Unoptimized Conditions Research Articles

A Statistical Approach for Production Improvement of a Neutral Protease From a Newly Isolated Strain of Aeromonas Hydrophila

Neutral proteases have a large variety of industrial applications. Therefore, the optimization of their production for the large-scale commercial purpose is critical. The present study was aimed to the optimization of nutritional and physical conditions affecting neutral protease production of Aeromonas hydrophila MSB16, not previously reported for this genus. Four key determinants (pH, temperature, glucose and peptone concentration), were selected based on the results of the one-factor-at-a-time (OFAT) method and then optimized by the response surface methodology (RSM). Maximum enzyme production (229.72 U/ml) which was 2.3 folds higher than that of the OFAT method (99.25 U/ml) and 3.15 times higher than unoptimized conditions (72.86 U/ml) was observed at 30 °C, pH 7, 4.5% w/v glucose and 4.5%w/v peptone by using a rotatable central composite design (RCCD). The results showed that the OFAT and RSM strategies were a useful screening and optimization methods for enhancing the protease production of the strain MSB16, respectively.

High-Performance Forward Osmosis Membranes Used for Treating High-Salinity Oil-Bearing Wastewater

Forward osmosis (FO), which is an emerging osmotic driving membrane separation process, can be directly used in high-salinity oil-bearing (HSOB) wastewater treatment. In this study, the thin film composite (TFC) polyamide FO membranes were fabricated for HSOB wastewater treatment. The impacts of the draw solution (DS) concentration, cross-flow rate, stirring rate, and membrane orientation on membrane performances were researched. The results indicated that the FO operation mode was better to achieve a stable water flux of TFC membranes as 29 L/(m2 h) under a cross-flow rate of 15 cm/s and 3 M NaCl as the DS, which was 61% higher than the water flux in the unoptimized operating conditions (18 L/(m2 h)). The rejection rate of salt and oil was 99% and 96%. In addition, the effect of different oil concentrations of HSOB wastewater was studied, and the results showed that FO performed well to treat HSOB wastewater when the oil concentration was <700 mg/L.

Enhanced production of xylanase by Fusarium sp. BVKT R2 and evaluation of its biomass saccharification efficiency.

Growth of Fusarium sp. BVKT R2, a potential isolate of forest soils of Eastern Ghats on birchwood xylan in mineral salts medium (MSM) under un-optimized conditions of 30°C, pH of 5.0, 150rpm and inoculum size of 5 agar plugs for 7days, yielded titer of 1290U/mL of xylanase (EC 3.2.1.8). The effect of various operating parameters such as different substrates and their concentration, additional carbon and nitrogen sources, incubation temperature, initial pH, agitation and inoculum size on the production of xylanase by Fusarium sp. BVKT R2 was studied in shake flask culture by one factor at a time approach. The same culture exhibited higher production of xylanase (4200U/mL) when grown on birch wood xylan in MSM under optimized conditions with an additional carbon source-sorbitol (1.5%) nitrogen source-yeast extract (1.5%) temperature of 30°C, pH of 5.0, agitation of 200rpm and inoculum of 6 agar plugs for only 5days. There was enhancement in xylanase production under optimized conditions by 3.2 folds over yields under un-optimized conditions. Growth of BVKT R2 culture on locally available lignocelluloses-sawdust, rice straw and cotton stalk-in MSM for 5days released soluble sugars to the maximum extent of 52.76% with respect to sawdust indicating its greater importance in saccharification essential for biotechnological applications.

Optimization of physicochemical parameters influencing the production of prodigiosin from Serratia nematodiphila RL2 and exploring its antibacterial activity.

In the present study, role of various physicochemical parameters influencing the production of antimicrobial pigment prodigiosin from Serratia nematodiphila RL2 was determined and optimized. The pigment-producing strain was isolated and based on molecular characterization (16S rRNA sequencing), was identified as S. nematodiphila RL2. The pigment produced by S. nematodiphila RL2 was characterized by thin layer chromatography (Rf 0.94), spectrophotometrically (λmax 535nm) and identified as prodigiosin. Optimization of production parameters of prodigiosin revealed, nutrient broth medium supplemented with lactose and yeast extract at 1% concentration each, have a positive effect on the bacterial growth (10.25-4.6mg/ml DCW) as well as pigment production (0.46-0.6mg/ml). Prodigiosin production (0.64mg/ml) increases optimally after 46-48h of incubation, at 35°C at pH between 6 and 7 with addition of metal ions such as Uranyl acetate. An increase of 65% in prodigiosin production (0.46-0.76mg/ml) was observed after optimizing the various production parameters than unoptimized conditions. Antimicrobial activity of the prodigiosin was also evaluated and found to be effective antimicrobial agent against bacterial pathogens including Listeria sp., Pseudomonas sp., Yersinia sp. and Shigella sp. Present study indicate that S. nematodiphila RL2 is a potent source of pigment prodigiosin which can be further explored for production of prodigiosin.

