Enhanced Production of Xylitol from Poplar Wood Hydrolysates Through a Sustainable Process Using Immobilized New Strain <Emphasis Type="Italic">Candida tropicalis</Emphasis> UFMG BX 12-a

A new strain, Candida tropicalis UFMG BX 12-a, was found to produce higher yields of xylitol on poplar wood hemicellulose hydrolysate. The hemicellulose hydrolysate liquor was detoxified using a novel method we developed, involving vacuum evaporation and solvent separation of inhibitors which made the hydrolysate free of toxins while retaining high concentrations of fermentable sugars. The effect of the detoxification method on the fermentation was also reported and compared to well-known methods reported in literature. In this study, the new strain C. tropicalis UFMG BX 12-a was used on the detoxified hydrolysate to produce xylitol. It was also compared to Candida guilliermondii FTI 20037, which has been reported to be one of the best strains for fermentative production of xylitol. To further improve the efficiency of the fermentation process, these strains were immobilized in calcium alginate beads. The yield (0.92 g g^?1) and productivity (0.88 g L^?1 h^?1) obtained by fermenting the wood hydrolysate detoxified by our new detoxification technique using an immobilized new Candida strain were found to be higher than the values reported in literature.     

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.     

Succinic Acid Production from Corn Cob Hydrolysates by Genetically Engineered Corynebacterium glutamicum

Corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. In the present work, we constructed a xylose metabolic pathway in C. glutamicum by heterologous expression of the xylA and xylB genes coming from Escherichia coli. Dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant C. glutamicum NC-2. The results indicated that the available activated charcoal pretreatment in dilute-acid hydrolysates of corn cobs could be able to overcome the inhibitory effect in succinic acid production. Succinic acid was shown to be efficiently produced from corn cob hydrolysates (55 g l^?1 xylose and 4 g l^?1 glucose) under oxygen deprivation with addition of sodium carbonate. Succinic acid concentration reached 40.8 g l^?1 with a yield of 0.69 g g^?1 total sugars within 48 h. It was the first report of succinic acid production from corn cob hydrolysates by metabolically engineered C. glutamicum. This study suggested that dilute-acid hydrolysates of corn cobs may be an alternative substrate for the efficient production of succinic acid by C. glutamicum.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L^?1) and was stimulated by moderate pH (4.5) and agitation (800 rev min^?1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L^?1 of PA with volumetric productivity of 0.90 g L^?1 h^?1. The yield of PA biosynthesis reached a high level of 1.03 g g^?1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.     

Screening of <Emphasis Type="Italic">Isochrysis</Emphasis> Strains and Utilization of a Two-Stage Outdoor Cultivation Strategy for Algal Biomass and Lipid Production

Isochrysis is a genus of marine algae without cell wall and capable of accumulating lipids. In this study, the lipid production potential of Isochrysis was assessed by comparing 15 Isochrysis strains with respect to their growth rate, lipid production, and fatty acid profiles. Three best strains were selected (lipid productivity, 103.0~121.7 mg L^?1 day^?1) and their lipid-producing capacities were further examined under different controlled parameters, e.g., growth phase, medium nutrient, and light intensity in laboratory cultures. Furthermore, the three Isochrysis strains were monitored in outdoor panel photobioreactors with various initial cell densities and optical paths, and the strain CS177 demonstrated the superior potential for outdoor cultivation. A two-stage semi-continuous strategy for CS177 was subsequently developed, where high productivities of biomass (1.1 g L^?1 day^?1) and lipid (0.35 g L^?1 day^?1) were achieved. This is a comprehensive study to evaluate the lipid-producing capability of Isochrysis strains under both indoor and outdoor conditions. Results of the present work lay a solid foundation for the physiological and biochemical responses of Isochrysis to various conditions, shedding light on the future utilization of this cell wall-lacking marine alga for biofuel production.     

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.     

