Production of Childinan SF-2 as Bioactive Compound from Daldinia childiae: A Survey on Antioxidant, Antibacterial and Antitumor Properties.

Background: Fungal extracts have received increased attention due to their great medicinal applications including antitumor, immune-modulating, antioxidant, radical scavenging, antiviral, antibacterial, antifungal and detoxificating properties. Objectives: This study is the first report on a novel bioactive compound, namely Childinan SF-2 which was isolated from soil ascomycete fungus. The significant antibacterial, antioxidant and cytotoxic properties of the extract may lead to development of novel, safe and useful substances. Materials and Methods: The isolate was identified on the basis of molecular approach. Spore suspension was inoculated in the culture medium and the bioactive compound was isolated from the viscous fermented broth via ethanol precipitation of the extracellular compound. The basic chemical composition of the extract including protein, carbohydrate, sulfate radical and uronic acid content were measured. FTIR (Fourier-transform infrared spectroscopy) and GC-MS (Gas chromatography-mass spectrometry) analysis were used for further structural characterization. The extract was utilized for treatment of AGS and MDA cell lines to assess the cell cycle and apoptosis. The antioxidant activity was examined using DPPH, hydroxyl radicals scavenging, ß-carotene bleaching inhibition and ferric reducing power assay methods. The extract was tested for evaluation of antibacterial activity using different Gram-positive and Gram-negative bacterial strains. Results: The fungal isolate was identified as the new strain Daldinia childiae SF-2. Initial biochemical characterization of the extract showed that the fungal biopolymer was composed of total sugars, protein, uronic acids and sulfated groups with values of 91.6%, 2.15%, 2.25% and 1.05% (w/w), respectively. FTIR and GC-MS analysis revealed that Childinan SF-2 might be mainly constructed from D-glucose, D-mannitol and D-galactofuranose. The in vitro experiments revealed that Childinan SF-2 enhanced the percentage of necrosis and apoptosis of cancer cells and blocked the cell cycle progression as shown by flowcytometry. Childinan SF-2 represented a considerable antioxidant and antibacterial activity. Conclusions: These results indicated that Childinan SF-2 can serve as a potential source in medicinal applications. Keywords Exopolysaccharides; Fungal Biopolymers; Mycelial Secretions; Natural Pharmaceuticals; Xylariaceae.

Biological function and molecular properties of Pyrenaican SF-1 as biological macromolecule extracted from Daldinia pyrenaica.

Molecular properties and biological functions of Pyrenaican SF-1 as a novel biological macromolecule extracted from a fungal isolate were studied. The isolate was identified as Daldinia pyrenaica on the basis of 5.8S rDNA sequencing. Pyrenaican SF-1 was obtained from the culture filtrate of the fungal isolate. The partial characterization of biochemical structure of Pyrenaican SF-1 was conducted. The fungal extract was also tested for the treatment of AGS, MDA and HeLa cell lines to assess cells proliferation, cells cycle and apoptosis. Furthermore, Pyrenaican SF-1 extract was tested for its antibacterial and antioxidant activity. Initial chemical analysis revealed that Pyrenaican SF-1 extract was composed of various monosaccharides such as d-glucose, D- mannitol, D-arabinose and ß-D-ribopyranose. In vitro study indicated that Pyrenaican SF-1 could effectively elevate percentage of apoptosis and necrosis of cancer cells and block cell cycle phase of the control group. The fungal extract could inhibit proliferation of Hela and MDA cell up to 67% and 56%, respectively. Moreover, Pyrenaican SF-1 represented a strong antioxidant activity compared to that one obtained from vitamin C. On the other hand, Pyrenaican SF-1 exhibited growth inhibitory effects against different Gram-negative and Gram-positive bacterial strains. Pyrenaican SF-1 can be considered as a bioactive macromolecule with promising application in pharmaceutical and medical sectors.

Utilization of sugarcane straw for production of ß-glucan biopolymer by Lasiodiplodia theobromae CCT 3966 in batch fermentation process.