Development of fermented oat flour beverage as a potential probiotic vehicle

Dairy products have conventionally been used as carriers of probiotics. However, lactose intolerance, cholesterol, allergenic milk proteins, and the trend towards vegetarianism motivated the search for non-dairy products as potential probiotic carriers. Cereals may represent an excellent choice due to their high nutritional value and consumption all around the world. In the present study, an oat based fermented product ‘probiotic fermented oat flour’ (PFOF) was developed using a probiotic strain Lactobacillus plantarum M-13 and honey. The bacterial isolate L. plantarum M-13 has previously been characterized for several probiotic functional attributes. For PFOF development, process variables, i.e., concentrations of oat flour (8.0% w/v) and honey (3.0% w/v), and incubation time (48 h) were optimized based on a Box-Behnken design. Optimization enhanced the viable cell count of L. plantarum M-13 in PFOF from 14.4 log cfu/ml (unoptimized conditions) to 16.9 log cfu/ml, i.e. by 17.4%. With respect to linear terms, the variable incubation time had the most substantial positive influence on viable cell count of L. plantarum M-13, while with respect to interactive terms, the variables incubation time and honey had the maximum effect. Good viability of L. plantarum M-13 was observed in PFOF over a period of three wk of storage at room temperature and with refrigeration. Furthermore, sugar content decreased and lactic acid content increased during storage. The PFOF remained free of Enterobacteriaceae contamination, although fungal and yeast contaminants were found.

Turning oil palm empty fruit bunch waste into substrate for optimal lipase secretion on solid state fermentation by Trichoderma strains

Abstract This study is aimed to improving extracellular lipase production by Trichoderma strains using oil palm empty fruit bunch (OPEFB) medium on solid state fermentation with the assistance of statistical optimization. Two selected Trichoderma strains (Trichoderma sp.1 and Hypocrea neorufa.1) with high lipase producing potential were employed in a stepwise optimization. Initially, the influence of nutritional (carbon, nitrogen sources, and inducers) and environmental (moisture and temperature) factors on lipase production was evaluated using One-Factor-At-A-time (OFAT) method. Next, three most influential factors (Glucose, Urea and Olive oil) resulting from OFAT were optimized for lipase production by Trichoderma sp.1 using the central composite design under response surface methodology. Experiments conducted under optimum conditions (10.38% (w/w) glucose, 0.86% (w/w) urea, and 7.38% (v/w) olive oil) were further improved the lipase production from 0.195 to 0.390 Ug−1, which is about 2 folds greater than un-optimized condition. This study demonstrated that OPEFB can be utilized as inexpensive substrate for improving the lipase secretion by Trichoderma sp.1.

Statistical Optimization of the Production of alpha-Amylase from Bacillus licheniformis MTCC 1483 Using Paddy Straw as Substrate

?-Amylase has been in increasing demand in industries due to its hydrolytic nature. Solid state fermentation (SSF) is a cost effective method for increasing the enzyme production. In the present study, amylase from Bacillus licheniformis MTCC 1483 was produced in large quantity by solid state fermentation using paddy straw as substrate. Response surface methodology is a useful tool for optimizing many parameters at a time and is used for increasing the amylase production. 8523 IUg-1 of enzyme activity was obtained under optimized conditions which lead to 35 fold increase in the yield of amylase from unoptimized condition.

Identification and characterization of Streptomyces flavogriseus NJ-4 as a novel producer of actinomycin D and holomycin.

