In this study, the dried biomass of four marine algae, namely Porphyra sp., Gracilaria bursa-pastoris, Undaria pinnatifida and Laminaria sp., were screened for their ability to remove methylene blue (MB) dye from aqueous solutions. Statistical approaches of the Plackett-Burman Design (PBD) and Box-Behnken Design (BBD) were applied to optimize different environmental conditions in order to achieve the maximum MB removal percentage by Gracilaria bursa-pastoris. The biosorbent was characterized before and after adsorption process using FTIR, XRD and SEM analysis. Additionally, isotherms, kinetics and thermodynamics studies were conducted to investigate the adsorption behavior of the adsorbent. The results showed that Gracilaria bursa-pastoris achieved the highest dye removal efficiency (98.5 %) compared to 96.5 %, 93.5 % and 93.9 % for Undaria pinnatifida, Porphyra sp. and Laminaria sp., respectively. PBD analysis revealed that the agitation speed, pH, and biomass dose were found to be the significant parameters affecting MB removal onto Gracilaria dried biomass. According to the BBD results, the maximum dye removal percentage (99.68 %) was obtained at agitation speed of 132 rpm, pH 7 and biomass dose of 7.5 g/L. FTIR, XRD and SEM analysis demonstrated the participation of several functional groups in the adsorption process and changes in the cell surface morphology of the adsorbent following the dye adsorption. The adsorption isotherms showed better fit to Freundlich model (R2 = 0.9891) than the Langmuir, Temkin, and Dubinin-Radushkevich models. The adsorption kinetics were best described by the pseudo-second-order model (R2 = 0.9999), suggesting the chemical interactions between dye ions and the algal biomass. The thermodynamic parameters indicated that the adsorption of MB onto Gracilaria dried biomass was spontaneous, feasible, endothermic and random. These results indicate that dried biomass of Gracilaria bursa-pastoris is an attractive, environmentally friendly, cheap and effective agent for MB dye removal from environmental discharges.

Oleaginous yeast can produce lipids while degrading phenol in wastewater treatment. In this study, a Plackett-Burman Design (PBD) was adopted to identify key factors of phenol degradation and lipid production using R toruloides 9564T. While temperature, inoculum size, and agitation were significant for both the processes (p < 0.05), pH and incubation were significant for lipid production, and phenol removal, respectively. Results from four factors (pH, temperature, inoculum size, and incubation period) central composite design (CCD) experiment were used to formulate quadratic and genetic algorithm-optimized ANN models. The reduced quadratic model for phenol degradation (R2: 0.993) and lipid production (R2: 0.958) were marginally inferior to ANN models (R2: 0.999, 0.982, respectively) on training sets. Multi-objective optimization with equal importance suggests phenol degradation between 106.4 and 108.76%, and lipid production of 0.864-0.903 g/L, by polynomial and ANN models. Complete phenol degradation (100%) and 3.35-fold increment (0.918 g/L) in lipid production were obtained at pH 6.07, inoculum size 14.68% v/v, at 29.5 °C in 92.17 h experimentally.

Ammonia recovery from wastewater has positive environmental benefits, avoiding eutrophication and reducing production energy consumption, which is one of the most effective ways to manage nutrients in wastewater. Specifically, ammonia recovery by membrane distillation has been gradually adopted due to its excellent separation properties for volatile substances. However, the global optimization of direct contact membrane distillation (DCMD) operating parameters to maximize ammonia recovery efficiency (ARE) has not been attempted. In this work, three key operating factors affecting ammonia recovery, i.e., feed ammonia concentration, feed pH, and DCMD running time, were identified from eight factors, by a two-level Plackett-Burman Design (PBD). Subsequently, Box-Behnken design (BBD) under the response surface methodology (RSM) was used to model and optimize the significant operating parameters affecting the recovery of ammonia though DCMD identified by PBD and statistically verified by analysis of variance (ANOVA). Results showed that the model had a high coefficient of determination value (R2 = 0.99), and the interaction between NH4Cl concentration and feed pH had a significant effect on ARE. The optimal operating parameters of DCMD as follows: NH4Cl concentration of 0.46 g/L, feed pH of 10.6, DCMD running time of 11.3 h, and the maximum value of ARE was 98.46%. Under the optimized conditions, ARE reached up to 98.72%, which matched the predicted value and verified the validity and reliability of the model for the optimization of ammonia recovery by DCMD process.

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme\'s catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60-80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

A reliable and greener alternative to the usual extraction methods is reported for the determination of pesticide residues in soybeans. This novel approach combines the classical QuEChERS extraction method with a DLLME (dispersive liquid-liquid microextraction) step, utilizing a deep eutectic solvent (DES) - camphor: hexanoic acid (1:1 molar ratio) - as the microextraction solvent. This DES has never been employed in pesticide analysis by gas chromatography-mass spectrometry of complex matrices like soybeans. A Plackett-Burman screening design was employed to optimize sample preparation variables of QuEChERS (amount of sodium chloride and magnesium sulfate, and amount of PSA and C18 sorbents) and DLLME (pH of medium, amount of sodium chloride, and volume of microextraction solvent). This design allowed for a systematic evaluation of the impact of each parameter on the method\'s performance. The optimized method was evaluated using a certified reference material and commercial samples of soybeans. The method exhibited high accuracy and precision for most of the analytes under study, demonstrating its applicability for pesticide residue analysis in soybeans. To assess the greenness and practicality of the developed method, the Analytical Greenness (AGREE) and Blue Applicability Grade Index (BAGI) metric systems were employed, respectively. Overall, the proposed QuEChERS-DLLME method using a DES solvent is a reliable and greener alternative to conventional extraction methods for the determination of pesticide residues in soybeans. Its high performance, coupled with its environmental friendliness, makes it a promising tool for food safety analysis.