Improved Bioethanol Production Using Fusants of Saccharomyces cerevisiae and Xylose-Fermenting Yeasts

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose?Cxylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8?±?0.31?g L^?1), ethanol productivity (1.06?g L^?1 h^?1) and ethanol yield (0.458?g g^?1) by fermentation of glucose?Cxylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08?±?0.142?g L^?1, ethanol yield of 0.44?g g^?1, productivity of 0.45?g L^?1?h^?1 and biomass yield of 0.40?g g^?1.     

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.     

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.     

Xanthan Gum Production by <Emphasis Type="Italic">Xanthomonas campestris pv. campestris</Emphasis> IBSBF 1866 and 1867 from Lignocellulosic Agroindustrial Wastes

This study aimed to evaluate the properties of xanthan gum produced by Xanthomonas campestris pv. campestris 1866 and 1867 from lignocellulosic agroindustrial wastes. XG was produced using an orbital shaker in a culture medium containing coconut shell (CS), cocoa husks (CH), or sucrose (S) minimally supplemented with urea and potassium. The XG production results varied between the CS, CH, and S means, and it was higher with the CH in strains 1866 (4.48 g L^?1) and 1867 (3.89 g L^?1). However, there was more apparent viscosity in the S gum (181.88 mPas) and the CS gum (112.06 mPas) for both 1866 and 1867, respectively. The ability of XG_CS and XG_CH to emulsify different vegetable oils was similar to the ability of XG_S. All gums exhibited good thermal stability and marked groups in the elucidation of compounds and particles with rough surfaces.     

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.     

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.     

Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant,Thermostable β-Xylosidase from a Tropical Strain of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> CBS 135684

From three cell-associated β-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60–70 °C) over a pH range of 5–9 with a specific activity of 163.3 units (U) mg^?1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a K _i value of 18 mM. The K _m and V _max values against p-nitrophenyl-β-d-xylopyranoside were 5.57 ± 0.27 mM and 137.0 ± 4.8 μmol min^?1 mg^?1 protein, respectively. When combining this β-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812 ± 0.015 g g^?1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6 U g^?1 β-xylosidase and 16 U g^?1 xylanase after incubation for 4 h at 70 °C and pH 6.0.     

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.     

Contribution of Tris Buffer on Xylitol Enzymatic Production

Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L^?1 h^?1 xylitol volumetric productivity with 99% xylose–xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.     

Characterization of Truncated <Emphasis Type="Italic">dsz</Emphasis> Operon Responsible for Dibenzothiophene Biodesulfurization in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. FUM94

Numerous desulfurizing bacteria from the Rhodococcus genus harbor conserved dsz genes responsible for the degradation of sulfur compounds through 4S pathway. This study describes a newly identified desulfurizing bacterium, Rhodococcus sp. FUM94, which unlike previously identified strains encodes a truncated dsz operon. DNA sequencing revealed a frameshift mutation in the dszA gene, which led to an alteration of 66 amino acids and deletion of other C-terminal 66 amino acids. The resulting DszA polypeptide was shorter than DszA in Rhodococcus sp. IGTS8 reference strain. Despite the truncation, desulfurizing activity of the operon was observed and attributed to the removal of an overlap of dszA and dszB genes, and lack of active site in the altered region. Desulfurization experiments resulted in specific production rate of 6.3 mmol 2-hydroxy biphenyl (kgDCW)^?1 h^?1 at 2 g l^?1 biocatalyst concentration and 68.8% biodesulfurization yield at 20 g l^?1 biocatalyst concentration, both at 271 μM dibenzothiophene concentration which is comparable to similar wild-type biocatalysts.     

