Plackett-Burman Statistical Design Research Articles

Identification and statistical optimization of a novel alginate polymer extracted from newly isolated Synechocystis algini MNE ON864447 with antibacterial activity

Cyanobacteria are a potential source of promising secondary metabolites with different biological activities, including antibacterial, antiviral, antifungal, antiprotozoal, and anticancer activities. To combat the emergence of antibiotic resistance, there is an urgent requirement for new drugs, and cyanobacteria metabolites can constitute alternative new antibacterial medication. The chemical complexity of their exopolysaccharides indicates that they have the potential to be bioactive molecules with many biological activities. The present study aimed to produce and optimise a novel alginate polymer from a newly isolated cyanobacterium, S. algini MNE ON864447, in addition to its promising antibacterial activity. We successfully isolated a new cyanobacterium strain, S. algini MNE ON864447 from the Nile River, which produces alginate as an extracellular polymeric substance. The isolated cyanobacterial alginate was identified using a set of tests, including FTIR, TLC, HPLC, GC–MS, and 1H NMR. Plackett–Burman statistical design showed that working volume (X1), the incubation period (X2), and inoculum size (X3) are the most significant variables affecting the production of alginate. The highest alginate production (3.57 g/L) was obtained using 4% inoculum size in 400 mL medium/L conical flask after 20 days of the incubation period. The extracted alginate showed potent antibacterial activity against both Gram-negative and Gram-positive bacteria and Streptococcus mutants (NCTC10449) are the most sensitive tested pathogen for purified cyanobacterial alginate with inhibition zone diameters of 34 ± 0.1 mm at 10 mg/mL of purified alginate while Vibro cholera (NCTC 8021) the lowest sensitive one and showed inhibition zone diameters of 22.5 ± 0.05 mm at the same cyanobacterial alginate concentration. This antibacterial activity is a critical step in the development of antibacterial drugs and presents a new challenge to fight against multi-resistant bacteria.

Mutagenesis enhances gellan gum production by a novel Sphingomonas spp.: upstream optimization, kinetic modeling, and structural and physico-functional evaluation.

Gellan gum (GG) has gained tremendous attention owing to its diversified applications. However, its high production and hence market cost are still a bottleneck in its widespread utilization. In the present study, high GG producing mutant of Sphingomonas spp. was developed by random mutagenesis using ethyl methylsulphonate (EMS) for industrial fermentation and identified as Sphingomonas trueperi after 16S rRNA and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. The fermentation conditions such as pH, temperature, and inoculum ratio were optimized by one factor at a time (OFAT) followed by screening of medium components by the Plackett-Burman statistical design. The most critical nutrients were further optimized by response surface methodology for maximizing GG production. The effect of dissolved oxygen tension in bioreactor on cell growth, substrate consumption, GG production, and batch productivity was elucidated. The highest GG titer (23 ± 2.4g/L) was attained in optimized medium at 10% inoculum (6.45 ± 0.5 log cfu/mL) under controlled fermentation conditions of pH (7), temperature (30°C), agitation (300-600rpm), and aeration (0.5-2.0 SLPM) at 22 ± 2% dissolved oxygen tension in a 10-L bioreactor. Kinetic modeling of optimized batch process revealed that logistic growth model could best explain biomass accumulation, while GG formation and substrate consumption were best explained by Luedeking-Piret and exponential decay model, respectively. Structural and physico-functional features of GG produced by mutant Sphingomonas spp. were characterized by HPLC, FTIR, NMR, DSC, TGA, GPC, SEM, and rheological analysis. The higher productivity (0.51g/L/h) under optimized fermentation conditions suggests potential consideration of mutant and process for commercial utilization.

Design of a minimized and low-cost culture medium for high mannitol production by Fructobacillus sp. CRL 2054 and F. tropaeoli CRL 2034

Mannitol is a natural polyol used in the food, medical and pharmaceutical industries. In this work, a simplified low-cost culture medium using fructose syrup as a carbon source was formulated for mannitol production by Fructobacillus sp. CRL 2054 and F. tropaeoli CRL 2034. To this end, the effect of different medium components in bacterial growth and mannitol synthesis was assessed by applying the Plackett-Burman statistical design. The formulated FSYP-min was used for fermentations at constant pH of 5.0 in a 2 L bioreactor, where two concentrations of fructose syrup (10 and 20 %, w/v, of total sugars) were evaluated. High mannitol concentrations (ca. 90 g/L) and volumetric productivities (3.7 g/L∗h) were achieved by both strains using 20 % (w/v) of sugars; these mannitol values are one of the highest reported for LAB to date. Finally, high-purity mannitol crystals were successfully extracted after fermentation with the formulated culture medium.