In this study, polymeric ionic liquids featuring different functional moieties were applied as sorbent coatings in direct-immersion solid-phase microextraction (DI-SPME) for the extraction of 2-methylimidazole (2-MI) and 4-methylimidazole (4-MI) from açaí-based food products followed by gas chromatography-mass spectrometry (GC-MS) analysis. The analytical method was optimized using a sequential experimental design. Variables used in GC-MS such as desorption time, as well as for SPME-DI, including extraction time, extraction temperature, incubation time of extraction, amount of NaCl in the extract, and stirring rate, were optimized. The fitness-for-purpose of the method was verified by the linearity of matrix-matched calibration curves (R2 ≥ 0.9921), adequate recoveries (81.7-89.7 %), and precision (relative standard deviations ≤11.2 %). The method was applied to twenty-five samples of açaí-based food products. 4-MI was found in four samples whereas 2-MI was not detected above the limit of detection. The method was found to be suitable for quality control analysis.

Cellulolytic bacteria were isolated from yak dung samples collected from different habitats of Sikkim, India. Isolate YE16 from the Yumthang Valley sample showed highest enzyme activity of 7.68 U/mL and was identified as Bacillus sp., which has a sequence similarity of 96.15% with B. velezensis. One factor at a time (OFAT) analysis revealed that an acidic pH of 5 with 37 °C temperature was optimum for maximum enzyme production after 36 hrs of incubation (13.88 U/mL), which was further increased after statistical optimization (34.70 U/mL). Media optimization based on response surface methodology predicted that Carboxymethyl cellulose (CMC) and MgSO4 at concentrations of 30 g/L and 0.525 g/L, respectively, at pH 5.5 to show CMCase activity of 30.612 U/mL, which was consistent with the observed value of 30.25 U/mL and confirmed the model. The crude enzyme also efficiently hydrolyzed alkaline pretreated sugarcane bagasse, releasing 7.09 g/L of glucose equivalent with an ethanol production of 3.05 g.

This study aimed to produce enzymes (beta (β)-mannanase using a recombinant Aspergillus sojae AsT3 and inulinase using Aspergillus niger A42) and oligosaccharides (mannooligosaccharides (MOS), fructooligosaccharides (FOS)) using coffee waste, ground coffee, and coffee extract by solid-state fermentation (SSF). Plackett-Burman Design (PBD) was used to create a design for enzyme production with four different parameters (temperature, pH, solid-to-liquid ratio (SLR), and mix with coffee wastes and ground coffee). The highest β-mannanase and inulinase activities were 71.17 and 564.07 U/mg of protein respectively. Statistical analysis showed that the temperature was statistically significant for the production of both enzymes (P < 0.05). The produced enzymes were utilized in French Pressed coffee extracts to produce oligosaccharides. As a result of the enzymatic hydrolyzation, the highest mannobiose, mannotriose, mannotetraose, and total MOS levels were 109.66, 101.11, 391.02, and 600.64 ppm, respectively. For the FOS production, the maximal 1,1,1-kestopentaose was 38.34 ppm. Consequently, this study demonstrates that a recombinant Aspergillus sojae AsT3 β-mannanase and Aspergillus niger A42 inulinase produced from coffee wastes and ground coffee can be used in coffee extracts to increase the amount of oligosaccharides in coffee extracts.

Mycelia products enhance edible mushrooms in alignment with future sustainability trends. To meet forthcoming market demands, the morphology of mycelial pellets was optimized for direct consumption. Among ten commercial edible mushrooms in Taiwan, Pleurotus sp. was selected for its rapid growth and was identified via an internal transcribed spacer sequence. A combination of Plackett-Burman design and Taguchi\'s L9(34) orthogonal table revealed the optimal formula as potato dextrose broth (2.4%), olive oil (2%), calcium carbonate (0.5%), yeast extract (0.75%), and soy flour (0.5%). This led to a biomass increase to 19.9 ± 1.1 g/L, resulting in a 2.17-fold yield increase. To refine morphology, image processing by ImageJ quantified spherical characteristics. The addition of 0.2 to 1.0% Tween 80 enhanced pellet compaction by over 50%. Dilution of the medium improved uniformity (0.85) and conversion rate (42%), yielding mycelial pellets with 2.10 ± 0.52 mm diameters and a yield of 15.1 ± 0.6 g/L. These findings provide an alternative evaluation and application of edible mycelial pellets as future food.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration, and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the central composite design. The strain produced 20 IU mL-1 of pectinase after 24 h under optimized conditions. The enzyme production kinetics data also confirmed that 24 h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of >65 kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations.