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La nanotecnología es considerada como la revolución industrial del siglo XXI, por esto la obtención y aplicación de nanopartículas (NPs) es de vital interés para muchos rubros de la ciencia e industria. El uso de las NPs-Fe3O4 obtenidas por síntesis verde se extiende desde la cosmética, medicina, remediación ambiental, eléctrica y electrónica, debido a sus características térmicas, ópticas y magnéticas, sin mencionar el bajo costo de producción. En esta revisión se analizan los factores con mayor efecto durante la síntesis de NPs-Fe3O4 como lo son el pH, la temperatura, el tiempo, la cantidad de precursores metálicos y cantidad de metabolitos secundarios; variables que afectan de manera directa el tamaño, forma y morfología de las NPs-Fe3O4.

Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory. Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.

Microbial production of aroma compounds is a promising alternative to extracting plants or chemical synthesis. The yeast Pichia fermentans ITD-00165 has demonstrated a high potential to produce isoamyl acetate. The yeast was cultured in a bioreactor on a chemically defined medium to evaluate the influence of airflow (0.5, 0.75, and 1.0 vvm) and agitation speed (150, 175, and 200 rpm) on the isoamyl acetate production. Isoamyl acetate was retained using an in situ entrapping system, which was obtained, adding a cap of n-decane in a ratio of 80:20 (medium:decane). This work demonstrated the relevance of well-controlled aeration to enhance isoamyl acetate production using isoamyl alcohol as a precursor. Culture operating near to 0.71 vvm and 168 rpm (kLa = 0.16 min-1) achieved production of 2.14 g L-1, which means an almost 2.5-fold increase in respect to the higher value previously reported. On the other hand, the minimum production occurred at the best aeration conditions (kLa = 0.22 min-1), meaning that excessive aeration could negatively affect the aroma production. Nevertheless, it appears that the concomitant ethanol production negatively affects aroma production.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low β-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized β-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The β-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the β-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, β-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

This review presents important information about general aspects of the mining process as a source of environmental pollution, economic and health impact, microorganisms involved in the microenvironments resulting from ore exploitation, and resistance mechanisms to As and Zn, as a global vision of topics of interest to researchers or students involved in this field. The exploitation of mineral resources generates ideal but extreme microenvironments, where the growth of microorganisms adapted to high concentration levels of heavy metals in comparison to common soil occurs; tailings, contaminated soils, and acid mine drains are sources of culturable heavy metal–resistant microorganisms that naturally grow under stressful conditions. The present review focuses on As and Zn as heavy metal pollutants, due to their presence in high concentrations in mining wastes, regardless of geographic location and mineral processes, in addition to their toxicity; the low profile of Zn as a pollutant even at the alarming levels can be found in mining wastes and consequently in the environment; the impact of ore processing is also included and provides an overview of some of the data on the relationships of mining microbiology highlighting the potential of microorganisms to eliminate these compounds discharged by any means into the environment. The relevance of this compendium is to highlight the interaction of native microorganisms present in mining wastes with high concentrations of heavy metals (As and Zn) as a biological treatment to remove such contaminants as an alternative to conventional technologies.

Recently, biotechnological opportunities have been found in non-Saccharomyces yeasts because they possess metabolic characteristics that lead to the production of compounds of interest. It has been observed that Kluyveromyces marxianus has a great potential in the production of esters, which are aromatic compounds of industrial importance. The genetic bases that govern the synthesis of esters include a large group of enzymes, among which the most important are alcohol acetyl transferases (AATases) and esterases (AEATases), and it is known that some are present in K. marxianus, because it has genetic characteristics like S. cerevisiae. It also has a physiology suitable for biotechnological use since it is the eukaryotic microorganism with the fastest growth rate and has a wide range of thermotolerance with respect to other yeasts. In this work, the enzymatic background of K. marxianus involved in the synthesis of esters is analyzed, based on the sequences reported in the NCBI database.