Cryptococcus albidus D24'ten Lipaz Üretimi Üzerindeki En Etkili Besin Maddelerinin Öneminin Belirlenmesi

In this study, the optimization of the medium components used for the production of lipase enzyme from Cryptococcus albidus D24 was performed using the Plackett-Burman statistical design method (PBD), and the most important nutrients affecting the production of lipase enzyme from D24 strain were determined as the first step. According to PBD, the highest lipase activity (19.34 U/ml/min) was obtained with medium including Tween 80 (X2) 2.5% (v/v), and (g/L) Peptone (X4) 8.0, Yeast Extract (X6) 7.5, Beef Extract (X7) 7.5, Malt Extract (X8) 7.5, NH4Cl (X9) 6.0, NaNO3 (X10) 1.5, (NH4)NO3 (X12) 6.0, (NH4)HCO3(X13) 6.0, MgSO4.7H2O (X15) 1.0, and KH2PO4 (X16) 2.0 at the end of 144 h cultivation. Regarding the concentration effect (CE) values obtained from PBD, NH4Cl (CE=7.1587), olive oil (CE=3.5544), (NH4)HCO3 (CE=3.0747), and tryptone (CE=2.1427) were evaluated as the more effective nutrients among the sixteen compounds studied. After that, the optimum concentrations of these effective compounds were experimented with Response Surface Methodology (RSM). Experimental results showed that the medium containing olive oil (X3) 1.5% (v/v), and (g/L) tryptone (X5) 3.0, NH4Cl (X9) 7.5, and (NH4)HCO3 (X13) yielded maximum lipase activity (12.03 U/ml/min) compared to other studied compounds. Although the maximum lipase activity obtained with RSM methodology was lower than that obtained by PBD, the cost of the nutrients used to produce one-unit enzyme is 0.104 Euro in the PBD, while only 0.0277 Euro is spent in RSM. In other words, the production of lipase using compounds coded X3, X5, X9, and X13 provides a cost-effective process.

Agarase Production by Marine Pseudoalteromonas sp. MHS: Optimization, and Purification

Agar is an essential polysaccharide that has been utilized in numerous fields. Many kinds of literature have been published regarding agarolytic microorganisms’ isolation and agarases biochemical studies. In this search, a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp. MHS. The agarase production was parallel to the growth of Pseudoalteromonas sp. MHS as cells displayed a lag phase (2 h), subsequently an exponential growth that prolonged till 10 h where maximum growth (OD550nm = 3.9) was achieved. The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth. Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment; the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL. Pseudoalteromonas sp. MHS agarase was partially purified and its molecular weight (MW) was determined by SDSPAGE (15-25 kDa). Agarase showed approximately 94% of its activity at 40 °C. The enzyme stability decreased as the temperature increased; the enzyme could retain about 98, 90, 80, 75, and 60% of its activity at 20, 30, 40, 50, and 60 °C, respectively. Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp. MHS; the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.

Optimization of immobilization of Pseudomonas cepacia lipase on multiwalled carbon nanotubes functionalized with glycyrrhizin and Tween 80.

In the present study, multiwalled carbon nanotubes (MWCNTs) were functionalized with glycyrrhizin and Tween 80 and applied for immobilization of Pseudomonas cepacia lipase (PcL). Characterization of f-MWCNTs was performed through Fourier-transform infrared spectroscopy, thermal gravimetric, field emission scanning electron microscopy, and energy-dispersive X-ray spectroscopy analysis. The optimum specific activity of immobilized PcL (studied by Plackett-Burman statistical design) occurred at 0.3 mg/mL of f-MWCNTs, 25 mM of phosphate buffer (pH 6.0), 15 min sonication time, 8 U/mL of enzyme concentration, and 24 h immobilization time at 4 °C in the absence of glutaraldehyde. In these conditions, the specific activity was 16.57 ± 0.71 U/mg, which was very close to the predicted amount (16.62 ± 0.64 U/mg). The results of thermal and pH stability showed that the stability of immobilized PcL was higher than that of the free PcL. The activity of immobilized PcL on f-MWCNTs held 93% after being incubated for 60 min at 70 °C. Moreover, the immobilized PcL on f-MWCNTs retained about 65% of its initial activity after 30 days of storage at 25 °C. In addition, about 50% of initial activity of immobilized PcL retained after 10 cycles of uses. Therefore, f-MWCNTs could be introduced as suitable support for enzymes immobilization.

