Single-cell Protein and Xylitol Production by a Novel Yeast Strain <Emphasis Type="Italic">Candida intermedia</Emphasis> FL023 from Lignocellulosic Hydrolysates and Xylose

Yeasts are good candidates to utilize the hydrolysates of lignocellulose, the most abundant bioresource, for bioproducts. This study aimed to evaluate the efficiencies of single-cell protein (SCP) and xylitol production by a novel yeast strain, Candida intermedia FL023, from lignocellulosic hydrolysates and xylose. This strain efficiently assimilated hexose, pentose, and cellubiose for cell mass production with the crude protein content of 484.2 g kg^?1 dry cell mass. SCP was produced by strain FL023 using corncob hydrolysate and urea as the carbon and nitrogen sources with the dry cell mass productivity 0.86 g L^?1 h^?1 and the yield of 0.40 g g^?1 sugar. SCP was also produced using NaOH-pretreated Miscanthus sinensis straw and corn steep liquor as the carbon and nitrogen sources through simultaneous saccharification and fermentation with the dry cell productivity of 0.23 g L^?1 h^?1 and yield of 0.17 g g^?1 straw. C. intermedia FL023 was tolerant to 0.5 g L^?1 furfural, acetic acid, and syringaldehyde in xylitol fermentation and produced 45.7 g L^?1 xylitol from xylose with the productivity of 0.38 g L^?1 h^?1 and the yield of 0.57 g g^?1 xylose. This study provides feasible methods for feed and food additive production from the abundant lignocellulosic bioresources.     

Enhanced l-Lactic Acid Production from Biomass-Derived Xylose by a Mutant Bacillus coagulans

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient l-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for l-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l^?1? l-lactic acid was obtained from 100 g l^?1 xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l^?1? l-lactic acid was obtained in 36 h and the yield was 83.09 %.     

In Situ Biphasic Extractive Fermentation for Hexanoic Acid Production from Sucrose by Megasphaera elsdenii NCIMB 702410

Hexanoic acid production by a bacterium using sucrose as an economic carbon source was studied under conditions in which hexanoic acid was continuously extracted by liquid–liquid extraction. Megasphaera elsdenii NCIMB 702410, selected from five M. elsdenii strains, produced 4.69 g l^?1 hexanoic acid in a basal medium containing sucrose. Production increased to 8.19 g l^?1 when the medium was supplemented by 5 g l^?1 sodium butyrate. A biphasic liquid–liquid extraction system with 10 % (v/v) alamine 336 in oleyl alcohol as a solvent was evaluated in a continuous stirred-tank reactor held at pH 6. Over 90 % (w/w) of the hexanoic acid in a 0.5 M aqueous solution was transferred to the extraction solvent within 10 h. Cell growth was not significantly inhibited by direct contact of the fermentation broth with the extraction solvent. The system produced 28.42 g l^?1 of hexanoic acid from 54.85 g l^?1 of sucrose during 144 h of culture, and 26.52 and 1.90 g l^?1 of hexanoic acid was accumulated in the extraction solvent and the aqueous fermentation broth, respectively. The productivity and yield of hexanoic acid were 0.20 g l^?1 h^?1 and 0.50 g g^?1 sucrose, respectively.     

Efficient Biotransformation of Phytosterols to Dehydroepiandrosterone by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

In this study, a method for the efficient production of dehydroepiandrosterone (DHEA) from phytosterols in a vegetable oil/aqueous two-phase system by Mycobacterium sp. was developed. After the 3-hydroxyl group of phytosterols was protected, they could be converted into DHEA with high yield and productivity by Mycobacterium sp. NRRL B-3683. In a shake flask biotransformation, 15.05 g l^?1 of DHEA and a DHEA yield of 85.39% (mol mol^?1) were attained after 7 days with an initial substrate concentration of 25 g l^?1. When biotransformation was carried out in a 30-l stirred bioreactor with 25 g l^?1 substrate, the DHEA concentration and yield was 16.33 g l^?1 and 92.65% (mol mol^?1) after 7 days, respectively. The results of this study suggest that inexpensive phytosterols could be utilized for the efficient production of DHEA.     

Mixed Food Waste as Renewable Feedstock in Succinic Acid Fermentation

Mixed food waste, which was directly collected from restaurants without pretreatments, was used as a valuable feedstock in succinic acid (SA) fermentation in the present study. Commercial enzymes and crude enzymes produced from Aspergillus awamori and Aspergillus oryzae were separately used in hydrolysis of food waste, and their resultant hydrolysates were evaluated. For hydrolysis using the fungal mixture comprising A. awamori and A. oryzae, a nutrient-complete food waste hydrolysate was generated, which contained 31.9 g L^?1 glucose and 280 mg L^?1 free amino nitrogen. Approximately 80–90 % of the solid food waste was also diminished. In a 2.5 L fermentor, 29.9 g L^?1 SA was produced with an overall yield of 0.224 g g^?1 substrate using food waste hydrolysate and recombinant Escherichia coli. This is comparable to many similar studies using various wastes or by-products as substrates. Results of this study demonstrated the enormous potential of food waste as renewable resource in the production of bio-based chemicals and materials via microbial bioconversion.     

