María del Carmen Montes-Horcasitas

Cloning and expression of a novel, moderately thermostable xylanase-encoding gene (Cfl xyn11A) from Cellulomonas flavigena

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.

Alternative non-chromatographic method for alcohols determination in Clostridium acetobutylicum fermentations

An economic, simple, quantitative, and non-chromatographic method for the determination of alcohols using microdiffusion principle has been adapted and validated for acetone-butanol-ethanol (ABE) fermentation samples. This method, based on alcohols oxidation using potassium dichromate in acid medium, and detection by spectrophotometry, was evaluated varying, both, temperature (35°C, 45°C, and 55°C) and reaction time (0 to 125min). With a sample analysis time of 90min at 45°C, a limit of detection (LOD), and a limit of quantification (LOQ) of 0.10, and 0.40g/L, respectively. The proposed method has been successfully applied to determine butanol and ethanol concentrations in ABE fermentation samples with the advantage that multiple samples can be analyzed simultaneously. The measurements obtained with the proposed method were in good agreement with those obtained with the Gas Chromatography Method (GCM). This proposed method is useful for routine analysis of alcohols and screening samples in laboratories and industries.

Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture.

Plasmid DNA (pDNA) production has recently increased as a result of advances in DNA vaccines. The practical development of pDNA vaccines requires high yield and productivity of supercoiled plasmid DNA (sc-pDNA). The yield and productivity are influenced by the host strain, the plasmid, the production process, and especially by growth conditions, such as the culture type and medium. We evaluated different strategies to increase pDNA production by Escherichia coli DH5α in batch culture. The strategies were driven by the development of a four single-factor experimental design and were based on the change of culture media composition in terms of carbon and nitrogen and the modification of the pH control by using NaOH or NH4OH. The results revealed the carbon (50g/L of glycerol) and nitrogen (8.34g/L of YESP) concentration in the culture medium and starting pH control with NH4OH when most of the organic nitrogen was consumed. Under these conditions, we obtained a volumetric yield of 213mg pDNA/L, a specific yield of 10mg pDNA/g DCW (dry cell weight), 92% of sc-pDNA and a productivity of 17.6mg pDNA/(Lh). The pDNA productivities reached were 42% higher than the productivities reported by other authors applying similar conditions.

Removal of phenanthrene in aqueous solution containing photon competitors by TiO2–C–Ag film supported on fiberglass

ABSTRACT Surface interactions with pollutants and photons are key factors that affect the applications of TiO2 in environmental remediation. In this study, the solubilizing agents dimethylsulfoxide and polyoxyethylene sorbitan monooleate, which act as photon competitors, had no effect on the photocatalytic activity of TiO2–C–Ag film in phenanthrene (PHE) removal. Fiberglass with TiO2–C–Ag coating removed 91.1 ± 5.2 and 99.7 ± 0.4% of PHE in treatments using UVA (365–465 nm) and UVC (254 nm) irradiation, respectively. The use of fiberglass as a support increased the superficial area, thus allowing PHE sorption. C and Ag, which are electrically active impurities in TiO2, enhanced its photocatalytic activity and thus the attraction of the pollutant to its surface. The use of high-frequency UV light (UVC) decreased the amount of carbon species deposited on the TiO2CAg film surface. X-ray photoelectron spectroscopy of the TiO2–C–Ag film revealed extensive oxidation of the carbon deposited on the film under UVC light and loss of electrons from Ag clusters by conversion of Ag0 to Ag3+.

Production of a halotolerant biofilm from green coffee beans immobilized on loofah fiber (Luffa cylindrica) and its effect on phenanthrene degradation in seawater

ABSTRACT A biofilm developed from low quality green coffee beans was tested for its capacity to degrade the polynuclear aromatic hydrocarbon (PAH), phenanthrene (Phe), in seawater. Microorganisms were immobilized on two types of Luffa cylindrica (with three and four placental cavities), and the effects of moisture content (20, 30 and 40% of water holding capacity) and particle size (<0.42 mm, 0.42–0.86 mm and 0.86–2.0 mm) of green coffee beans on microbial activity were considered. Biofilm growth determined by respirometry showed a highest microbial activity at a moisture content of 40% and particle size of 0.42–0.86 mm. The loofah fiber with three placental cavities showed the highest adherence of microorganisms. The kinetics of microbial growth in both seawater and distilled water and the scanning electron microscopies indicated that the microorganisms associated with green coffee beans are halotolerant. In fact, I-GCB-SW-G biofilm degraded 67.56% of Phe (50 mg L−1) in seawater, at a significantly higher rate than in distilled water (I-GCB-DW-W).

Halotolerant Biofilm in Coffee Beans for Phenanthrene Degradation Under Selected Culture Conditions through a Plackett-Burman Experimental Design

A halotolerant biofilm was developed on green coffee beans by sequential enrichment in the presence of diesel in order to degrade phenanthrene. Experiments were performed under several combined culture conditions using a Plackett-Burman experimental design. The microorganisms composing the biofilm were identified. These included three fungi, Aspergillus niger, Fusarium solani and Fusarium oxysporum, identified by the Fungal Biodiversity Centre of Holland by macroscopic and microscopic morphological evaluation and by molecular techniques (18s-ITS1-5.8SITS2- 28s rRNA); two yeasts, Candida orthopsilosis and Rhodotorula mucilaginosa; and two bacteria, Pseudomonas putida and Klebsiella variicola, identified by the Mexican Collection of Microbial Cultures (Coleccion Mexicana de Cultivos Microbianos, CINVESTAV) according to colony morphology and biochemical [API 20C AUX and API20 (NE and E), respectively] and molecular (fragments ITS1-5.8S-16s-26s and 16s rRNA, respectively) tests. Each microorganism and biofilm were tested for their tolerance to diesel and salinity conditions and their capacity to degrade phenanthrene; degradation capacity was affected significantly (α<0.05) at low concentrations of NaNO3 and KH2 PO4 and at high concentrations of peptone, CaCO3 and FeSO4 (R2=0.95; C=24.97). Two of the tested conditions, T2 and T5, degraded 91.92% and 89.33%, respectively, of 180 mg/L of phenanthrene. Thus, the biofilms developed from microorganisms of green coffee beans preadapted to salinity conditions presented the metabolic capability to remove phenanthrene from seawater under selective culture conditions.