The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g(-1) and 211 ± 0.3% U g(-1) of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.
This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g(-1)), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g(-1)) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid).
The culture conditions for gibberellic acid (GA3) production by the fungus Penicillium variable (P. variable) was optimized using a statistical tool, response surface methodology (RSM). Interactions of culture conditions and optimization of the system were studied using Box-Behnken design (BBD) with three levels of three variables in a batch flask reactor. Experimentation showed that the model developed based on RSM and BBD had predicted GA3 production with R(2) = 0.987. The predicted GA3 production was optimum (31.57 mg GA3/kg substrate) when the culture conditions were at 7 days of incubation period, 21% v/w of inoculum size, and 2% v/w of olive oil concentration as a natural precursor. The results indicated that RSM and BBD methods were effective for optimizing the culture conditions of GA3 production by P. variable mycelia.
An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.
The response surface method was applied in this study to improve cellulase production from oil palm empty fruit bunch (OPEFB) by Botryosphaeria rhodina. An experimental design based on a two-level factorial was employed to screen the significant environmental factors for cellulase production. The locally isolated fungus Botryosphaeria rhodina was cultivated on OPEFB under solid-state fermentation (SSF). From the analysis of variance (ANOVA), the initial moisture content, amount of substrate, and initial pH of nutrient supplied in the SSF system significantly influenced cellulase production. Then the optimization of the variables was done using the response surface method according to central composite design (CCD). Botryosphaeria rhodina exhibited its best performance with a high predicted value of FPase enzyme production (17.95 U/g) when the initial moisture content was at 24.32%, initial pH of nutrient was 5.96, and 3.98 g of substrate was present. The statistical optimization from actual experiment resulted in a significant increment of FPase production from 3.26 to 17.91 U/g (5.49-fold). High cellulase production at low moisture content is a very rare condition for fungi cultured in solid-state fermentation.
Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates which could be used in designing enzyme-based sensors for detecting OP compounds. In the present work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme-chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased Km value by over 50%, increased Vmax and Kcat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2-12) and temperature (25-90°C) was also achieved. The method worked in the 0 to 1050 nM concentration ranges, and had a detection limit as low as 5 × 10(-11) M.
Hydrogen production by cyanobacteria could be one of the promising energy resources in the future. However, there is very limited information regarding the kinetic modeling of hydrogen production by cyanobacteria available in the literature. To provide an in-depth understanding of the biological system involved during the process, the Haldane's noncompetitive inhibition equation has been modified to determine the specific hydrogen production rate (HPR) as a function of both dissolved CO2 concentration (CTOT) and oxygen production rate (OPR). The highest HPR of 15 [Formula: see text] was found at xCO2 of 5% vol/vol and the rate consequently decreased when the CTOT and OPR were 0.015 k mol m(-3) and 0.55 mL h(-1), respectively. The model provided a fairly good estimation of the HPR with respect to the experimental data collected.
Statistically designed experiments were used in developing a low-serum medium for the production of a diagnostic monoclonal antibody against congenital adrenal hyperplasia using hybridoma 192. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate, and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco's modified Eagle's medium (DMEM; containing 4 mM L-glutamine, 1% antibiotic-antimycotic agent) supplemented with 0.4% by volume fetal bovine serum (FBS), 311.8 mM ferric citrate, 17.3 nM sodium selenite, and 4.5 mM zinc sulfate (LSD) was found to support the growth of the hybridoma. Specific cell growth rate in the LSD (0.033 ± 0.001/h) was slightly lower than in the control medium (i.e., basal DMEM supplemented with 2% FBS; 0.0045 ± 0.003/h). Nevertheless, the specific MAb production rate for LSD was higher (0.057 ± 0.015 pg/cell · h versus 0.004 ± 0.002 pg/cell · h in LSD and control, respectively). The antibody produced in the LSD showed high specificity and no cross-reactivity with the other structural resemblance's steroid hormones, revealing no structural changes owing to the new medium formulation developed. The new medium formulation effectively reduced the medium cost by up to 64.6%.
Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K2HPO4 along with three additional nutrients (yeast extract, MgSO4, and NH4Cl) were identified to form the basal medium which was further analyzed by Plackett-Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH4Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600 nm) cell density of 7.5 using 23.7 g/L of tryptone, 8.8 g/L of yeast extract, and 0.7 g/L of NH4Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.
This study aimed to investigate the effects of the ultrasound-assisted extraction conditions on the yield, specific activity, temperature, and storage stability of the pectinase enzyme from guava peel. The ultrasound variables studied were sonication time (10-30 min), ultrasound temperature (30-50 °C), pH (2.0-8.0), and solvent-to-sample ratio (2:1 mL/g to 6:1 mL/g). The main goal was to optimize the ultrasound-assisted extraction conditions to maximize the recovery of pectinase from guava peel with the most desirable enzyme-specific activity and stability. Under the optimum conditions, a high yield (96.2%), good specific activity (18.2 U/mg), temperature stability (88.3%), and storage stability (90.3%) of the extracted enzyme were achieved. The optimal conditions were 20 min sonication time, 40 °C temperature, at pH 5.0, using a 4:1 mL/g solvent-to-sample ratio. The study demonstrated that optimization of ultrasound-assisted process conditions for the enzyme extraction could improve the enzymatic characteristics and yield of the enzyme.