Bioprocess development for enhanced endoglucanase production by newly isolated bacteria, purification, characterization and in-vitro efficacy as anti-biofilm of Pseudomonas aeruginosa

Endoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett–Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO4.7H2O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T1/2) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 μg.

Improving lipid production capacity of new natural oleaginous yeast: Pichia cactophila firstly

Biodiesel is a useful alternative source of energy and can be used as a support for oil-source diesel. Microbial lipids are considered a promising raw material for biodiesel production. In a previously completed study, Pichia cactophila was found to be the best lipid-producing yeast by lipid screening studies among 107 yeast isolates. In this study, the lipid production conditions of P. cactophila, which was chosen as a lipid producer for the first time, were partially optimized by using response surface methodology in crude glycerol. Plackett-Burman statistical experiment design was used to determine the effect of glycerol, yeast extract, peptone, urea, (NH4)2SO4, NH4Cl, NH4NO3 factors on lipid production. In order to determine the optimum levels of the most effective parameters by the result of screening (glycerol, yeast extract, urea, NH4NO3), Box-Behnken experimental design method was used. The optimum production conditions were determined as 39 g/L glycerol, 20 g/L yeast extract, 5 g/L urea, and 20 g/L NH4NO3, and the result of the experiments carried out under these conditions the lipid value (%) was determined as 91.43. Lipid content was increased 1.408-fold (40.79%) by two-step optimization with the response surface methodology and reached a very high value. The fatty acid composition obtained under optimum conditions is compatible with vegetable oils. This suggests that the microbial lipid of P. cactophila can be used as a raw material for biodiesel production and is promising for further studies.

Decolorization of Remazol Brilliant Blue Dye by Escherichia Coli NG188 under Optimized Conditions

The objective of this study was carried out to degrade Remazol Brilliant Blue (RB) textile dye by using Escherichia coli NG188 isolated from the industrial wastewater effluent. The optimum RB degradation (%) was obtained at; 40°C, inoculum size 5%, and pH 8 under static condition. The influence of nutritional factors on dye degradation via Plackett-Burman statistical design was investigated. On the basis of the calculated t-values, p-values and confidence level: glucose and dye concentration had confidence levels above 90% and hence were considered the significant parameters that influence dye degradation by Escherichia coli NG188. A verification experiment was carried out to evaluate the accuracy of the applied Plackett-Burman statistical design. The decolorization percentage showed a 1.24 fold increase in the degradation rate of the dye compared to the average of the basal condition results. The complete decolorization of the RB dye was reported using the Response Surface Methodology (RSM). The biodegradation of RB was confirmed by UV–visible spectroscopic and FTIR analysis.

Optimization of Poly-β-Hydroxybutyrate (PHB) Production by an Egyption strain of Rhizobium fabae F44 using Response Surface Methodology

The present work is aimed to isolate, purify, identify a newly isolate Rhizobium fabae from different regions in Egypt then, produce and optimize Poly-β-Hydroxybutyrate polymer (PHB) production by the selected isolate Rhizobium fabae F44 using a statistical approach of response surface methodology (RSM). Rhizobium fabae F44 isolate was picked up from Sharkia Governorate in Egypt and then identified by the 16SrRNA gene as Rhizobium fabae. Eleven different variables which affect the production of PHB polymer were screened by Plackett–Burman statistical design approach. Maximization of PHB production was adjusted by the terms of The Face Central Composite Design (FCCD) of RSM and assessed at three coded levels (–1, 0, +1). One way Anova was used to statistically analyze all obtained results in relation to post hoc multiple comparison analysis performed by Tukey’s HSD. Appulses of nutritional and physical factors (two carbon sources, two nitrogen sources, mineral salts, pH, temperature, incubation time, inoculum size and agitation rate) were studied using Plackett-Burman design method. 58 isolates were obtained from the different 9 governorates with 100 % infection plant technique. The 16SrRNA showed a similarity of 99.56 (%) to Rhizobium fabae F44. Out of all tested significant media components, sucrose, yeast extract, agitation rate had the highest significant effects on the response as for PHB production, with confidence level > 98% and were further optimized using FCCD. Predicted Maximum PHB production was observed as (78.51%) which in near the mid-point (0) values (concentrations) of sucrose which reached (25 g/l) and yeast extract of (0.5 g/l) for 48 hrs. at 150 rpm agitation rate. The observed experimental value reached 87.5u/ml was very close result to the predicted one validating the model. So, Response Surface Methodology is an effective statistical approach which can substitute the use of one variable at a time approach due to its adequacy and efficiency to optimize PHB production by Rhizobium fabae F44.

