Bioethanol Production from Ipomoea Carnea Biomass Using a Potential Hybrid Yeast Strain

The paper deals with the exploitation of Ipomoea carnea as a feedstock for the production of bioethanol. Dilute acid pretreatment under optimum conditions (3 %H₂SO₄, 120 °C for 45 min) produced 17.68 g L^?1 sugars along with 1.02 g L^?1 phenolics and 1.13 g L^?1 furans. A combination of overliming and activated charcoal adsorption facilitated the removal of 91.9 % furans and 94.7 % phenolics from acid hydrolysate. The pretreated biomass was further treated with a mixture of sodium sulphite and sodium chlorite and, a maximum lignin removal of 81.6 % was achieved. The enzymatic saccharification of delignified biomass resulted in 79.4 % saccharification with a corresponding sugar yield of 753.21 mg g^?1. Equal volume of enzymatic hydrolysate and acid hydrolysate were mixed and used for fermentation with a hybrid yeast strain RPRT90. Fermentation of mixed detoxified hydrolysate at 30 °C for 28 h produced ethanol with a yield of 0.461 g g^?1. A comparable ethanol yield (0.414 g g^?1) was achieved using a mixture of enzymatic hydrolysate and undetoxified acid hydrolysate. Thus, I. carnea biomass has been demonstrated to be a potential feedstock for bioethanol production, and the use of hybrid yeast may pave the way to produce bioethanol from this biomass.     

Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules

Biotransformation of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) was investigated with a newly isolated Alcaligenes faecalis ZJUTBX11 strain showing nitrilase activity in the immobilized form. To reduce the mass transfer resistance and to increase the toleration ability of the microorganisms to the toxic substrate as well as to enhance their ability to be reused, encapsulation of the whole cells in alginate-chitosan-alginate (ACA) membrane liquid-core capsules was attempted in the present study. The optimal pH and temperature for nitrilase activity of encapsulated A. faecalis ZJUTBX11 cells were 7.5°C and 35°C, respectively, which is consistent with free cells. Based on the Michaelis-Menten model, kinetic parameters of the conversion reaction with IDAN as the substrate were: K _m = (17.6 ± 0.3) mmol L^?1 and V _max = (97.6 ± 1.2) μmol min^?1 g^?1 of dry cell mass for encapsulated cells and (16.8 ± 0.4) mmol L^?1 and (108.0 ± 2.7) μmol min^?1 g^?1 of dry cell mass for free cells, respectively. After being recycled ten times, the whole cells encapsulated in ACA capsules still retained 90 % of the initial nitrilase activity while only 35 % were retained by free cells. Lab scale production of IDA using encapsulated cells in a bubble column reactor and a packed bed reactor were performed respectively.     

Simultaneous Analysis of Isoniazid and Its Impurities by CZE

An alternative methodology for simultaneous determination of isoniazid, isonicotinic acid, 4-cyanopyridine, ethyl isonicotinate and isonicotinamide by capillary zone electrophoresis, within an analysis time of 11 min, is proposed. An electrolyte composed by 50 mmol L⁻¹ of acetic acid/sodium acetate buffer and 12.5 mmol L⁻¹ of Cu²⁺, an analyte dilution in 1 mmol L⁻¹ of Brij 35 and 12.5 mmol L⁻¹ of Cu²⁺ and +20 kV voltage were optimized. After evaluating some figures of merit, such as linearity, precision, recovery and quantification limit, the method was successfully applied to the isoniazid analysis in tablets. The contents found were 99.3 mg of isoniazid and 0.3 mg of isonicotinic acid, corresponding to 0.3 % of impurity regarding the content of the active ingredient in the pharmaceutical formulation.