ß-Glucans as emerging biopolymer are widely produced by microorganisms in fermentation processes using commercial sugars which make process non-economic. Lignocellulosic substances are inexpensive carbon sources, which could be exploited for sustainable production of ß-glucans. In this study, a lignocellulosic material, namely sugarcane straw (SCS) was utilized for the production of extracellular ß-glucan by Lasiodiplodia theobromae CCT3966. SCS was subjected to acid and subsequent alkaline pretreatment, followed by enzymatic saccharification using cellulase enzyme. Quantity of 48.65 g/L glucose was released after enzymatic hydrolysis. ß-Glucan production was performed by cultivation of fungal strain in SCS hydrolysate at 28 °C and initial culture pH 7. Highest ß-glucan yield and productivity of 0.047 gg-1 and 0.014 gL-1h-1, respectively was obtained at 72 h fermentation time. Kinetic study of ß-glucan production revealed experimental biosynthesis of ß-glucan from SCS hydrolysate followed the trend generated by Logistic and Luedeking-Piret models. Chemical structure of biopolymer produced showed ß-glucan constitution.

Enhanced butanol production by optimization of medium parameters using Clostridium acetobutylicum YM1.

A new isolate of the solvent-producing Clostridium acetobutylicum YM1 was used to produce butanol in batch culture fermentation. The effects of glucose concentration, butyric acid addition and C/N ratio were studied conventionally (one-factor-at-a-time). Moreover, the interactions between glucose concentration, butyric acid addition and C/N ratio were further investigated to optimize butanol production using response surface methodology (RSM). A central composite design was applied, and a polynomial regression model with a quadratic term was used to analyze the experimental data using analysis of variance (ANOVA). ANOVA revealed that the model was highly significant (p < 0.0001) and the effects of the glucose and butyric acid concentrations on butanol production were significant. The model validation experiment showed 13.82 g/L butanol was produced under optimum conditions. Scale up fermentation in optimized medium resulted in 17 g/L of butanol and 21.71 g/L of ABE. The experimental data of scale up in 5 L bioreactor and flask scale were fitted to kinetic mathematical models published in the literature to estimate the kinetic parameters of the fermentation. The models used gave the best fit for butanol production, biomass and glucose consumption for both flask scale and bioreactor scale up.

Enhancement of bioplastic polyhydroxybutyrate P(3HB) production from glucose by newly engineered strain Cupriavidus necator NSDG-GG using response surface methodology.

This study aimed to enhance production of polyhydroxybutyrate P(3HB) by a newly engineered strain of Cupriavidus necator NSDG-GG by applying response surface methodology (RSM). From initial experiment of one-factor-at-a-time (OFAT), glucose and urea were found to be the most significant substrates as carbon and nitrogen sources, respectively, for the production of P(3HB). OFAT experiment results showed that the maximum biomass, P(3HB) content, and P(3HB) concentration of 8.95 g/L, 76 wt%, and 6.80 g/L were achieved at 25 g/L glucose and 0.54 g/L urea with an agitation rate of 200 rpm at 30 °C after 48 h. In this study, RSM was applied to optimize the three key variables (glucose concentration, urea concentration, and agitation speed) at a time to obtain optimal conditions in a multivariable system. Fermentation experiments were conducted in shaking flask by cultivation of C. necator NSDG-GG using various glucose concentrations (10-50 g/L), urea concentrations (0.27-0.73 g/L), and agitation speeds (150-250 rpm). The interaction between the variables studied was analyzed by ANOVA analysis. The RSM results indicated that the optimum cultivation conditions were 37.70 g/L glucose, 0.73 g/L urea, and 200 rpm agitation speed. The validation experiments under optimum conditions produced the highest biomass of 12.84 g/L, P(3HB) content of 92.16 wt%, and P(3HB) concentration of 11.83 g/L. RSM was found to be an efficient method in enhancing the production of biomass, P(3HB) content, and P(3HB) concentration by 43, 21, and 74%, respectively.

Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol.