Evaluation of different variables on the supercritical CO2 extraction of oat (Avena sativa L.) oil; main fatty acids, polyphenols, and antioxidant content

Six variables (temperature, pressure, static time, dynamic time, chemical pretreatment, and particle size) were evaluated in the supercritical CO2 extraction of oil from oats (Avena sativa L.), by the responses obtained using a Plackett–Burman statistical design, including the mass of oil extracted, main fatty acids (GC–MS), polyphenols (HPLC), antioxidant activity (ORAC), and total phenolic content (TPC). For the mass of oil extracted, pressure (55 MPa) and particle size (>250 μm) were significant, with a highest yield of 0.800 g/20 g oat. Oleic acid was the most abundant fatty acid (45–53%), followed by linoleic (36–42%) and palmitic (12–16%). Fatty acid composition was significantly affected by particle size, pretreatment, and temperature. The polyphenols vanillin (43.33 μg/g oil), ferulic acid (53.6 μg/g oil), vanillic acid (0.78 μg/g oil), and coumaric acid (2.2 μg/g oil) were detected. Pretreatment and high temperature were significant for increasing the polyphenol content of the oil fractions. TPC was also increased by temperature and pretreatment (97.1 mg gallic acid equivalents/100 g oil) and showed a good correlation with polyphenols (R2 = 82%). Additionally, ORAC antioxidant activity (265 mmol Trolox equivalents/g oil) was significantly affected by pretreatment, temperature (80 °C), and particle size (>250 μm).

A Study on effect of formulation variable on durg relese pattern of extended release matrix tablet by using Plackett burman statistical design

A Study on effect of formulation variable on durg relese pattern of extended release matrix tablet by using Plackett burman statistical design

Optimization of enzymatic saccharification of fucoidan and alginate from brown seaweed using fucoidanase and alginate lyase from the marine fungus Dendryphiella arenaria

Fucoidanase and alginate lyase are promising biocatalysts for several biotechnological applications. The sequentially extracted fucoidan and alginate from the brown macroalgae Sargassum latifolium were used for the optimization of a cost-effective culture medium for fucoidanase and alginate lyase production by the marine fungus Dendryphiella arenaria. Plackett–Burman statistical design was conducted for initial determination of the importance of 11 independent variables on enzyme potentiation, and the significant variables were further optimized using Box–Behnken design. The optimum conditions for fucoidanase production were fucoidan (1.5% w/v), NaCl (1.5%), urea (0.3%), and incubation period (2 days), which gives ~ 4 U mL−1 of crude fucoidanase. While, alginate (1.5% w/v), NaCl (4%), NH4Cl (0.3%), and incubation period (6 days) were the optimum conditions that enhanced alginate lyase production to ~ 24 U mL−1. Additionally, a new protocol for the enzymatic saccharification of fucoidan and alginate was optimized using Box–Behnken design with respect to substrate concentration, enzyme dosage, and temperature. The enzymatic saccharification of citric acid-extracted fucoidan gave a maximum yield of reducing sugar 365 mg g−1 fucoidan, while the alkali-extracted alginate gave 439.66 mg g−1 alginate. The results showed that the two enzymes could be exploited for the efficient production of reducing sugars from fucoidan and alginate, which are the key substrate for producing biofuels from brown macroalgal biomass.

Optimization of Manufacturing Process of Traditional Algerian Jben Cheese Using Response Surface Methodology (RSM)

The Aim of this study was to develop an optimized method for manufacturing process of traditional Algerian Jben cheese, using response surface methodology (RSM). In order to develop the objective method of making this traditional cheese, several factors have been studied and a Plackett-Burman statistical design was applied. The effects of the four screened factors (enrichment with milk powder, 10 - 20 g/l; pH of milk, 5.75 - 6.75, enzymatic extract dose, 0.5 - 1.5 ml and coagulation temperature 40 - 60 °C) on the response were investigated, using a Box-Behnken statistical design. Multiple regression analysis was used so that experimental data fits to a second-order polynomial equation. This multiple analysis showed that the model explains about 90.73% of the variation. Based on statistical results, it can be noticed that enrichment with milk powder and pH of milk (Ë‚0.0001***) were highly significant factor influincing cheese yield. The optimal production parame-ters that maximized cheese product (20 g/l enrichment with milk powder, 5.75 pH of milk, 1.29 ml enzymatic extract dose and 60°C coagulation temperature) and the maximal predicted cheese yield (52.68 % ) were found out through response surface methodology. Under these conditions, a verification experiment was carried out and cheese yield was found to be 49.46 %. The overall percentage of agreement for the experimental results (more than 93 % validity) with the predicted values indicates the validation of the statistical model and the success of the optimization process.