The removal of total petroleum hydrocarbons (TPHs) from contaminated mining soil was carried out under in vitro conditions. The aerobic consumption of TPH in the slow bioremediation stage via biostimulation with native microorganisms and biostimulation-bioaugmentation with autochthonous fungal isolates was evaluated. The initial TPH concentration was 70,880 ± 975 mg TPH/kg soil, soil was amended with nutrients at a C:N:P ratio of 100:15:1, the water content was adjusted to the soil field capacity, and batch microcosm reactors were incubated at room temperature (20.5 ± 3.1°C) for 90 days. The bioaugmentation process was tested using four hydrocarbonoclastic fungal strains isolated from the same contaminated mining soil individually and a mixed culture of the four isolates. The molecular characterization of the isolated fungi was based on sequence analysis of 18S rRNA, and the fungi were identified as Aspergillus niger MT786339.1, Aspergillus fumigatus MT786338.1, Aspergillus terreus MT786341.1, and Aspergillus flavus MT786340.1. The best TPH removal was achieved by inoculation with the fungal consortium (57 ± 1.97%) at 45 days (slow stage) after initiating the biostimulation process, followed by inoculation with Aspergillus niger (49 ± 1.2%), Aspergillus terreus (44 ± 0.67%), Aspergillus fumigatus (35 ± 0.98%), and Aspergillus flavus (32 ± 0.38%), while the degradation rate achieved with native microorganisms was only 21.6 ± 1.5%; statistical analysis of the results showed significant differences.

Bioleaching is suggested as a methodology with benefits in ex situ/in situ bioremediation for reducing metal contamination in dredged sediments. The process of bioleaching assisted by microorganisms in a stirred tank is widely used, among other biohydrometallurgical techniques, for the efficient extraction of metals of interest if the recovery costs of valuable metals allow it. Measurement of the mass transfer capacity of oxygen in an aerobic bioreactor is of vital importance; this factor determines the productivity of the system, since it promotes microbial growth and hence enables a bioconversion process at the mineral surface takes place. The main objective of the present research was to determine the volumetric oxygen transfer coefficient (kLa) during the manganese bioleaching process in a stirred tank reactor using silicone oil as a vector to improve the transfer of gaseous substrates. Experiments were carried out in triplicate with a 22 factorial design analyzing temperature (30 and 40 °C) and stirring speed (300 and 500 rpm). The effect of viscosity on the system was also determined by modifying the concentrations of mining waste (30, 40, and 50%), ferrous sulfate (FeSO4 · 7H2O) (2, 4, 8, and 14%), and silicone oil (0, 5, and 10%) at 0 and 24 h of mixing. The results showed an increase in the value of kLawith respect to time, from kLa(0 h, 30 °C, 500 rpm) = 8.75 s−1 to kLa(24 h, 40 °C, 500 rpm) = 18.18 s−1; ANOVA statistical analysis was performed with the Fisher and Tukey tests and showed that there were statistically significant differences.

The search for microbial enzymatic activities applied to wastewater treatment is an important task in environmental biotechnology. Microbial enzymes have been previously explored in hostile habitats. They are increasingly important in extreme habitats; biological wastewater from the pulp and paper mill industry can harbor microorganisms with valuable enzymatic capabilities that can improve the efficiency for the same process of depuration. This study was performed to characterize and evaluate cellulolytic, amylolytic, and lipolytic activities of bacteria isolated from a pulp and paper effluent. The enzymatic activities were evaluated by the formation of a clear halo around the colonies in defined substrate media. By the use of a sequence analysis of 16S rDNA libraries, isolates were identified. The 16S rDNA libraries belong to the Bacillus subtilis, B. megaterium, B. licheniformis, B. pumilus, B. thuringiensis, B. cereus, Chryseobacterium daecheongense, and Microbacterium sediminis (an alkali-tolerant bacteria which has only been isolated from deep-sea sediment). B. cereus was the best strain for cellulose and lipase activities; moreover, C. daecheongense was best for amylase activity. The present study shows that the aerated lagoons from the pulp and paper industry are a promising source of bacterial with different enzyme activities. This data is relevant for industrial applications.