Catalytic depolymerization of mannan composition of palm kernel cake (PKC) by mannanase was optimized to enhance the release of mannan-derived monomeric sugars for further application in acetone-butanol-ethanol (ABE) fermentation. Efficiency of enzymatic hydrolysis of PKC was studied by evaluating effects of PKC concentration, mannanase loading, hydrolysis pH value, reaction temperature and hydrolysis time on production of fermentable sugars using one-way analysis of variance (ANOVA). The ANOVA results revealed that all factors studied had highly significant effects on total sugar liberated (P<0.01). The optimum conditions for PKC hydrolysis were 20% (w/v) PKC concentration, 5% (w/w) mannanase loading, hydrolysis pH 4.5, 45°C temperature and 72h hydrolysis time. Enzymatic experiments in optimum conditions revealed total fermentable sugars of 71.54±2.54g/L were produced including 67.47±2.51g/L mannose and 2.94±0.03g/L glucose. ABE fermentation of sugar hydrolysate by Clostridium saccharoperbutylacetonicum N1-4 resulted in 3.27±1.003g/L biobutanol.

Batch culture and repeated-batch culture of Cunninghamella bainieri 2A1 for lipid production as a comparative study.

This research was performed based on a comparative study on fungal lipid production by a locally isolated strain Cunninghamella bainieri 2A1 in batch culture and repeated-batch culture using a nitrogen-limited medium. Lipid production in the batch culture was conducted to study the effect of different agitation rates on the simultaneous consumption of ammonium tartrate and glucose sources. Lipid production in the repeated-batch culture was studied by considering the effect of harvesting time and harvesting volume of the culture broth on the lipid accumulation. The batch cultivation was carried out in a 500 ml Erlenmeyer flask containing 200 ml of the fresh nitrogen-limited medium. Microbial culture was incubated at 30 °C under different agitation rates of 120, 180 and 250 rpm for 120 h. The repeated-batch culture was performed at three harvesting times of 12, 24 and 48 h using four harvesting cultures of 60%, 70%, 80% and 90%. Experimental results revealed that nitrogen source (ammonium tartrate) was fully utilized by C. bainieri 2A1 within 24 h in all agitation rates tested. It was also observed that a high amount of glucose in culture medium was consumed by C. bainieri 2A1 at 250 rpm agitation speed during the batch fermentation. Similar results showed that the highest lipid concentration of 2.96 g/L was obtained at an agitation rate of 250 rpm at 120 h cultivation time with the maximum lipid productivity of 7.0 × 10(-2) mg/ml/h. On the other hand, experimental results showed that the highest lipid concentration produced in the repeated-batch culture was 3.30 g/L at the first cycle of 48 h harvesting time using 70% harvesting volume, while 0.23 g/L gamma-linolenic acid (GLA) was produced at the last cycle of 48 h harvesting time using 80% harvesting volume.

Saccharification of polysaccharide content of palm kernel cake using enzymatic catalysis for production of biobutanol in acetone-butanol-ethanol fermentation.

In this work, hydrolysis of cellulose and hemicellulose content of palm kernel cake (PKC) by different types of hydrolytic enzymes was studied to evaluate monomeric sugars released for production of biobutanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in acetone-butanol-ethanol (ABE) fermentation. Experimental results revealed that when PKC was hydrolyzed by mixed ß-glucosidase, cellulase and mannanase, a total simple sugars of 87.81±4.78 g/L were produced, which resulted in 3.75±0.18 g/L butanol and 6.44±0.43 g/L ABE at 168 h fermentation. In order to increase saccharolytic efficiency of enzymatic treatment, PKC was pretreated by liquid hot water before performing enzymatic hydrolysis. Test results showed that total reducing sugars were enhanced to 97.81±1.29 g/L with elevated production of butanol and ABE up to 4.15±1.18 and 7.12±2.06 g/L, respectively which represented an A:B:E ratio of 7:11:1.

Production of butanol by Clostridium saccharoperbutylacetonicum N1-4 from palm kernel cake in acetone-butanol-ethanol fermentation using an empirical model.

Palm kernel cake (PKC) was used for biobutanol production by Clostridium saccharoperbutylacetonicum N1-4 in acetone-butanol-ethanol (ABE) fermentation. PKC was subjected to acid hydrolysis pretreatment and hydrolysates released were detoxified by XAD-4 resin. The effect of pH, temperature and inoculum size on butanol production was evaluated using an empirical model. Twenty ABE fermentations were run according to an experimental design. Experimental results revealed that XAD-4 resin removed 50% furfural and 77.42% hydroxymethyl furfural. The analysis of the empirical model showed that linear effect of inoculums size with quadratic effect of pH and inoculum size influenced butanol production at 99% probability level (P<0.01). The optimum conditions for butanol production were pH 6.28, temperature of 28°C and inoculum size of 15.9%. ABE fermentation was carried out under optimum conditions which 0.1g/L butanol was obtained. Butanol production was enhanced by diluting PKC hydrolysate up to 70% in which 3.59g/L butanol was produced.