Enhanced halophilic lipase secretion by Marinobacter litoralis SW-45 and its potential fatty acid esters release.

An approach was made to enhance the halophilic lipase secretion by a newly isolated moderate halophilic Marinobacter litoralis SW-45, through the statistical optimization of Plackett-Burman (PB) experimental design and the Face Centered Central Composite Design (FCCCD). Initially, PB statistical design was used to screen the medium components and process parameters, while the One-factor-at-a-time technique was availed to find the optimum level of significant parameters. It was found that MgSO4 · 7H2 O, NaCl, agitation speed, FeSO4 · 7H2 O, yeast extract and KCl positively influence the halophilic lipase production, whereas temperature, carbon source (maltose), inducer (olive oil), inoculum size, and casein-peptone had a negative effect on enzyme production. The optimum level of halophilic lipase production was obtained at 3.0 g L-1 maltose, 1% (v/v) olive oil, 30 °C growth temperature and 4% inoculum volume (v/v). Further optimization by FCCCD was revealed 1.7 folds improvement in the halophilic lipase production from 0.603 U ml-1 to 1.0307 U ml-1 . Functional and biochemical characterizations displayed that the lipase was significantly active and stable in the pH ranges of 7.0-9.5, temperature (30-50 °C), and NaCl concentration (0-21%). The lipase was maximally active at pH 8.0, 12% (w/v) NaCl, and 50 °C temperature. Besides, M. litoralis SW-45 lipase was found to possess the promising industrial potential to be utilized as a biocatalyst for the esterification.

Determining the effect of trace elements on biohydrogen production from fruit and vegetable wastes

Trace elements are one of the important parameters for dark fermentative H2 production because they work as co-factors in H2 formation biochemistry. Lack or excess of trace element and its concentrations could be an important reason for the low yield of H2 production. In this study, the effects of 11 different trace elements (Fe, Ni, Zn, Co, Cu, Mn, Al, B, Se, Mo and W) were tested at two levels in terms of biohydrogen production from Fruit and Vegetable Wastes (FVW) with Biochemical Hydrogen Potential (BHP) Tests using Plackett-Burman statistical design. 1.1–2.8 times enhancement of biohydrogen production was determined with its addition. The most effective trace elements were found as Zn and Ni. In order to reveal the resident microbial flora, Denaturing Gradient Gel Electrophoresis (DGGE) analysis was carried out on all BHP effluent samples. Results of DGGE analysis, four microbial sequences evaluated as Clostridium sp., Clostridium baratii, Uncultured bacterium, Uncultured Streptococcus sp., and their similarity rates were 99%, 100%, 89%, 98%, respectively.

Optimization of the in situ transesterification step for biodiesel production using biomass of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil

Abstract The in situ (one-step) acid-catalyzed transesterification step for conversion to biodiesel of biomass from oleaginous yeast Yarrowia lipolytica grown on waste cooking oil (WCO) is studied. The process yield of biodiesel was optimized by investigating effects of various parameters, namely, biomass, methanol, chloroform, catalyst, temperature, time and sonication. A Plackett-Burman statistical design of experiments revealed that biomass is the most significant factor influencing biodiesel (FAME, fatty acid methyl ester) production. Subsequently, a one variable design (OVD) of experiments for increased biomass loadings showed higher yields of FAME with no additional requirement of reactants, solvents or special equipment. The biomass grown on WCO had a lipid productivity of 0.042 g L-1 h−1 and 4 g of this loading gave a high FAME yield of 0.88 g in 8 h at 50 °C with methanol: chloroform (10:1) and acid catalyst (0.2 M H2SO4,1.0 ml g−1). The FAME profile had desirable amounts of saturated (32.81%), monounsaturated (36.41%), polyunsaturated (30.59%) methyl esters. The predicted and experimentally determined physico-chemical properties of FAME were found in accordance with specified international standards. Thus, the direct one-pot in situ transesterification reaction using Y. lipolytica biomass grown on WCO provides a high yield of biodiesel with potential applicability while simultaneously addressing the management of this pollutant.