This paper is the first public report that Streptomyces flavogriseus can produce both actinomycin D and holomycin. The actinomycete strain NJ-4 isolated from the soil of Nanjing Agricultural University was identified as S. flavogriseus. This S. flavogriseus strain was found for the first time to produce two antimicrobial compounds that were identified as actinomycin D and holomycin. GS medium, CS medium and GSS medium were used for the production experiments. All three media supported the production of actinomycin D, while holomycin was detected only in GS medium and was undetectable by HPLC in the CS and GSS media. The antimicrobial activity against B. pumilus, S. aureus, Escherichia coli, F. moniliforme, F. graminearum and A. niger was tested using the agar well diffusion method. Actinomycin D exhibited strong antagonistic activities against all the indicator strains. Holomycin exhibited strong antagonistic activities against B. pumilus, S. aureus and E. coli and had antifungal activity against F. moniliforme and F. graminearum but had no antifungal activity against A. niger. The cell viability was determined using an MTT assay. Holomycin exhibited cytotoxic activity against A549 lung cancer cells, BGC823 gastric cancer cells and HepG2 hepatocellular carcinoma cells. The yield of actinomycin D from S. flavogriseus NJ-4 was 960 mg/l. S. flavogriseus NJ-4 exhibits a distinct capability and has the industrial potential to produce considerable yields of actinomycin D under unoptimized conditions.

Xylanase production from Thermomyces lanuginosus VAPS-24 using low cost agro-industrial residues via hybrid optimization tools and its potential use for saccharification

The xylanase production from Thermomyces lanuginosus VAPS-24 has been optimized using OFAT (One factor at a time) approach using agro-industrial substrates. Further, central composite design (CCD) has been employed to optimize various process parameters such as temperature (45–55°C), carbon source concentration (1.5–2.5%), fermentation time (72–120h) and production medium pH (6−8). Maximum xylanase yield after RSM optimization was approximately double (119.91±2.53UmL−1) than un-optimized conditions (61.09±0.91UmL−1). Several hybrid statistical tools such as Genetic Algorithm-Response Surface Methodology (GA-RSM), Artificial Neural Network (ANN), Genetic Algorithm-Artificial Neural Network (GA-ANN) were employed to obtain more optimized process parameters to maximize the xylanase production and observed an increase of 10.50% xylanase production (132.51±3.27UmL−1) as compared to RSM response (119.91±2.53UmL−1). The various pretreated and untreated agricultural residues were subjected to saccharification by using crude xylanase in which the pretreated rice straw yielded maximum fermentable sugars 126.89mgg−1.

Lipase mediated synthesis of rutin fatty ester: Study of its process parameters and solvent polarity

Lipophilization of antioxidants is recognized as an effective strategy to enhance solubility and thus effectiveness in lipid based food. In this study, an effort was made to optimize rutin fatty ester synthesis in two different solvent systems to understand the influence of reaction system hydrophobicity on the optimum conditions using immobilised Candida antartica lipase. Under unoptimized conditions, 52.14% and 13.02% conversion was achieved in acetone and tert-butanol solvent systems, respectively. Among all the process parameters, water activity of the system was found to show highest influence on the conversion in each reaction system. In the presence of molecular sieves, the ester production increased to 62.9% in tert-butanol system, unlike acetone system. Under optimal conditions, conversion increased to 60.74% and 65.73% in acetone and tert-butanol system, respectively. This study shows, maintaining optimal water activity is crucial in reaction systems having polar solvents compared to more non-polar solvents.

Enhanced β-fructofuranosidase biosynthesis by Rhodotorula glutinis using Taguchi robust design method

The aim of this study was to produce β-fructofuranosidase enzyme by Rhodotorula glutinis SO28, using sugar beet (Beta vulgaris) as carbon source due to its high sucrose content and easy availability. β-Fructofuranosidase production was carried out in submerged fermentation. Taguchi orthogonal array (OA) design of experiment (DOE) method was employed for optimization process of β-fructofuranosidase production by R. glutinis SO28. An OA layout of L16 was constructed with five influential factors on β-fructofuranosidase biosynthesis namely, carbon source (sugar beet), initial pH, incubation temperature, agitation speed and incubation time. The average results of β-fructofuranosidase yield obtained from the determined 16 batches were processed with Minitab® 16.2.3 software at “larger is better” as quality character. The results showed that the maximum β-fructofuranosidase activity was obtained as 21.11 ± 0.47 U/mL, which was close to the predicted result (21.78 ± 0.43 U/mL). Consequently, sugar beet can be suggested as an economical substrate for β-fructofuranosidase production. Besides, use of Taguchi DOE enhanced enzyme activity about 3-fold when compared with unoptimized condition.

Alkyl Formate Ester Synthesis by a Fungal Baeyer-Villiger Monooxygenase.

We investigated Baeyer-Villiger monooxygenase (BVMO)-mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ∼80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space-time yield (STY) of 4.2 g L-1 d-1 (3.4 g L-1 d-1 extracted product).