Recently, biotechnological opportunities have been found in non-Saccharomyces yeasts because they possess metabolic characteristics that lead to the production of compounds of interest. It has been observed that Kluyveromyces marxianus has a great potential in the production of esters, which are aromatic compounds of industrial importance. The genetic bases that govern the synthesis of esters include a large group of enzymes, among which the most important are alcohol acetyl transferases (AATases) and esterases (AEATases), and it is known that some are present in K. marxianus, because it has genetic characteristics like S. cerevisiae. It also has a physiology suitable for biotechnological use since it is the eukaryotic microorganism with the fastest growth rate and has a wide range of thermotolerance with respect to other yeasts. In this work, the enzymatic background of K. marxianus involved in the synthesis of esters is analyzed, based on the sequences reported in the NCBI database.

Two varieties of common beans (Phaseolus vulgaris L.), Bayo Victoria and Negro 8025, were evaluated to determine the effect on cellular viability and mechanisms involved in apoptosis pathways, using a cellular model with HT-29 cells. Aqueous methanolic (50:50) extracts from cooked beans were analyzed for phenolic composition, identifying greater diversity of phenolic compounds in Bayo Victoria extracts. However, Negro 8025 showed greater phenolic content and cytotoxicity effects at lower media inhibitory concentrations, and greater effectiveness to activate apoptotic pathways. Proteins related to the arrest of cell cycle were modulated by both bean cultivars. Qualitative analysis by HPLC-PAD and HPLC-MS systems of phenolic compounds in common bean extracts showed mainly hydroxybenzoic and hydroxycinnamic acids, flavonols, and monomeric flavan-3-ols. Bioactive phenolics such as catechin, kaempferol, and ferulic acid were found in both cultivars as well anticancer phytochemicals such as quercetin, protocatechuic acid, myricetin, naringenin and their derivatives, and procyanidins., This work was financially supported by the Tecnológico Nacional de Mexico (TecNM) (Project grant No. 5737.16‐P). We acknowledge to the Mexican Council of Science and Technology (CONACyT) for the analytical equipment acquired by means of two project grants (No. 253333 and 224651). Author RLB also recognizes graduate scholarship from CONACyT.

The aim of this study was to identify cellulolytic fungal strains capable of degrading cellulose from an aerated lagoon in a pulp and paper mill. Four fungal strains that were found to be highly active were isolated on carboxymethyl cellulose (CMC) and suggested to be CMCase/endoglucanase. The identified strains were Aspergillus niger, Penicillium sp., Aspergillus fumigatus, and Mucor sp. All the strains were studied in terms of cultural morphological characteristics and microscopic examinations. The endoglucanase with the highest isolate production was Penicillium sp., which also showed the highest qualitative endoglucanase activity (1.3 cm), in addition to the main activity of endoglucanase with 297 mmol/mg.min after 116 h. The results indicated that CMC is able to induce endoglucanase enzyme production and that the fungal isolates showed significant cellulose degradation properties.

Pulsed electric field (PEF) is a promising non-thermal food preservation technology. The objective was to study inactivation of yeasts in PEF-treated kombucha analogues prepared from Quercus obtusata infusions. Fermentation conditions of infusions from Q. obtusata were time (7 days), sugar (10%), starting culture (10%), and inoculum (2.5%, at 25 °C). The PEF treatment considered using square waves, an electric field strength (37.3–53.4 kV/cm), PEF processing time (445.3–1979.2 μs), an output temperature (18.31 ± 0.98 °C), an input energy (21.2 - 136.5 KJ/L), and two feed flow rates (51.42 and 102.85 L/h). pH, °Brix, color determinations, microbiological testing, total phenolic, flavonoid content, DPPH test, and UPLC/ESI/MS/MS analysis were done. No changes at different PEF conditions were observed for pH and °Brix. Higher color changes were observed at higher specific energies. Acid-acetic bacteria were more sensitive to PEF than yeasts. Lower specific energies render products with higher polyphenolic content and antioxidant capacity. Industrial relevance Pulse electric field is an interesting alternative to preserve kombucha analogues from oak leave infusions with minimal changes in physicochemical characteristics, antioxidant activity and bioactive compounds. The present work describes the effect of feed flow and specific energy on the several characteristics of fermented beverages, determining conditions for best processing.

This study was conducted with a fungal strain isolated from a mining soil contaminated with total petroleum hydrocarbons (TPH) and properly identified by pol...

