable oils at various concentrations with shaking at 150 rpm for 8 days at 25°C. The maximum lipase production was obtained using castor oil (30.5±0.6 U mL^?1), followed in order by coconut oil (20.8±0.4 U mL^?1), olive oil (20.8±0.4 U mL^?1) and cottonseed oil (20.6±0.4 U mL^?1). The highest lipase activity (37.7±0.4 U mL^?1) was obtained when castor oil was used at a concentration of 4% (v/v) of basal medium. When the surfactant Tween 80 was added at the fourth day rather than at the beginning of incubation, a maximum lipase activity of 44.9±3.5 U mL^?1 was obtained. The optimal temperature and pH for lipase production were 25°C and pH 8.0, respectively. This is the first report on lipase production by the biocontrol fungus N. rileyi.     

Production of laccase from Trametes versicolor by solid-state fermentation using olive leaves as a phenolic substrate

The aim of the present study was to investigate whether olive leaves were feasible as a substrate for laccase production by the white-rot fungus Trametes versicolor FPRL 28A INI under solid-state fermentation conditions. Different experiments were conducted to select the variables that allow obtaining high levels of laccase activity. In particular, the effects of the initial moisture content, substrate particle size, supplementation with inorganic and organic nitrogen sources were evaluated. Highest laccase activity (276.62 ± 25.67 U/g dry substrate) was achieved with 80 % initial moisture content and 1.4–1.6 mm particle size of the substrate supplemented with yeast extract (1 % (w/w) nitrogen). Such a high activity was obtained without any addition of inducers.