Repeated batch fermentation biotechnology for the biosynthesis of lipid and gamma-linolenic acid by Cunninghamella bainieri 2A1.

The biosynthesis of biomedical products including lipid and gamma-linolenic acid (GLA) by Cunninghamella bainieri 2A1 was studied in repeated batch fermentation. Three key process variables, namely, glucose concentration, ammonium tartrate concentration, and harvesting time, were optimized using response surface methodology. Repeated batch fermentation was carried out by the cultivation of Cunninghamella bainieri 2A1 in nitrogen-limited medium with various nitrogen concentration (1-4 g/L) and glucose concentration (20-40 g/L) at three time intervals (12 h, 24 h, and 48 h). Experimental results showed that the highest lipid concentration of 6.2 g/L and the highest GLA concentration of 0.4 g/L were obtained in optimum conditions, where 20.2 g/L glucose, 2.12 g/L ammonium tartrate, and 48 h harvesting time were utilized. Statistical results showed that the interaction between glucose and ammonium tartrate concentration had highly significant effects on lipid and GLA biosynthesis (P < 0.01). Moreover, harvesting time had a significant interaction effect with glucose and ammonium tartrate concentration on lipid production (P < 0.05).

Enhanced butanol production by Clostridium acetobutylicum NCIMB 13357 grown on date fruit as carbon source in P2 medium.

The production of biobutanol was studied by the cultivation of Clostridium acetobutylicum NCIMB 13557 in P2 medium including date fruit as the sole substrate. The effect of P2 medium and the effect of different concentrations of date fruit ranging from 10 to 100 g/L on biobutanol production were investigated. Anaerobic batch culture was carried out at 35 °C incubation temperature and pH 7.0 ± 0.2 for 72 h. Experimental results showed that the lowest yield of biobutanol and acetone-butanol-ethanol (ABE) was 0.32 and 0.35 gram per gram of carbohydrate consumed (g/g), respectively, when an initial date fruit concentration of 10 g/L was utilized. At this fruit date concentration a biobutanol production value of 1.56 g/L was obtained. On the other hand, the maximum yield of biobutanol (0.48 g/g) and ABE (0.63 g/g) was produced at 50 g/L date fruit concentration with a biobutanol production value as high as 11 g/L. However, when a higher initial date fruit concentration was used, biobutanol and ABE production decreased to reach the yield of 0.22 g/g and 0.35 g/g, respectively, where 100 g/L date fruit was used. Similar results also revealed that 10.03 g/L biobutanol was produced using 100 g/L date fruit.

Optimization of aeration and agitation rate for lipid and gamma linolenic acid production by Cunninghamella bainieri 2A1 in submerged fermentation using response surface methodology.

The locally isolated filamentous fungus Cunninghamella bainieri 2A1 was cultivated in a 5 L bioreactor to produce lipid and gamma-linolenic acid (GLA). The optimization was carried out using response surface methodology based on a central composite design. A statistical model, second-order polynomial model, was adjusted to the experimental data to evaluate the effect of key operating variables, including aeration rate and agitation speed on lipid production. Process analysis showed that linear and quadratic effect of agitation intensity significantly influenced lipid production process (P < 0.01). The quadratic model also indicated that the interaction between aeration rate and agitation speed had a highly significant effect on lipid production (P < 0.01). Experimental results showed that a lipid content of 38.71% was produced in optimum conditions using an airflow rate and agitation speed of 0.32 vvm and 599 rpm, respectively. Similar results revealed that 0.058(g/g) gamma-linolenic acid was produced in optimum conditions where 1.0 vvm aeration rate and 441.45 rpm agitation rate were used. The regression model confirmed that aeration and agitation were of prime importance for optimum production of lipid in the bioreactor.

Utilization of palm kernel cake for production of beta-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor.

The production of beta-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on beta-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of beta-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5 degrees C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of beta-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of beta-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of beta-mannanase were incubation temperature of 32 degrees C, initial moisture level of 59% and aeration rate of 0.5 l/min. A beta-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.