Simultaneous saccharification and fermentation of alkali-pretreated corncob under optimized conditions using cold-tolerant indigenous holocellulase

The present investigation was aimed towards pretreatment optimization of corncob to maximize cellulose and hemicellulose recovery, followed by substrate selection for holocellulase production using psychrotolerant Aspergillus niger SH3. Dilute alkali pretreatment (1.5% NaOH) resulted in higher recovery of cellulose (59.66%) and hemicellulose (28.34%) from corncob, while corn stover proved to be the best substrate for holocellulase production. Further, saccharification was optimized by Box-Behnken design to select the suitable conditions for maximum sugar release from pretreated corncob. The optimum conditions for maximum sugar release were 8% (w/v) substrate loading, 11 FPU/gds enzyme loading at temperature 38 °C and pH 3.0 which resulted in 114.5% higher sugar yield (912 mg/gds of pretreated biomass) as compared with un-optimized conditions (425.35 mg/gds). Theoretical yield of 48.8% ethanol was achieved through simultaneous saccharification and fermentation (SSF) using pretreated corncob. This study illustrates the potential of different corn residues as a promising substrate for bioethanol production.

Optimization of bioprocess variables for production of a thermostable and wide range pH stable carboxymethyl cellulase from Bacillus subtilis MS 54 under solid state fermentation

Vast application spectrum of cellulases especially for bioconversion of lignocellulosics into simple sugars for production of biofuel, chemicals, and other industrial products in an environmentally benign manner, faces bottleneck due to unavailability of process‐apt cellulases, and high production cost of enzymes. Solid state fermentation (SSF) offers several advantages over submerged fermentation (SmF) including the cost‐effective production of enzymes particularly using agricultural residues as substrates. Bacillus subtilis MS 54, a recently isolated bacterium was previously shown to produce thermostable and wide range pH stable cellulase under SmF. This study reports the Design of experiments (DoE) based optimization of process variables for cellulase production from B. subtilis MS 54 under SSF. Plackett–Burman (PB) design and response surface methodology (RSM) were used, respectively, for selection of significant process variables, and for optimization of the selected variables. Three process variables, that is, moisture content, maize bran and almond shell, earmarked based on PB‐design, were optimized by RSM to achieve cellulase yield enhancement by 2.02‐fold as compared with that under unoptimized conditions. Application of maize bran and almond shells as substrates for bacterial cellulase production under SSF is being reported for the first time. Solid‐state fermentation coupled with DoE tools represents a cost‐effective strategy for production of industrial enzymes. © 2017 American Institute of Chemical Engineers Environ Prog, 36: 1123–1130, 2017

Outdoor Growth Characterization of an Unknown Microalga Screened from Contaminated Chlorella Culture.

Outdoor microalgae cultivation process is threatened by many issues, such as pest pollution and complex, changeable weather. Therefore, it is difficult to have identical growth rate for the microalgae cells and to keep their continuous growth. Outdoor cultivation requires the algae strains not only to have a strong ability to accumulate oil, but also to adapt to the complicated external environment. Using 18S rRNA technology, one wild strain Scenedesmus sp. FS was isolated and identified from the culture of Chlorella zofingiensis. Upon contamination by Scenedesmus sp., the species could quickly replace Chlorella zofingiensis G1 and occupy ecological niche in the outdoor column photobioreactors. The results indicated that Scenedesmus sp. FS showed high alkali resistance. It also showed that even under the condition of a low inoculum rate (OD680, 0.08), Scenedesmus sp. FS could still grow in the outdoor raceway pond under a high alkaline environment. Even under unoptimized conditions, the oil content of Scenedesmus sp. FS could reach more than 22% and C16–C18 content could reach up to 79.68%, showing that this species has the potential for the biodiesel production in the near future.

Optimization of cultural conditions for polygalacturonase production by a newly isolated Aspergillus fumigatus R6 capable of retting kenaf

Sequential optimization strategy was used to enhance the production of extracellular polygalacturonase by a newly isolated Aspergillus fumigatus R6 using rice bran as a substrate in solid state conditions. Three significant variables influencing the polygalacturonase production were identified as initial moisture level, temperature and incubation time (P<0.0001). The model established by face-centered central composite design was significant (P<0.05) with high R2 (0.98). The model validity was verified and the optimum conditions were at an initial moisture level of 49.6%, 33°C and 129h of incubation time with the maximum polygalacturonase activity of 565U/g, resulted in 2.65 fold increase in polygalacturonase activity compare to the unoptimized conditions. Kenaf stem treated with A. fumigatus R6 polygalacturonase enzyme at 72h produced high strength of kenaf bast fibers (287MPa) with high Young’s modulus (10404MPa) and the color is in satisfactory.