Prospecting fungal ligninases using corncob lignocellulosic fractions

Microorganisms play an important role in the bioconversion of organic residues and have therefore become promising for obtaining value-added enzymes. In an attempt to take advantage of the by-products and residues of bioconversion, this work sought to use lignocellulosic fractions extracted from corncob as fermentation substrate for ligninase induction by Pleurotus sajor-caju. To obtain the corncob lignocellulosic fractions, biomass was submitted to treatment by alkaline extraction (NaOH 0.75 mol L^?1, 55 °C for 2 h) and organosolv (40% ethanol/water, 185 °C for 20 min). The in natura biomass and lignocellulosic fractions were used as substrates in the subsequent fermentation processes: 2% in natura corncob; 2% cellulose–lignin complex fraction; 2% lignin-enriched fraction; 1% lignin-enriched fraction; and synthetic medium fungal (SMF) as standard. Chemical and physical–chemical analyses indicated the effectiveness of the lignocellulosic extraction process. According to the results, the developed system promoted the induction of ligninases by P. sajor-caju. The enzymatic analysis showed laccase production (768 U L^?1) using the 1% lignin-enriched fraction as substrate. Manganese peroxidase production was 1050 U L^?1 with the use of the 2% lignin-enriched fraction. The presence of lignocellulosic fractions extracted from corncob’s lignin-enriched fraction in the culture medium favored the induction of ligninases in comparison to the use of residue alone.     

Improvement on <Emphasis Type="SmallCaps">d</Emphasis>-xylose to Xylitol Biotransformation by <Emphasis Type="Italic">Candida guilliermondii</Emphasis> Using Cells Permeabilized with Triton X-100 and Selected Process Conditions

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by d-xylose and MgCl₂·6H₂O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % d-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the d-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.     

Synthesis,antifungal and insecticidal activity of novel [1,2,4]triazolo[4,3-<Emphasis Type="Italic">a</Emphasis>]pyridine derivatives containing a sulfide substructure

A series of [1,2,4]triazolo[4,3-a]pyridine derivatives bearing a sulfide substructure was designed, synthesized and characterized via ¹H·NMR, ¹³C·NMR, IR and elemental analyses. Bioassay Results indicated some of the derivatives displayed good fungicidal activity on Rhizoctonia cerealis, moderated insecticidal activity against Plutella xylostella and good insecticidal activity on Helicoverpa armigera. The inhibitory effects of compounds 4g and 4u against Rhizotonia cerealis were 70.9% at 50 μg mL^?1; the IC₅₀ values of compounds 4d and 4s against Plutella xylostella were 43.87 and 50.75 μg mL^?1, respectively. And the IC₅₀ values of compounds 4d, 4q, and 4s on Helicoverpa armigera were 58.3, 77.14 and 65.31 μg mL^?1, respectively, which were better than that of commercial chlorpyrifos (103.77 μg mL^?1).     

PVA-Hydrogel Entrapped <Emphasis Type="Italic">Candida Guilliermondii</Emphasis> for Xylitol Production from Sugarcane Hemicellulose Hydrolysate

Viable cells of Candida guilliermondii were immobilized by inclusion into polyvinyl alcohol (PVA) hydrogel using the freezing–thawing method. Entrapment experiments were planned according to a 2³ full factorial design, using the PVA concentration (80, 100, and 120 g L⁻¹), the freezing temperature (−10, −15, and −20 °C), and the number of freezing-thawing cycles (one, three, and five) as the independent variables, integrated with three additional tests to estimate the errors. The effectiveness of the immobilization procedure was checked in Erlenmeyer flasks as the pellet capability to catalyze the xylose-to-xylitol bioconversion of a medium based on sugarcane bagasse hemicellulosic hydrolysate. To this purpose, the yield of xylitol on consumed xylose, xylitol volumetric productivity, and cell retention yield were selected as the response variables. Cell pellets were then used to perform the same bioconversion in a stirred tank reactor operated at 400 rpm, 30 °C, and 1.04 vvm air flowrate. At the end of fermentation, a maximum xylitol concentration of 28.7 g L⁻¹, a xylitol yield on consumed xylose of 0.49 g g⁻¹ and a xylitol volumetric productivity of 0.24 g L⁻¹ h⁻¹ were obtained.