     

Biosynthesis of Benzoylformic Acid from Benzoyl Cyanide with a New Bacterial Isolate of Brevibacterium sp. CCZU12-1

Brevibacterium sp. CCZU12-1 with high nitrilase activity could effectively hydrolyze benzoyl cyanide into benzoylformic acid. After the culture optimization, the preferred carbon sources, nitrogen sources, and inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (2.5 g/L), and ε-caprolactam (2.0 mM), respectively. After the reaction optimization, the optimum reaction temperature, reaction pH, organic cosolvent, and metal ion were 30 °C, 7.0, ethanol (2 %, v/v), and Ca²⁺ (0.1 mM), respectively. At biotransformation of 120-mM benzoyl cyanide for 24 h, the yield of benzoylformic acid reached 91.8 %. Moreover, the microbial nitrilase from Brevibacterium sp. CCZU12-1 could hydrolyze various nitriles, and it significantly exhibited high nitrilase activity against benzoyl cyanide, 3-cyanopyridine, and α-cyclohexyl-mandelonitrile.     

Production of <Emphasis Type="Italic">R</Emphasis>-Mandelic Acid Using Nitrilase from Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Cells Immobilized with Tris(Hydroxymethyl)Phosphine

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L^?1 day^?1 and the space-time productivity of 143.2 mmol L^?1 h^?1 g^?1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Impact of Nitrate Dose on Toluene Degradation under Denitrifying Condition

In this study, we investigated the impact of nitrate dose on toluene degradation by Pseudomonas putida to elucidate the upper limit of nitrate concentration and whether an optimum ratio of nitrate to toluene concentration exists. Batch microcosm studies were conducted in order to monitor toluene degradation for various ratios (2–20) of nitrate to toluene with nitrate concentrations ranging from 0 to 700 mg?L^?1 for a given toluene concentration of 50 and 25 mg?L^?1 during 4-day (short term) and 14-day (long term) incubation time, respectively. The short-term study revealed that nitrate concentration of 500 mg?L^?1 was toxic to bacteria and the optimum concentration was 300 mg?L^?1 yielding the highest toluene degradation rate (0.083 mg?L^?1?h^?1). In the batch study of long term, toluene degradation was limited to 6 days after which the nitrate at 50 mg?L^?1 was depleted, indicating that nitrate was a necessary electron acceptor. For both batch studies, an optimum ratio of 6 was found yielding the highest toluene degradation rate. This indicates that an appropriate nitrate dose is essential for efficient degradation of toluene when bioremediation of groundwater contaminated with toluene is under consideration.     

Bioflocculant Exopolysaccharide Production by Azotobacter indicus Using Flower Extract of Madhuca latifolia L

Efficacy of Azotobacter indicus ATCC 9540 strain for production exopolysaccharide (EPS) bioflocculant was investigated. Mahua flower extract (Madhuca latifolia L), a natural substrate at the concentration of 20 g L^?1, gave maximum recovery of EPS followed by sucrose and mannitol as compared to other carbon sources after 172 h. Yeast extract was found to be the most effective nitrogen source as compared to beef extract, sodium nitrate, ammonium sulfate, casein hydrolysate, and urea for the production of EPS. EPS production was increased in presence of nitrogen (5.51 g L^?1) as compared to nitrogen-free medium (3.51 g L^?1), and fermentation time was also reduced by 28 h. Maximum EPS production (6.10 g L^?1) was found in the presence of 20 g L^?1 flower extract and 0.5 g L^?1 yeast extract containing Ashby’s media with 180 rpm at 30 °C at 144 h, under controlled conditions in 2.5 L fermenter using optimized medium. The isolated EPS showed cation-dependent flocculating activity. Concentration of EPS played an important role in bioflocculating activity which increased in a concentration-dependent manner up to a certain limit, with the maximum flocculation of 72% at 500 mg L^?1 concentration but remained almost static after this concentration. Extracted polymer was characterized by different chemical tests, FT-IR spectroscopy, and TLC which showed presence of uronic acids, O-acetyl groups, and Orcinol with suggestive indication of alginate like polymer. This study suggests that use of M. latifolia L. flowers can be a potential alternative bioresource for production of exopolysaccharide.     

