Enhanced production of poly-3-hydroxybutyrate by recombinant Escherichia coli containing NAD kinase and phbCAB operon

With the help of Tn5 transposon technique, gene yfjB encoding NAD kinase in Escherichia coli (E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate (PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfjB gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions.     

Evaluation of the Nitric Oxide and Nitrite Scavenging Capability,N-Nitrosamine Formation Inhibitory Activity,and Optimization of Ultrasound-Assisted Aqueous Two-Phase System Extraction of Total Saponins from <Emphasis Type="Italic">Coreopsis tinctoria</Emphasis> Flowering Tops by Response Surface Methodology

Coreopsis tinctoria flowering tops (CTFs) is a popular health product as herbal tea or as a traditional medicinal herb that is rich in saponins and exerts substantial biological activity. In this study, an ultrasound-assisted aqueous two-phase system (ATPS) was utilized to extract total saponins from CTFs and optimize the extraction process by response surface methodology. Moreover, the nitric oxide and nitrite scavenging capability, and N-nitrosamine formation inhibitory activity of total saponins were evaluated. Results showed that the optimal conditions for total saponins were 37.76% (w/w) ammonium sulfate and 35.62% (w/w) ethanol in ATPS coupled with ultrasonic-assisted extraction. Under the optimal conditions, the maximum yield of total saponins of 33.4 g/kg can be obtained from the CTFs raw material. The nitric oxide radical scavenging, nitrite scavenging, and N-nitrosamine inhibitory activities (SC₅₀) were 287.92 ± 7.42, 191.63 ± 7.69, and 1787.4 ± 51.26 μg/mL, respectively. The total saponins has a certain nitric oxide and nitrite scavenging capability, and N-nitrosamine formation inhibitory activity in vitro. Given these activities, research on saponins from CTFs provides profound and lasting implications for the novel applications of C. tinctoria.     

Production of Poly (3-Hydroxybutyric Acid) by <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> in a Biocalorimeter and its Thermokinetic Studies

Bioplastic production from microbial sources is an emerging area which provides opportunities even to convert the wastes into bioplastics. Poly (3-hydroxybutyric acid), commonly called as PHB, is a bioplastic, which is stored as intracellular cytoplasmic inclusions in microorganisms. The objectives of this study are to calorimetrically monitor the PHB production and evaluate the thermokinetic data in a bioreaction calorimeter (BioRC1e). Thus, a well-known PHB-producing bacteria Ralstonia eutropha was selected for batch process in a bioreaction calorimeter. The metabolic heat generated was found to be correlated with the biomass, substrate consumption, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and PHB production. The OUR pattern explained the oxidative metabolism of the strain R. eutropha. The heat yields due to biomass and glucose consumption during PHB production were found to be 12.56 and 13.56 kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80 kJ/mol of O₂. The concentration of PHB obtained in BioRC1e was 4.33 g/L with a production rate of 0.09 g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and ¹H and ¹³C nuclear magnetic resonance (NMR) analysis.     

The Feasibility of Thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> as a Platform for Efficient PHB Production

Recently, poly(3-hydroxybutyrate) (PHB) has been found in a few thermophilic strains where several advantages can be gained from running fermentation at high temperatures. Caldimonas manganoxidans, a thermophilic gram-negative bacterium, was investigated for the feasibility as a PHB-producing strain. It is suggested that the best fermentation strategy for achieving the highest PHB concentration of 5.4?±?1.1 g/L (from 20 g/L glucose) in 24 h is to use the fermentation conditions that are favored for the bacterial growth, yet temperature and pH should be chosen at conditions that are favored for the PHB content. Besides, the above fermentation conditions produce PHB that has a high molecular weight of 1274 kDa with a low polydispersity index (PDI) of 1.45, where the highest Mw of PHB of 1399?kDa (PDI of 1.32) is obtained in this study. To the best knowledge of authors, C. manganoxidans has the best PHB productivity among the thermophiles and is comparable to those common PHB-producing mesophiles.     

Experimental and computational studies on molecularly imprinted solid-phase extraction for gonyautoxins 2,3 from dinoflagellate <Emphasis Type="Italic">Alexandrium minutum</Emphasis>

An innovative and effective extraction procedure based on molecularly imprinted solid-phase extraction (MISPE) was developed for the isolation of gonyautoxins 2,3 (GTX2,3) from Alexandrium minutum sample. Molecularly imprinted polymer microspheres were prepared by suspension polymerization and and were employed as sorbents for the solid-phase extraction of GTX2,3. An off-line MISPE protocol was optimized. Subsequently, the extract samples from A. minutum were analyzed. The results showed that the interference matrices in the extract were obviously cleaned up by MISPE procedures. This outcome enabled the direct extraction of GTX2,3 in A. minutum samples with extraction efficiency as high as 83 %, rather significantly, without any need for a cleanup step prior to the extraction. Furthermore, computational approach also provided direct evidences of the high selective isolation of GTX2,3 from the microalgal extracts.     

Harnessing the Effect of pH on Lipid Production in Batch Cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> SKY7

The objective of this research was to investigate the kinetics of lipid production by Yarrowia lipolytica SKY7 in the crude glycerol-supplemented media with and without the control of pH. Lipid and citric acid production were improved with the pH control condition. There was no significant difference observed in the biomass concentration with or without the pH control. In the pH-controlled experiments, the biomass and lipid concentration reached 18 and 7.78 g/L, (45.5% w/w), respectively, with lipid yield (Yp/s) of 0.179 g/g at 60 h of fermentation. The lipid production was directly correlated with growth and the process was defined as growth associated. After 60 h of fermentation, the lipid degradation was noticed in the pH-controlled reactor whereas it occurred after 84 h in the pH-uncontrolled reactor. Apart from lipid, citric acid was produced as the major extracellular product in both fermentations but the much lower concentration in uncontrolled pH. Based on the experimental results, it is evident that controlling the pH will enhance the lipid production by 15% compared to pH-uncontrolled fermentation.     

Elucidating the Effect of Glycerol Concentration and C/N Ratio on Lipid Production Using <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> SKY7

The high demand for renewable energy and increased biodiesel production lead to the surplus availability of crude glycerol. Due to the above reason, the bio-based value addition of crude glycerol into various bioproducts is investigated; among them, microbial lipids are attractive. The present study was dedicated to find the optimal glycerol concentration and carbon/nitrogen (C/N) ratio to produce maximum lipid using Yarrowia lipolytica SKY7. The glycerol concentration (34.4 to168.2 g/L) and C/N ratio (25 to 150) were selected to investigate to maximize the lipid production. Initial glycerol concentration 112.5 g/L, C/N molar ratio of 100, and with 5 % v/v inoculum supplementation were found to be optimum for biomass and lipid production. Based on the above optimal parameters, lipid concentration of 43.8 % w/w with a biomass concentration of 14.8 g/L was achieved. In the case of glycerol concentration, the maximum Yp/s (0.192 g/g); Yx/s (0.43 g/g) was noted when the initial glycerol concentration was 112.5 g/L with C/N molar ratio 100 and inoculum volume 5 % v/v. The glycerol uptake was also noted to increase with the increase in glycerol concentration. At low C/N ratio, the glycerol consumption was found to be high (79.43 g/L on C/N 25) whereas the glycerol consumption was observed to decrease when the C/N ratio was raised to 150 (40.8 g/L).     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

Gas-chromatographic determination of trace <Emphasis Type="Italic">O</Emphasis>-isobutyl-2-<Emphasis Type="Italic">S</Emphasis>-(<Emphasis Type="Italic">N,N</Emphasis>-diethylamino)ethyl ester of methylthiophosphonic acid (VX-type compounds) in slime

A procedure was proposed for the gas-chromatographic determination of trace O-isobutyl-S-2-(N,N-diethylamino)ethyl ester of methylthiophosphonic acid (mixed ester, ME) in burning products formed after the detoxication of elements in building constructions, personal protection equipment, waste degassing solutions, and other production wastes (slime) at a level of (1.0–10.0) × 10^?8 mg/g. The procedure is based on the extraction of ME from an analyzed material with a mixture of aqueous solutions of monoethanolamine and NaOH, its back extraction to hexane, the evaporation of the hexane layer (with the addition of HCl) to dryness, the transformation of ME into methyl ester of O-isobutylmethylphosphonic acid with the use of methanol in the presence of AgNO₃, and the chromatography of the derivative on an open tubular column with the chemically modified HP-INN OWax stationary phase with the use of a flame-photometric detector.     

Thermal polymerization of styrene: New data on the conversion dependence of the initiation rate

With the aid of new acceptors of free radicals usable at high temperatures (T > 100°C), the rate of initiation w _i has been measured experimentally for the thermal polymerization of styrene at 122.5°C in a wide range of conversions C = 0–80%. It has been shown that the value of w _i tends to increase in the course of polymerization transformation in agreement with the w _i = f(C) relationship calculated from the kinetic data on the thermal polymerization of styrene in the absence of counters of free radicals. Hypothetical reasons for this non-trivial tendency have been formulated. The experimental dependence w _i = f(C) has been measured for the first time and has been invoked to refine currently available mathematical models for the thermal polymerization of styrene that assume that w _i remains invariable in the course of polymerization transformation.     

Microplate-Based Evaluation of the Sugar Yield from Giant Reed,Giant Miscanthus and Switchgrass after Mild Chemical Pre-Treatments and Hydrolysis with Tailored <Emphasis Type="Italic">Trichoderma</Emphasis> Enzymatic Blends

Giant reed, miscanthus, and switchgrass are considered prominent lignocellulosic feedstocks to obtain fermentable sugars for biofuel production. The bioconversion into sugars requires a delignifying pre-treatment step followed by hydrolysis with cellulase and other accessory enzymes like xylanase, especially in the case of alkali pre-treatments, which retain the hemicellulose fraction. Blends richer in accessory enzymes than commercial mix can be obtained growing fungi on feedstock-based substrates, thus ten selected Trichoderma isolates, including the hypercellulolytic strain Trichoderma reesei Rut-C30, were grown on giant reed, miscanthus, or switchgrass-based substrates. The produced enzymes were used to saccharify the corresponding feedstocks, compared to a commercial enzymatic mix (6 FPU/g). Feedstocks were acid (H₂SO₄ 0.2–2%, w/v) or alkali (NaOH 0.02–0.2%, w/v) pre-treated. A microplate-based approach was chosen for most of the experimental steps due to the large number of samples. The highest bioconversion was generally obtained with Trichoderma harzianum Or4/99 enzymes (78, 89, and 94% final sugar yields at 48 h for giant reed, miscanthus, and switchgrass, respectively), with significant increases compared to the commercial mix, especially with alkaline pre-treatments. The differences in bioconversion yields were only partially caused by xylanases (maximum R ² = 0.5), indicating a role for other accessory enzymes.     

Acetone–Butanol–Ethanol Production from Waste Seaweed Collected from Gwangalli Beach,Busan, Korea,Based on pH-Controlled and Sequential Fermentation Using Two Strains

The optimal conditions for acetone–butanol–ethanol (ABE) production were evaluated using waste seaweed from Gwangalli Beach, Busan, Korea. The waste seaweed had a fiber and carbohydrate, content of 48.34%; these are the main resources for ABE production. The optimal conditions for obtaining monosaccharides based on hyper thermal (HT) acid hydrolysis of waste seaweed were slurry contents of 8%, sulfuric acid concentration of 138 mM, and treatment time of 10 min. Enzymatic saccharification was performed using 16 unit/mL Viscozyme L, which showed the highest affinity (K_m?=?1.81 g/L). After pretreatment, 34.0 g/L monosaccharides were obtained. ABE fermentation was performed with single and sequential fermentation of Clostridium acetobutylicum and Clostridium tyrobutyricum; this was controlled for pH. A maximum ABE concentration of 12.5 g/L with Y_ABE 0.37 was achieved using sequential fermentation with C. tyrobutyricum and C. acetobutylicum. Efficient ABE production from waste seaweed performed using pH-controlled culture broth and sequential cell culture.     

Solution Thermodynamics of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis><Superscript>/</Superscript>-Ethylenebis-(salicylideneiminato)-diaquochromium(III) Chloride in Aqueous Dimethylsulphoxide

The densities, viscosities and refractive indices of N,N ^/-ethylene-bis(salicylideneiminato)-diaquochromium(III) chloride, [Cr(salen)(H₂O)₂]Cl, in aqueous dimethylsulfoxide (DMSO) with different mass fractions (w ₂ = 0.20, 0.40, 0.60, 0.80 and 1.00) of DMSO were determined at 298.15, 308.15 and 318.15 K under atmospheric pressure. From measured densities, viscosities and refractive indices the apparent molar volumes (V _φ ), standard partial molar volume (V _φ ⁰ ), the slope (S _V ^* ), standard isobaric partial molar expansibility (φ _E ⁰ ) and its temperature dependence (?φ _E ⁰ /?T) _p , the viscosity B-coefficient, its temperature dependence (?B/?T), solvation number (S _n ) and apparent molar refractivity (R _D ^φ ), etc., were calculated and discussed on the basis of ion–ion and ion–solvent interactions. These results revealed that the solutions are characterized by ion–solvent interactions rather than by ion–ion interactions and the complex behaves as a long range structure maker. Thermodynamics of viscous flow was discussed in terms of transition state theory.     

Supramolecular 2D structure of [Co(2-Me-Pyz)<Subscript>2</Subscript>(H<Subscript>2</Subscript>O)<Subscript>4</Subscript>](NO<Subscript>3</Subscript>)<Subscript>2</Subscript>

The complex [Co(2-Me-Pyz)₂(H₂O)₄](NO₃)₂ is synthesized and its structure is determined. The crystals are monoclinic: space group P2₁/n, a = 10.685(2) Å, b = 6.837(1), c = 12.515(3) Å, β = 91.84(3)°, V = 913.8(3) ų, ρ_calcd = 1.042 g/cm ³, Z = 2. The Co²⁺ ion (in the inversion center) is coordinated at the vertices of the distorted octahedron by two nitrogen atoms of methylpyrazine and four oxygen atoms of the water molecules (Co(1)–N(1) 2.180(3), average Co(1)–O(w) 2.079(3) Å, angles at the Co atom 87.9(1)–92.1(1)°). Supramolecular pseudometallocycles are formed in the structure through the O(w)–H…N(1) hydrogen bonds between the coordinated H₂O molecules and the terminal nitrogen atoms of the 2-methylpyrazine molecules. Their interaction results in the formation of supramolecular layers joined by the NO₃ groups into a three-dimensional framework.     

The Influence of Nonionic Surfactant Adsorption on Enzymatic Hydrolysis of Oil Palm Fruit Bunch

Nonionic surfactants have been utilized to improve the enzymatic hydrolysis of lignocellulosic materials. However, the role of surfactant adsorption affecting enzymatic hydrolysis has not been elaborated well. In this work, nonionic surfactants differing in their molecular structures, namely the polyoxyethylene sorbitan monooleate (Tween 80), the secondary alcohol ethoxylate (Tergitol 15-S-9), and the branched alcohol ethoxylate (Tergitol TMN-6), were studied for their effects on the enzymatic hydrolysis of palm fruit bunch (PFB). The PFB was pretreated with a 10% w/v sodium hydroxide solution and then hydrolyzed using the cellulase enzyme from Trichoderma reesei (ATCC 26921) at 50 °C and pH 5. The optimal conditions providing similar yields of reducing sugar required Tween 80 and Tergitol TMN-6 at 0.25% w/v, while Tergitol 15-S-9 was required at 0.1% w/v. All the surfactants improved the enzymatic conversion efficiency and reduced unproductive binding of the enzyme to lignin. In addition, the adsorption isotherm of cellulase was fit well by the Freundlich isotherm, while adsorption of the three nonionic surfactants agreed well with the Langmuir isotherm. Adsorption capacities of the three nonionic surfactants were consistent with their enhancement efficiencies in hydrolysis. The critical micelle concentration was observed as a key property of nonionic surfactant for adsorption capacity.     

Cotton Stalk Pretreatment Using <Emphasis Type="Italic">Daedalea flavida</Emphasis>, <Emphasis Type="Italic">Phlebia radiata</Emphasis>, and <Emphasis Type="Italic">Flavodon flavus</Emphasis>: Lignin Degradation,Cellulose Recovery,and Enzymatic Saccharification

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.     