Cellulase Production from Bacillus subtilis SV1 and Its Application Potential for Saccharification of Ionic Liquid Pretreated Pine Needle Biomass under One Pot Consolidated Bioprocess

Pretreatment is the requisite step for the bioconversion of lignocellulosics. Since most of the pretreatment strategies are cost/energy intensive and environmentally hazardous, there is a need for the development of an environment-friendly pretreatment process. An ionic liquid (IL) based pretreatment approach has recently emerged as the most appropriate one as it can be accomplished under ambient process conditions. However, IL-pretreated biomass needs extensive washing prior to enzymatic saccharification as the enzymes may be inhibited by the residual IL. This necessitated the exploration of IL-stable saccharification enzymes (cellulases). Current study aims at optimizing the bioprocess variables viz. carbon/nitrogen sources, medium pH and fermentation time, by using a Design of Experiments approach for achieving enhanced production of ionic liquid tolerant cellulase from a bacterial isolate Bacillus subtilis SV1. The cellulase production was increased by 1.41-fold as compared to that under unoptimized conditions. IL-stable cellulase was employed for saccharification of IL (1-ethyl-3-methylimidazolium methanesulfonate) pretreated pine needle biomass in a newly designed bioprocess named as “one pot consolidated bioprocess” (OPCB), and a saccharification efficiency of 65.9% was obtained. Consolidated bioprocesses, i.e., OPCB, offer numerous techno-economic advantages over conventional multistep processes, and may potentially pave the way for successful biorefining of biomass to biofuel, and other commercial products.

Production and Properties of a Thermostable, pH-Stable Exo-Polygalacturonase Using Aureobasidium pullulans Isolated from Saharan Soil of Algeria Grown on Tomato Pomace.

Polygalacturonase is a valuable biocatalyst for several industrial applications. Production of polygalacturonase using the Aureobasidium pullulans stain isolated from Saharan soil of Algeria was investigated. Its capacity to produce polygalacturonase was assessed under submerged culture using tomato pomace as an abundant agro-industrial substrate. Optimization of the medium components, which enhance polygalacturonase activity of the strain Aureobasidium pullulans, was achieved with the aid of response surface methodology. The composition of the optimized medium was as follows: tomato pomace 40 g/L, lactose 1.84 g/L, CaCl20.09 g/L and pH 5.16. Practical validation of the optimum medium provided polygalacturonase activity of 22.05 U/mL, which was 5-fold higher than in unoptimized conditions. Batch cultivation in a 20 L bioreactor performed with the optimal nutrients and conditions resulted in a high polygalacturonase content (25.75 U/mL). The enzyme showed stability over a range of temperature (5–90 °C) with an optimum temperature of 60 °C with pH 5.0, exhibiting 100% residual activity after 1h at 60 °C. This enzyme was stable at a broad pH range (5.0–10). The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of polygalacturonic acid. Moreover, the exo-polygalacturonase was able to enhance the clarification of both apple and citrus juice. As a result, an economical polygalacturonase production process was defined and proposed using an industrial food by-product.

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

ABSTRACTCost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728 U ml−1), which was followed by gram husk (714 U ml−1), mustard cake (680 U ml−1), and soybean meal (653 U ml−1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020 U ml−1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.

Optical monitoring of CH3NH3PbI3 thin films upon atmospheric exposure

CH3NH3PbI3 perovskite films of interest for photovoltaic (PV) devices have been prepared by (i) vapor deposition and (ii) solution processing. Complex dielectric function (ε = ε1 + iε2) spectra and structural parameters of the films have been extracted using near infrared to ultraviolet spectroscopic ellipsometry. In situ real time spectroscopic ellipsometry (RTSE) over a 48 h period has been performed on vapor deposited CH3NH3PbI3 after the deposition in normal atmospheric laboratory ambient conditions. Analysis of RTSE data for vapor deposited CH3NH3PbI3 film prepared under un-optimized conditions identifies phase segregated PbI2 and CH3NH3I at the substrate/film interface and unreacted PbI2 and CH3NH3I on the film surface. This analysis also provides the time dependence of the effective thicknesses of perovskite film, unreacted components, and phase segregated layers to track CH3NH3PbI3 decomposition.