Determination of Chlorophenoxy Acid Herbicides in Water Samples by Suspended Liquid-Phase Microextraction�CLiquid Chromatography

In this study, directly suspended liquid-phase microextraction was investigated for the extraction and determination of five chlorophenoxy acid herbicides in water samples. The optimized parameters for extraction of chlorophenoxy acid herbicides were 1 M HCl concentration in sample solution, solution temperature 20 °C, 45-min extraction time, 1,000 rpm stirring rate, 25 ??L extracting solvent volume and without NaCl addition. Under the optimum conditions, the enrichment factor ranged from 192 to 390. Calibration curves yielded good linearity (R ² > 0.999) and the linear range was 5.0?C500.0 ??g L^?1, limit of detection was 0.3?C0.4 ??g L^?1 and limit of quantification was 1?C2 ??g L^?1 for analytes and the relative standard deviations were in the range of 3?C10% (n = 3). Finally, the proposed method was successfully applied to the quantification of five chlorophenoxy acid herbicides in water samples and recovery was in the range of 74?C110%.     

Steroselective Hydrolysis of DL‐Beta‐Acetylthioisobutyramide Catalyzed by Genetically Engineered E. Coli Immobilized on Celite 580 in a Packed Bed Bioreactor

Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl ‐β‐acetylthioisobutyramide (dl ‐ATIA) to form d ‐β‐acetylthioisobutyric acid (DAT), a key intermediate for synthesis of a series of angiotensin converting enzyme inhibitors. The esterase gene of Pseudomonas putida IFO12996 was cloned and expressed in Escherichia coli which was further immobilized and retained on a packed bed bioreactor filled with Celite 580. The packed bed bioreactor was used to conduct the stereoselective hydrolysis of dl ‐ATIA and to give DAT with a yield of 34.5%, enantiometric excess value of 97% and enantioselectivity value > 150. The optimal pH and temperature for the reaction were 9.0 and 57 °C ～ 67 °C, respectively. The kinetic constants (Km and Vmax) of immobilized cells were found to be 372.5 mM and 285.7 μmol min^?1 (g cell)^?1, respectively. The immobilized cells retained over 60% of the initial catalytic activity after 5 batch cycles of production. This paper presents a simple, practical and economical process of immobilization of genetically engineered E. coli on a novel packed bed bioreactor for production of DAT.     

A Study of the Effects of Aeration and Agitation on the Properties and Production of Xanthan Gum from Crude Glycerin Derived from Biodiesel Using the Response Surface Methodology

The effects of aeration and agitation on the properties and production of xanthan gum from crude glycerin biodiesel (CGB) by Xanthomonas campestris mangiferaeindicae 2103 were investigated and optimized using a response surface methodology. The xanthan gum was produced from CGB in a bioreactor at 28 °C for 120 h. Optimization procedures indicated that 0.97 vvm at 497.76 rpm resulted in a xanthan gum production of 5.59 g L^?1 and 1.05 vvm at 484.75 rpm maximized the biomass to 3.26 g L^?1. Moreover, the combination of 1.05 vvm at 499.40 rpm maximized the viscosity of xanthan at 0.5 % (m/v), 25 °C, and 25 s^?1 (255.40 mPa s). The other responses did not generate predictive models. Low agitation contributed to the increase of xanthan gum production, biomass, viscosity, molecular mass, and the pyruvic acid concentration. Increases in the agitation contributed to the formation of xanthan gum with high mannose concentration. Decreases in the aeration contributed to the xanthan gum production and the formation of biopolymer with high mannose and glucose concentrations. Increases in aeration contributed to increased biomass, viscosity, and formation of xanthan gum with greater resistance to thermal degradation. Overall, aeration and agitation of CGB fermentation significantly influenced the production of xanthan gum and its properties.     