The goal of this study was to evaluate the effect of high Tween 80 concentrations on hydrocarbon contaminants and hydrocarbonoclastic microorganisms present in contaminated mining soil during a bioremediation process. The independent variables included Tween 80 concentration and process time. The elected response variables included concentration of total petroleum hydrocarbons (TPH), the chemical composition of contaminants, viable microbial count, and CO2 production. These were measured at various time points during the bioremediation process, which was conducted at room temperature. Higher removal of pollutants occurred at low Tween 80 concentrations. An analysis of the normalized variables shows that the rate of TPH degradation depended on microbial growth rate, which depended on Tween 80 concentration. The addition of Tween 80 changed the concentration and type of the residual hydrocarbon contaminants present, as well as the count and genus of the hydrocarbonoclastic microorganisms that grew during the bioremediation process. Higher concentrations of Tween 80 increased the levels of the chemical compounds present in the bioremediated soil while reducing the viable count and microbial diversity during the bioremediation process. Conversely, low Tween 80 concentrations produced only monoethylhexyl phthalate, which is not a petroleum hydrocarbon derivative. Overall, these results prove that the removal of TPH is dependent on the count and genus of active indigenous hydrocarbonoclastic microorganisms in soil, and these conditions may be controlled by modulating the concentration of Tween 80 that is applied.

The dimeric enzyme β-glucosidase from Aspergillus niger has been immobilized on different amino-agarose beads at pH 5 and 7, exploiting the versatility of glutaraldehyde. The stability of the free enzyme depended on enzyme concentration. Immobilization via ion exchange improved enzyme stability/activity, depending on the immobilization pH. However, the enzyme was desorbed in 75 mM NaCl at pH 7 and some stability/enzyme concentration dependence still existed. Treatment: of these biocatalysts with glutaraldehyde increased enzyme stability (e.g. at pH 5, after incubation under conditions where the enzyme just ionically exchanged was fully inactivated, the activity of the glutaraldehyde treated enzyme remained unaltered). Immobilization on glutaraldehyde pre-activated supports yielded a higher increase in enzyme activity, but the stabilization was lower. While when measuring the enzyme activity at pH 4 there were no changes after immobilization, all immobilized enzymes were more active than the free enzyme at pH 6 and 7 (2–3 times). The Ki/Km ratio did not significantly decrease in any immobilized biocatalysts, and in some cases it worsened in a significant way (by a 9 fold factor using preactivated supports). The new biocatalysts are significantly more stable and avoid enzyme subunit desorption, being the immobilization pH a key point in their design.

Aerated lagoons are a main unit operation for wastewater treatment in the paper industry. Many such operations involve inoculation with bacterial formulations in which in situ effectiveness has not been proven; this can be translated into low efficiency in treatment and unnecessary investments. Lack of knowledge of bacterial biodiversity present in a lagoon limits the capacity to exploit the maximum degradation. To overcome such problems, various methods to identify and study these microorganisms have been developed. In this study, a PCR-DGGE analysis was performed to estimate the bacterial diversity and to verify the presence of bacteria present in a commercial inoculum in two aerated lagoons of a pulp and paper effluent. Phylogenetic affiliation of predominant member’s correspondent to γ- and β-proteobacteria and Firmicutes were found. The dominant bacteria present in lagoon 2 belonged to the following genus Microbacterium sp. Rhodocyclaceae sp., Eubacterium sp. and B. subtilis. In lagoon 1 the dominant genus included Microbacterium sp., Rhodocyclaceae sp. Tepidimonas sp., Acetanaerobacterium sp., and Flavobacteria sp. The two characterized lagoons were not similar to the commercial inoculum. In addition, non-dominant bacteria (less relative intensity) were composed mostly of bacteria of the commercial inoculum.

One of the processes most used in biotechnology today for handling industrial liquid wastes is biological wastewater treatment. The efficiency and quality of its operation depends on the composition and activity of the microbial community that is present. The application of traditional and molecular techniques has provided a glimpse into the “black box” and has given information to improve the wastewater treatment process. However, bleach pulp and paper mill effluents require a better understanding of the active bacterial population. For the study of these microorganisms, molecular techniques have been used for more than 15 years. However, there has been a lack of knowledge of the physiological requirements and relations with the environment, which seems to be very difficult to obtain involving profile on the diversity. Nowadays, high-throughput sequencing technology is a promising method that makes it possible to identify the entire profile of microbial communities. In combination with fingerprint methods, this approach allows the identification and analysis of the whole biodiversity of microbial communities. In this review, several identification techniques will be discussed.