Single Cell Oil Production from Hydrolysates of Inulin by a Newly Isolated Yeast <Emphasis Type="Italic">Papiliotrema laurentii</Emphasis> AM113 for Biodiesel Making

Microbial oils are among the most attractive alternative feedstocks for biodiesel production. In this study, a newly isolated yeast strain, AM113 of Papiliotrema laurentii, was identified as a potential lipid producer, which could accumulate a large amount of intracellular lipids from hydrolysates of inulin. P. laurentii AM113 was able to produce 54.6% (w/w) of intracellular oil in its cells and 18.2 g/l of dry cell mass in a fed-batch fermentation. The yields of lipid and biomass were 0.14 and 0.25 g per gram of consumed sugar, respectively. The lipid productivity was 0.092 g of oil per hour. Compositions of the fatty acids produced were C_14:0 (0.9%), C_16:0 (10.8%), C_16:1 (9.7%), C_18:0 (6.5%), C_18:1 (60.3%), and C_18:2 (11.8%). Biodiesel obtained from the extracted lipids could be burnt well. This study not only provides a promising candidate for single cell oil production, but will also probably facilitate more efficient biodiesel production.     

Optimization of High Solids Dilute Acid Hydrolysis of Spent Coffee Ground at Mild Temperature for Enzymatic Saccharification and Microbial Oil Fermentation

Soluble coffee, being one of the world’s most popular consuming drinks, produces a considerable amount of spent coffee ground (SCG) along with its production. The SCG could function as a potential lignocellulosic feedstock for production of bioproducts. The objective of this study is to investigate the possible optimal condition of dilute acid hydrolysis (DAH) at high solids and mild temperature condition to release the reducing sugars from SCG. The optimal condition was found to be 5.3 % (w/w) sulfuric acid concentration and 118 min reaction time. Under the optimal condition, the mean yield of reducing sugars from enzymatic saccharification of defatted SCG acid hydrolysate was 563 mg/g. The SCG hydrolysate was then successfully applied to culture Lipomyces starkeyi for microbial oil fermentation without showing any inhibition. The results suggested that dilute acid hydrolysis followed by enzymatic saccharification has the great potential to convert SCG carbohydrates to reducing sugars. This study is useful for the further developing of biorefinery using SCG as feedstock at a large scale.     

Ethanol Production from Starch Hydrolyzates using <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and Glucoamylase Entrapped in Polyvinylalcohol Hydrogel

The glucoamylase from Aspergillus niger, immobilized into poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats®, was used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis in free form. This system was stable in both the repeated batch and continuous mode of SSF. The microorganism was found to adsorb on the capsules with immobilized enzyme. This increased the ethanol productivity of the repeated batch system with 5% w/v of immobilized glucoamylase almost 2.1 times (7.2 g l^?1 h^?1) compared to free enzyme–free microorganism system (3.5 g l^?1 h^?1). The continuous SSF with the immobilized glucoamylase (11.5% w/v) tested for 15 days had productivity 10 g l^?1 h^?1, which is comparable to continuous experiments on semi-defined glucose medium (10 g l^?1 h^?1). These two systems were stable in both glucoamylase activity and microorganism productivity.     

Structure elucidation and X-ray crystallographic study of 3-(naphth-1-ylmethyl)indole

Synthesis and structure determination of 3-(naphth-1-ylmethyl)indole are reported. The molecular and crystal structures together with the molecular formula were determined by spectral and single crystal X-ray studies. X-ray crystallography revealed the presence of two conformers arising from the flipping of the naphthalene unit. The crystal of the compound belongs to the triclinic crystal system and space group \(P\bar 1\). Crystal data are as follows: a = 10.302(5) Å, b = 12.522(4) Å, c = 13.383(4) Å, α = 111.9(1)°, β = 116.86(6)°, γ = 71.65(5)°; V = 1726.429 ų; Z = 4. The final R and Rw are R = 0.0744 (on observed F′s); R = 0.0924 (all F data), R _w = 0.1757 (observed F ²) and R _w = 0.1834 (all F ² data).