A New Temperature Control Shifting Strategy for Enhanced Triterpene Production by <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> G0119 Based on Submerged Liquid Fermentation

Temperature control is a very important factor on triterpene productivity in submerged liquid fermentation. Temperature effects from 23 to 32 °C on triterpene production by Ganoderma lucidum G0119 were investigated in 6-L stirred fermentor. Logistic and Luedeking-Piret equations were used to estimate the mycelial growth and triterpene production kinetics by regression analysis. On that basis, a temperature-shifting fermentation control strategy was established. From 0 to 61 h, culturing was performed at 32 °C to get high specific mycelial growth rate. Between 62 and 127 h, the temperature was decreased stepwise from 31 to 30 °C to maintain high triterpene productivity. After 128 h, temperature was maintained at 29 °C to minimize triterpene production inhibition and sustain high productivity. Elevated triterpene level (0.269 g L^?1), yield (0.0101 g g^?1), and productivity (0.00207 g (L h)^?1) were achieved representing 27.32, 13.94, and 37.11 % higher than submerged liquid fermentation at constant temperature of 29 °C, respectively, feasible for the industrial scale.     

LC Analysis of Aliphatic Primary Amines and Diamines After Derivatization with 2,6-Dimethyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester

A method for sensitive simultaneous analysis of aliphatic primary amines and diamines has been developed and validated. The compounds were analyzed by reversed-phase high-performance liquid chromatography after pre-column derivatization with 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester as fluorescent probe. The derivatization reaction was performed at 50 °C for 40 min in 0.1 mol L^?1 borax buffer solution (pH 7.5). The resulting fluorophores were separated to baseline on a C₁₈ column and fluorimetrically detected at λ _ex/λ _em = 326/409 nm. Detection limits were in the range 0.50–0.02 nmol L^?1. The method was successfully used for analysis of aliphatic amines in water, human urine, and serum.     

Isolation of Novel Microalgae from Acid Mine Drainage and Its Potential Application for Biodiesel Production

Microalgae were selected and isolated from acid mine drainage in order to find microalgae species which could be cultivated in low pH condition. In the present investigation, 30 microalgae were isolated from ten locations of acid mine drainage in South Korea. Four microalgae were selected based on their growth rate, morphology, and identified as strains of KGE1, KGE3, KGE4, and KGE7. The dry biomass of microalgae species ranged between 1 and 2 g L^?1 after 21 days of cultivation. The growth kinetics of microalgae was well described by logistic growth model. Among these, KGE7 has the highest biomass production (2.05?±?0.35 g L^?1), lipid productivity (0.82?±?0.14 g L^?1), and C16–C18 fatty acid contents (97.6 %). These results suggest that Scenedesmus sp. KGE 7 can be utilized for biodiesel production based on its high biomass and lipid productivity.     

Enhanced Production of Poly-γ-glutamic Acid by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> TISTR 1010 with Environmental Controls

Bacillus licheniformis TISTR 1010 was used for glutamic acid-independent production of poly-γ-glutamic acid (γ-PGA). A fed-batch production strategy was developed involving feedings of glucose, citric acid, and ammonium chloride at specified stages of the fermentation. With the dissolved oxygen concentration controlled at ≥50% of air saturation and the pH controlled at ~7.4, the fed-batch operation at 37 °C afforded a peak γ-PGA concentration of 39.9 ± 0.3 g L^?1 with a productivity of 0.926 ± 0.006 g L^?1 h^?1. The observed productivity was nearly threefold greater than previously reported for glutamic acid-independent production using the strain TISTR 1010. The molecular weight of γ-PGA was in the approximate range of 60 to 135 kDa.     

Protein-Coated Microcrystals of Pseudomonas aeruginosa PseA lipase

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL PCMC) have been prepared by immobilization of protein onto K₂SO₄ as excipient solid support carrier and n-propanol as precipitating solvent. Transmission electron micrographs confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane, compared to free lipase. The V _max, K _m, and temperature optimum for PAL PCMC increased from 0.49 to 5.66 nmol min^?1 mg^?1, 589 to 679.8 mmol, and 40°C to 45°C, respectively. These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives at 45°C, 55°C, and 60°C compared to free P. aeruginosa PseA lipase. Their catalytic efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple and effective technique for obtaining stable and efficient lipase preparation for biocatalysis in nonaqueous medium.     