Mineral Supplementation Increases Erythrose Reductase Activity in Erythritol Biosynthesis from Glycerol by Yarrowia lipolytica

The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO₄·7H₂O (25 mg l^?1) increased erythritol production by Y. lipolytica by 14.5 %. In the bioreactor culture with manganese ion addition, 47.1 g l^?1 of erythritol was produced from 100.0 g l^?1 of glycerol, which corresponded to volumetric productivity of 0.87 g l^?1 h^?1. The addition of Mn²⁺ enhanced the intracellular activity of erythrose reductase up to 24.9 U g^?1 of dry weight of biomass (DW), hence, about 1.3 times more than in the control.     

Enzymatic Hydrolysis and Succinic Acid Fermentation from Steam-Exploded Corn Stalk at High Solid Concentration by Recombinant Escherichia coli

Steam-exploded corn stalk biomass was used as the substrate for succinic acid production via lignocellulose enzymatic hydrolysis and fermentation. Succinic acid fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). For the washed steam-exploded corn stalk at 30 % substrate concentration, i.e., 30 % water-insoluble solids (WIS), enzymatic hydrolysis yielded 97.5 g/l glucose solution and a cellulose conversion of 73.6 %, thus a high succinic acid level up to 38.6 g/l. With the unwashed steam-exploded corn stalk, though a cellulose conversion of 71.2 % was obtained in hydrolysis at 30 % solid concentration (27.9 % WIS), its hydrolysate did not ferment at all, and the hydrolysate of 25 % solid loading containing 3.8 g/l acetic acid and 168.2 mg/l furfural exerted a strong inhibition on succinic acid production.     

Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

The concentration of ethanol produced from lignocellulosic biomass should be at least 40 g l^?1 [about 5 % (v/v)] to minimize the cost of distillation process. In this study, the conditions for the simultaneous saccharification and fermentation (SSF) at fed-batch mode for the production of ethanol from alkali-pretreated empty palm fruit bunch fibers (EFB) were investigated. Optimal conditions for the production of ethanol were identified as temperature, 30 °C; enzyme loading, 15 filter paper unit g^?1 biomass; and yeast (Saccharomyces cerevisiae) loading, 5 g l^?1 of dry cell weight. Under these conditions, an economical ethanol concentration was achieved within 17 h, which further increased up to 62.5 g l^?1 after 95 h with 70.6 % of the theoretical yield. To our knowledge, this is the first report to evaluate the economic ethanol production from alkali-pretreated EFB in fed-batch SSF using S. cerevisiae.     

Controlled Production of Exopolysaccharides from Enterobacter A47 as a Function of Carbon Source with Demonstration of Their Film and Emulsifying Abilities

The bacterium Enterobacter A47 has demonstrated the ability to synthesise distinct exopolysaccharides (EPS) as a function of the substrate used. The culture's performance was evaluated in experiments using either glucose or xylose, as single carbon sources, and compared with the substrate (glycerol) used in previous studies. The highest EPS production (13.23 g L^?1) was obtained in the glucose fed assay, with a volumetric productivity of 3.38 g L^?1 day^?1. The use of xylose resulted in lower productivity (1.39 g L^?1 day^?1). The synthesised polymers have the same main sugar monomers (fucose, glucose, galactose and glucuronic acid), but their relative proportion varied with the substrate used. The acyl groups' content and composition were also affected by the substrate used. The polymers produced from glycerol (EPS-s) and glucose (EPS-g) had identical shear-thinning behaviour and good emulsion-stabilising capacity and their films had similar mechanical and water vapour properties. However, the emulsions stabilised with EPS-g were less stable and destabilised within short periods of time or when subjected to heat and freezing/thawing procedures. On the other hand, the polymer produced from xylose had little emulsion-stabilising capacity and lower apparent viscosity than EPS-s and EPS-g, but its films were considerably more elastic.     

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.     