This study aimed to work out a simple and high-yield procedure for the immobilization of horseradish peroxidase on silver nanoparticle. Ultraviolet-visible (UV-vis) and Fourier-transform infrared spectroscopy and transmission electron microscopy were used to characterize silver nanoparticles. Horseradish peroxidase was immobilized on β-cyclodextrin-capped silver nanoparticles via glutaraldehyde cross-linking. Single-cell gel electrophoresis (Comet assay) was also performed to confirm the genotoxicity of silver nanoparticles. To decrease toxicity, silver nanoparticles were capped with β-cyclodextrin. A comparative stability study of soluble and immobilized enzyme preparations was investigated against pH, temperature, and chaotropic agent, urea. The results showed that the cross-linked peroxidase was significantly more stable as compared to the soluble counterpart. The immobilized enzyme exhibited stable enzyme activities after repeated uses.
Microbial content formed in bioreactors plays a significant role in the anaerobic process. Therefore, the physicochemical characteristics of microbial content in a modified anaerobic inclining-baffled reactor (MAI-BR) treating recycled paper mill effluent (RPME) were investigated using Fourier transform infrared (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric (TG), and derivative thermogravimetric (DTG) analyses, scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), Brunauer-Emmett-Teller (BET), and surface area analyzer. FTIR spectra revealed that the microbial content had stronger characteristic peaks corresponding to alcohols, water, lipids carbohydrates, proteins, and mineral compounds. Calcite, muscovite, and lepidolite were the prevalent mineral phases found by XRD analysis. The elemental of these minerals like C, Ca, N, O, and Si was confirmed by XPS results. The microbial content samples from each compartment showed similar thermal behavior. SEM images showed that straight rod-shaped and Methanosaeta-like microorganisms were predominant, whereas C, O, and Ca were noticed by EDS on the surface of granules. The BET surface areas and pores of granules are found to decline throughout the reactor's compartment, where Compartment 1 had the largest values. Thus, the findings of this study establish further understanding of the physicochemical properties of microbial content formed in MAI-BR during the RPME treatment.
This research comprehends iron-oxide nanoparticle (IONP) production, the apparent metallic nanostructure with unique superparamagnetic properties. Durian-rind-extract was utilized to synthesize IONP and the color of reaction mixture becomes dark brown, indicated the formation of IONPs and the peak was observed at ∼330 nm under UV-visible spectroscopy. The morphological observation under high-resolution microscopies has revealed the spherical shape and the average size (∼10 nm) of IONP. The further support was rendered by EDX-analysis showing apparent iron and oxygen peaks. XRD results displayed the crystalline planes with (110) and (300) planes at 2θ of 35.73° and 63.53°, respectively. XPS-data has clearly demonstrated the presence of Fe2P and O1s peaks. The IONPs were successfully capped by the polyphenol compounds from durian-rind-extract as evidenced by the representative peaks between 1633 and 595 cm-1 from FTIR analysis. The antimicrobial potentials of IONPs were evidenced by the disk-diffusion assay. The obtained results have abundant attention and being actively explored owing to their beneficial applications.
The study was conducted to determine anti-tyrosinase and antioxidant activities of the extracted collagen hydrolysate (CH) derived from Malaysian jellyfish, Rhopilema hispidum. Collagen was extracted using 1:1 (w:v) 0.1 M NaOH solution at temperature 25 °C for 48 hr followed by treatment of 1:2 (w:v) distilled water for another 24 hr and freeze-dried. The extracted collagen was hydrolyzed using papain at optimum temperature, pH and enzyme/substrate ratio [E/S] of 60 °C, 7.0 and 1:50, respectively. CH was found to exhibit tyrosinase inhibitory activity, DPPH radical scavenging and metal ion-chelating assays up to 64, 28, and 83%, respectively, after 8 hr of hydrolysis process. The molecular weight of CH was found <10 kDa consisting of mainly Gly (19.219%), Glu (10.428%), and Arg (8.848%). The UV-visible spectrum analysis showed a major and minor peak at 218 and 276 nm, accordingly. The FTIR spectroscopy confirmed the amide groups in CH. The SEM images demonstrated spongy and porous structure of CH. In the cytotoxicity study, CH has no cytotoxicity against mouse embryonic 3T3 fibroblast cell line with IC50 value >500 µg/ml. Results revealed that the CH generated from this study has a potential to be developed as active ingredient in cosmeceutical application.