Co-production of Fructooligosaccharides and Levan by Levansucrase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> natto with Potential Application in the Food Industry

Fructooligosaccharides and levan have a wide range of applications in the food industry due to their physiological and functional properties. The enzymatic synthesis of these molecules exhibits great advantages when compared with microbial fermentation. In this study, the production of levansucrase from Bacillus subtilis natto and its utilization in fructooligosaccharides and levan syntheses using different reaction conditions were described. The best condition for levansucrase production was 420.7 g L^?1 of sucrose at pH 7.0, which reached 23.9 U ml^?1 of transfructosylation activity. In a bioreactor, the highest production of fructooligosaccharides was 41.3 g L^?1 using a medium containing 350 g L^?1 sucrose at 35 °C for 36 h. The enzymatic synthesis of levan resulted in 86.9 g L^?1 when conditions similar to those used for fructooligosaccharides synthesis were applied. These results indicate that the levansucrase from B. subtilis natto could be applied for the co-production of fructooligosaccharides and levan, which are biomolecules that have health benefits and are used successfully in the food industry.     

Effect of Organic Loading Rate and Fill Time on the Biohydrogen Production in a Mechanically Stirred AnSBBR Treating Synthetic Sucrose-Based Wastewater

This study investigated the feasibility to produce biohydrogen of a mechanically stirred anaerobic sequencing batch biofilm reactor (AnSBBR) treating sucrose-based synthetic wastewater. The bioreactor performance (30 °C) was evaluated as to the combined effect of fill time (2, 1.5, and 1 h), cycle length (4, 3, and 2 h), influent concentration (3,500 and 5,250 mg chemical oxygen demand (COD)?L^?1) and applied volumetric organic load (AVOL_CT from 9.0 to 27.0 g COD L^?1 d^?1). AVOLs were varied according to influent concentration and cycle length (t _C). The results showed that increasing AVOL_CT resulted in a decrease in sucrose removal from 99 to 86 % and in improvement of molar yield per removed load (MYRL_S.n) from 1.02 mol H₂?mol carbohydrate^?1 at AVOL_CT of 9.0 g COD L^?1 d^?1 to maximum value of 1.48 mol H₂?mol carbohydrate^?1, at AVOL_CT of 18.0 g COD L^?1 d^?1, with subsequent decrease. Increasing AVOL_CT improved the daily molar productivity of hydrogen (MPr) from 15.28 to 49.22 mol H₂?m^?3 d^?1. The highest daily specific molar productivity of hydrogen (SMPr) obtained was 8.71 mol H₂?kg TVS^?1 d^?1 at an AVOL_CT of 18.0 g COD L^?1 d^?1. Decreasing t _C from 4 to 3 h decreased sucrose removal, increased MPr, and improved SMPr. Increasing influent concentration decreased sucrose removal only at t _C of 2 h, improved MYRL_S,n and MPr at all t _C, and also improved SMPr at t _C of 4 and 3 h. Feeding strategy had a significant effect on biohydrogen production; increasing fill time improved sucrose removal, MPr, SMPr, and MYRL_S,n for all investigated AVOL_CT. At all operational conditions, the main intermediate metabolic was acetic acid followed by ethanol, butyric, and propionic acids. Increasing fill time resulted in a decrease in ethanol concentration.     

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml^?1 and 45.66 μmol min^?1 mg^?1 and 9.22 mg ml^?1 and 16.05 μmol min^?1 mg^?1, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.     

Nitrogen Removal by Chelatococcus daeguensis TAD1 and Its Denitrification Gene Identification

Chelatococcus daeguensis TAD1 was demonstrated to be an aerobic denitrifier. It can utilize not only nitrate and nitrite but also ammonium at high temperature (about 50 °C). The strain had the capability to remove 122.7 and 71.7 mg L^?1 NH₄ ⁺-N by 18 h at 50 and 30 °C, respectively. Triplicate heterotrophic nitrification experiments showed that 32.3 % of removed NH₄ ⁺-N was completely converted to nitrogen gas by 18 h at 50 °C. The denitrification genes involved in C. daeguensis TAD1 were identified and sequenced. It was found that the genes responsible for denitrification in TAD1 were napA, nirK, cnorB, and nosZ. Taken together, TAD1 can be an effective candidate for simultaneous nitrification and denitrificaton at high temperature.