Enhancing pIFN-α Production and Process Stability in Fed-Batch Culture of Pichia pastoris by Controlling the Methanol Concentration and Monitoring the Responses of OUR/DO Levels

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor using an in situ methanol electrode-based feeding process with the control level of methanol concentration linearly increased to 10 g l^?1 for the first 20 h and maintained at 10 g l^?1 for the rest of expression phase. With this two-stage control process, the highest pIFN-α concentration reached a level of 1.81 g l^?1, which was 1.5-fold of that in the previous constant 10 g l^?1 induction experiments. There is an improvement of the pIFN-α productivity from more distribution of carbon flux to protein expression. The pIFN-α expression stability could be further enhanced by a simple on-line fault diagnosis method for methanol overfeeding based on oxygen uptake rate changing patterns. By implementing corrective action of feeding glycerol after fault detection, the production yield increased to twice the amount it would have been without the diagnosis.     

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48?±?0.02 g g^?1), theoretical yield (93?±?3 %), and extracellular invertase productivity (1,430?±?50 IU l^?1 h^?1), respectively, when fermenting 180 g sugars l^?1 in molasses medium at 43 °C in 300 m³ working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M^?1) and entropy (ΔS*) (?202.88 J M^?1 K^?1) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l^?1 h^?1) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.     

Improvement of the Production Efficiency of l-(+)-Tartaric Acid by Heterogeneous Whole-Cell Bioconversion

An Escherichia coli-engineered bacterium with cis-epoxysuccinate hydrolase (ESH) activity was used to catalyze the stereospecific hydrolysis of cis-epoxysuccinic acid to l-(+)-tartaric acid. The effect of the substrate composition on the production efficiency of l-(+)-tartaric acid was investigated. Based on the sodium-type homogeneous substrate system, a heterogeneous substrate system, composed of 1.2 M sodium-type substrate and 1.8 M calcium-type substrate, was designed to improve ESH catalytic efficiency. After process optimization, a catalytic efficiency of 9.37?×?10^?3 g U^?1 h^?1 was obtained with fed-batch mode in the heterogeneous substrate system, about a twofold increase compared to the traditional bioconversion process with Nocardia tartaricans cells. The scale-up tests were carried out in a 15-m³ stirred tank reactor, which indicated that the heterogeneous substrate system had great application prospect for the l-(+)-tartaric acid industrial production.     

Production of the Biopesticide Azadirachtin by Hairy Root Cultivation of Azadirachta indica in Liquid-Phase Bioreactors

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l^?1dry weight and azadirachtin yield of 3.2 mg g^?1 leading to a volumetric productivity of azadirachtin as 1.14 mg l^?1 day^?1. The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).     

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l^?1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol^?1 added cysteine and a productivity of 6.1 ± 0.0 g l^?1 h^?1. This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.     

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.     

Flow-Injection Determination of Iron Based on Its Catalysis on the Oxidation Reaction of Xylenol Orange by Potassium Bromate

A flow injection system is proposed for catalytic kinetic spectrophotometric determination of trace iron(II + III). The involved reaction is based on the catalytic effect of iron(III) on oxidation reaction of xylenol orange by potassium bromate to form a blue-violet complex. Iron(II) is also determined, being oxidized to iron(III) by potassium bromate. The calibration graph is linear in the range of 0.02–10.0 µg l^?1 and 10.0–1100 µg l^?1. The relative standard deviation is 1.5% for 4.0 µg l^?1 iron(III) and 2.3% for 60.0µg l^?1 iron(III) (n = 11). The presented system was applied successfully to the determination of iron in natural waters.     

Optimization of the Extraction Conditions and Simultaneous Quantification of Six Flavonoid Glycosides in Flos Chrysanthemi by RP-LC

A sensitive, accurate and reliable reversed-phase liquid chromatographic method coupled with DAD (278 nm) was established for simultaneous quantification of six compounds in 20 cultivars of Flos Chrysanthemi. The method was carried out by using a Kromasil 100-5 C₁₈ column with methanol–acetonitrile—1.414 × 10^?2 mol L^?1 aqueous phosphoric acid as a gradient mobile phase. The contents of the six flavonoid glycosides in Flos Chrysanthemi could be determined within 120 min. The linear calibration ranges for these were 0.42–126.00, 11.44–220.00, 0.53–530.00, 4.80–195.00, 11.00–220.00, and 0.12–200.00 μg mL^?1. Their recoveries were 95.33–105.33% with RSDs from 0.10 to 2.00%. Their lower limits of quantification were 0.420, 1.144, 0.250, 0.480, 0.242, and 0.120 μg mL^–1. The method can be used for analysis of the six flavonoid glycosides in Flos Chrysanthemi.     

Simultaneous Determination of Acetaminophen, Caffeine, and Chlorphenamine Maleate in Paracetamol and Chlorphenamine Maleate Granules

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column. The mobile phase was methanol 0.05 mol L^?1 aqueous KH₂PO₄ solution, 45:55 (v/v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min^?1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL^?1 for acetaminophen, 5.0–120.0 μg mL^?1 for caffeine, and 9.6–230.4 μg mL^?1 for chlorphenamine maleate.     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.