Optimisation of critical medium components and culture conditions for enhanced biomass and lipid production in the oleaginous diatom Navicula phyllepta: a statistical approach.

Diatoms hold great promise as potential sources of biofuel production. In the present study, the biomass and lipid production in the marine diatom Navicula phyllepta, isolated from Cochin estuary, India and identified as a potential biodiesel feedstock, were optimized using Plackett-Burman (PB) statistical experimental design followed by central composite design (CCD) and response surface methodology (RSM). The growth analyses of the isolate in different nitrogen sources, salinities and five different enriched sea water media showed the best growth in the cheapest medium with minimum components using urea as nitrogen source at salinity between 25 and 40gkg-1. Plackett-Burman experimental analyses for screening urea, sodium metasilicate, sodium dihydrogen phosphate, ferric chloride, salinity, temperature, pH and agitation influencing lipid and biomass production showed that silicate and temperature had a positive coefficient on biomass production, and temperature had a significant positive coefficient, while urea and phosphate showed a negative coefficient on lipid content. A 24 factorial central composite design (FCCD) was used to optimize the concentration of the factors selected. The optimized media resulted in 1.62-fold increase (64%) in biomass (1.2±0.08gL-1) and 1.2-fold increase (22%) in estimated total lipid production (0.11±0.003gL-1) compared to original media within 12days of culturing. A significantly higher biomass and lipid production in the optimized medium demands further development of a two-stage strategy of biomass production followed by induction of high lipid production under nutrient limitation or varying culture conditions for large-scale production of biodiesel from the marine diatom.

Optimization of Cellulase Production by Halobacillus sp. QLS 31 Isolated from Lake Qarun, Egypt

A halophilic cellulase-producing bacterium was isolated from a sediment sample collected from Lake Qarun (Fayoum Province, Egypt). Molecular identification based on 16S rDNA amplification and sequencing revealed 99% homology with Halobacillus sp. and hence was designated as Halobacillus sp. QLS 31. Medium composition and culture conditions were optimized for enhancing the production of cellulase enzyme using the Plackett-Burman statistical design. Ten variables were evaluated for their influence on cellulase production. Carboxymethyl cellulose (CMC), zinc sulfate (ZnSO4), and inoculum size were found to exert a significant effect on cellulase productivity by Halobacillus sp. QLS 31. The maximum specific activity of cellulase enzyme was 48.08U/mg. Following the predicted conditions, a 7.5-fold increase in cellulase specific activity (175.47U/mg) was achieved compared to the basal medium (23.19U/mg) under the following optimized conditions: temperature (30°C), fermentation time (2days ), pH value (9), CMC concentration (1%), inoculum size (1%), yeast extract concentration (0.1%), ammonium sulfate ((NH3)2SO4) concentration (0.1%), sodium chloride (NaCl) concentration (20%), and metal inducers: ZnSO4 (0.1%) and Ca/Mg ratio (0.01%). Thus, the results of this study provide an important basis for more efficient, cheap industrial cellulase production from halophilic Halobacillus sp. QLS 31.

Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H

In this present study, a newly isolated strain, Streptomyces sp. NEAE-H, capable of producing high amount of black extracellular melanin pigment on peptone-yeast extract iron agar and identified as Streptomyces glaucescens NEAE-H. Plackett–Burman statistical design was conducted for initial screening of 17 independent (assigned) variables for their significances on melanin pigment production by Streptomyces glaucescens NEAE-H. The most significant factors affecting melanin production are incubation period, protease-peptone and ferric ammonium citrate. The levels of these significant variables and their interaction effects were optimized by using face-centered central composite design. The maximum melanin production (31.650 μg/0.1 ml) and tyrosinase activity (6089.10 U/ml) were achieved in the central point runs under the conditions of incubation period (6 days), protease-peptone (5 g/L) and ferric ammonium citrate (0.5 g/L). Melanin pigment was recovered by acid-treatment. Higher absorption of the purified melanin pigment was observed in the UV region at 250 nm. It appeared to have defined small spheres by scanning electron microscopy imaging. The maximum melanin yield was 350 mg dry wt/L of production medium. In vitro anticancer activity of melanin pigment was assayed against skin cancer cell line using MTT assay. The IC50 value was 16.34 ± 1.31 μg/ml for melanin and 8.8 ± 0.5 μg/ml for standard 5-fluorouracil.