A chemical method to synthesize amorphous silica nanoparticles from the incinerated paddy straw has been introduced. The synthesis was conducted through the hydrolysis by alkaline-acidic treatments. As a result, silica particles produced with the sizes were ranging at 60-90 nm, determined by high-resolution microscopy. The crystallinity was confirmed by surface area electron diffraction. Apart from that, chemical and diffraction analyses for both rice straw ash and synthesized silica nanoparticles were conducted by X-ray diffraction and Fourier-transform infrared spectroscopy. The percentage of silica from the incinerated straw was calculated to be 28.3. The prominent surface chemical bonding on the generated silica nanoparticles was with Si-O-Si, stretch of Si-O and symmetric Si-O bonds at peaks of 1090, 471, and 780 cm-1, respectively. To confirm the impurities of the elements in the produced silica, were analyzed using X-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy. The stability of silica nanoparticles was investigated using thermogravimetric analysis and zeta potential. The measured size from zeta potential analysis was 411.3-493 nm and the stability of mass reduction was located at 200 °C with final amount of mass reduced ∼88% and an average polydispersity Index was 0.195-0.224.
A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.
This study aimed at purification of phycocyanin (PC) from Phormidium tergestinum using an aqueous two-phase system (ATPS) comprised of polyethylene glycol (PEG) and salts. The partitioning efficiency of PC in ATPS and the effect of phase composition, pH, crude loading, and neutral salts on purification factor and yield were investigated. Results showed that PC was selectively partitioned toward bottom phase of the system containing potassium phosphate. Under optimum conditions of 20% (w/w) PEG 4000, 10% (w/w) potassium phosphate, 20% (v/v) crude load at pH 7, with addition of 0.5% (w/w) NaCl, PC from P. tergestinum was partially purified up to 5.34-fold with a yield of 87.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of PC was ∼19 kDa. Results from this study demonstrated ATPS could be used as a potential approach for the purification of PC from P. tergestinum.
Microalgal carbohydrate is considered one of the potential feedstock for biofuel produced via the bioconversion process. However, the current cultivation process using commercial medium exhibited low biomass production and its carbohydrate productivity which become a bottleneck for sustainable microalgal-carbohydrate-based biofuel production. Thus, the objective of this study is to assess the utilization of industrial waste including molasses and glycerol on the Halochlorella rubescens and Tetraselmis suecica growth as well as its carbohydrate content under different cultivation modes such as autotrophic, heterotrophic and photoheterotrophic conditions. From this study, the highest maximum biomass of H. rubenscens and T. suecica of 0.653 ± 0.009 and 0.669 ± 0.01gL-1 were obtained when the cultivation was performed under photoheterotrophic using molasses. High carbohydrate content of H. rubescens and T. seucica of 56.81 ± 0.39% and 71.52 ± 0.03% with glucose represent the dominant sugar was observed under this condition. The growth kinetic model of the analysis indicated that Huang and Gompertz Models described well the growth of H. rubescens and T. suecica under photoheteroptroph condition with a high significant R2 of 0.99. The information generated could be beneficial for the future development of low-cost microalgal cultivation media formulation for future microalgal carbohydrate-based products such as bioethanol.
Microalga biomass has been recognized as a sustainable bio-product to replace terrestrial biomass in biofuel production. The microalga industry has high operating costs, specifically on harvesting and biomass recovery. Therefore, the development of an efficient harvesting method is crucial to the minimization of production cost. A statistical analysis through response surface methodology was used to investigate the optimization of harvesting efficiency using alum and chitosan as a coagulant. Growth rate and biomass productivity were also determined. This research revealed that the harvesting efficiency using alum was 99.3%, with optimum dosage and pH of 177.74 mg L(-1) and 8.24, respectively. Chitosan achieved 94.2% biomass recovery at an optimal dosage of 169.95 mg L(-1) at pH of 12. Moreover, Botryococcus sp. achieved the maximum growth of 0.7551 µmax d(-1), with an average total biomass productivity of 9.81 mg L(-1) d(-1) in domestic wastewater. Overall, this study shows that both alum and chitosan coagulants have great potential for efficient microalgal biomass recovery. It suggests that domestic wastewater as a potential growth medium for the large-scale production of microalga biomass.
Antibiofilm polymers have the ability to inhibit bacterial biofilm formation, which is known to occur ubiquitously in the environment and pose risks of infection. In this study, production of P(3HB-co-4HB) copolymer and antimicrobial yellow pigment from Cupriavidus sp. USMAHM13 are enhanced through medium optimization. Before the improvement of yellow pigment production, screening for the best additional supplement was performed resulting in high-yield yellow pigmentation using yeast extract with optimum concentration of 2 g/L. Effects of different concentrations of 1,4-butanediol, ammonium acetate, and yeast extract were studied using central composite design. Under optimal conditions, 53 wt% of polyhydroxyalkanoate (PHA) content, 0.35 g/L of pigment concentration, and 5.87 g/L of residual biomass were achieved at 0.56 wt% C of 1,4-butanediol, 1.14 g/L of ammonium acetate, and 2 g/L of yeast extract. Antibiofilm tests revealed that the yellow pigment coated on P(3HB-co-4HB) copolymer had significant effect on the inhibition of bacteria proliferation and colonization from 6 hr onward reaching 100% inhibition by 12 hr, hence effectively inhibiting the biofilm formation.