A pilot-scale aerated lagoon was used for the aerobic treatment of pulp and paper mill effluent from September 1 to December 1, 2014. The aerated lagoon was installed at the chemistry laboratory in the Chemical Engineering Department at the Durango Institute of Technology and was fed with real pulp and paper mill effluent. The experimental work was run under various operating conditions. The operating parameters (total and volatile suspended solids (VSS) and dissolved oxygen concentration (DO)) and environmental variables (temperature, pH, COD, and BOD5 of influent water) were monitored daily. In all the experiments conducted, the aerated lagoon generated an effluent of optimal quality complying with the requirements of SEMARNAT (2003) and CONAGUA (2003). A model that explains the behavior of the system under realistic operating conditions was obtained. The model indicated an optimal DO of approximately 4 mg/L for concentrations up to 1000 mg/L, showing variations in concentrations above this value. This data indicate that the flexibility of the bacterial formulation and its ability to adapt to environmental changes play an important role in the stability of an aerated lagoon.

The kombucha beverage is typically prepared from black tea, sweetened with sucrose, inoculated with previously fermented liquid broth and/or tea fungus pellicle, and incubated. Alternative sources have been used for kombucha beverages. Guava leaves have been used for long time as traditional medicine. It is found in many commercially available botanical supplements in form of decoction, milled and used as comprises. They are rich in polyphenolic compounds. Several changes are produced during fermentation of the beverages. The main objective of the present work is to characterize fermentation process of guava leaves infusions by kombucha and studying possible chemical changes in their polyphenolic profile. Infusions from guava leaves were prepared and fermented by the kombucha consortium. The pH, titrable acidity, polyphenolic compounds, sugar consumption, organic acid along the fermentation was made by UPLC-ESI-MS. Kombucha from Camellia sinensis (CS) was made as a control. Higher rate of sucrose consumption was observed for Kombucha made with CS, also, higher production of organic acids (acetic and succinic acid) was observed too. Both behaviors were related to the content of glucose. The flavan-3-ols were diminishing along the fermentation time, with the exception of epigallocatechin in Camellia sinensis, Flavan-3-ol content in Guava leaves was low. Higher content of dicaffeoyl quinnic acid was observed for both systems in special for CS, falling after a maximum peak; minor constituents of hydroxycinnamic acids were stable along the fermentation for both systems.

Pulp and paper mills are a major source of pollution, generating huge amounts of intensely colored effluent that goes to the receiving end of a wastewater treatment plant. The biochemical oxygen demand test (BOD5) relies heavily on the microorganism metabolic capability added to the test as seeding material. The seeding material in the testing is obtained from sewage sampling or from commercial sources. Specific organic pollutants that are present in paper and pulp mill effluent can only be degraded by specific microbes; therefore, common sewage or synthetic seed may lead to erroneous BOD5 estimations. In this study, specific microbial species were selected to evaluate their degradation efficiency, both individually and in combination. The microorganisms selected in the formulated seed exhibit BOD5 in a reproducible and synergistic manner. The formulation of this specific microbial consortium can be used to develop bioremediation strategies.

Restarter yeast strains are used to consume residual sugar (mainly fructose) in stuck wine fermentations. Forty-three yeast strains were evaluated initially. Strain ITD-00068 showed the highest values for the maximum growth rate, maximum fructose consumption rate, and maximum ethanol production rate (0.143, 0.268, and 0.231 h−1, respectively). Therefore, it was selected for further molecular and kinetic analyses. RFLP analysis identified this isolate as Saccharomyces cerevisiae. Strain ITD-00068 consumed 100% and 36% of the fructose present (at 20°C and 30°C, respectively), when cultured in medium with 12% (v/v) ethanol. The fructose consumption rate was reduced by 97% at 30°C, when ethanol was initially present in the media. S. cerevisiae ITD-00068 was tested in stuck fermentation conditions and was able to complete fermentation in 144 h in a commercial red wine, demonstrating that S. cerevisiae ITD-00068 is a potential candidate for use as a restarter in wine stuck fermentation at 20°C.