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Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H2O instead of H2O2. The measurement of laccase activity (UL-1) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ420nm, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL-1 for rGILCC 1 and 5291.665 ± 45.83 UL-1 for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower Km values occurred for both enzymes (1.49 × 10-2 mM for rGILCC 1 and 3.72 × 10-2 mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10-2 mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1-ABTS and POXA 1B-ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model-ABTS interactions (GILCC 1-ABTS and POXA 1B-ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme-substrate interactions, offering potential applications in environmental substrate treatments.
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Lacasa , Simulación de Dinámica Molecular , Lacasa/metabolismo , Peróxido de Hidrógeno , Citratos , Oxidación-ReducciónRESUMEN
We produced and characterised biochar made from Caribbean pine sawdust as raw material. The biochar (BC500) was used as biocompatible support to co-inoculate phosphate solubilizing bacteria (PSB) (BC500/PSB) on Allium cepa L., plants at a greenhouse scale for four months. The three biomaterials study included proximate analysis, elemental analysis, aromaticity analysis, scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), adsorption studies at different pH and PSB stability as a function of time. The results indicated that BC500 is suitable as organic support or solid matrix to maintain the viability of PSB able to solubilise P from phosphate rock (PR). The biofertilizer (BC500/PSB) allows increasing germination, seedling growth, nutrient assimilation, and growth of Allium cepa L., because PSB immobilised on BC500 promoted nutrient mobilisation, particularly P, during cultivation of Allium cepa L., at pots scale. The two treatments to evaluate the biofertilizer (BC500/PSB) showed the highest concentrations of total P with 1.25 ± 0.13 and 1.38 ± 0.14 mg bulb-1 in A. cepa L. This work presents the benefits of a new product based on bacteria naturally associated with onion and an organic material (BC500) serving as a bacterial carrier that increases the adsorption area of highly reactive nutrients, reducing their leaching or precipitation with other nutrients and fixation to the solid matrix of the soil.
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Fosfatos , Pinus , Bacterias , Carbón Orgánico/química , Cebollas , Fosfatos/química , Suelo/químicaRESUMEN
The first traces of Tetracycline (TE) were detected in human skeletons from Sudan and Egypt, finding that it may be related to the diet of the time, the use of some dyes, and the use of soils loaded with microorganisms, such as Streptomyces spp., among other microorganisms capable of producing antibiotics. However, most people only recognise authors dating between 1904 and 1940, such as Ehrlich, Domagk, and Fleming. Antibiotics are the therapeutic option for countless infections treatment; unfortunately, they are the second most common group of drugs in wastewaters worldwide due to failures in industrial waste treatments (pharmaceutics, hospitals, senior residences) and their irrational use in humans and animals. The main antibiotics problem lies in delivered and non-prescribed human use, use in livestock as growth promoters, and crop cultivation as biocides (regulated activities that have not complied in some places). This practice has led to the toxicity of the environment as antibiotics generate eutrophication, water pollution, nutrient imbalance, and press antibiotic resistance. In addition, the removal of antibiotics is not a required process in global wastewater treatment standards. This review aims to raise awareness of the negative impact of antibiotics as residues and physical, chemical, and biological treatments for their degradation. We discuss the high cost of physical and chemical treatments, the risk of using chemicals that worsen the situation, and the fact that each antibiotic class can be transformed differently with each of these treatments and generate new compounds that could be more toxic than the original ones; also, we discuss the use of enzymes for antibiotic degradation, with emphasis on laccases.
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Antibacterianos , Lacasa , Contaminantes Químicos del Agua , Antibacterianos/análisis , Farmacorresistencia Microbiana , Aguas Residuales/química , Contaminantes Químicos del Agua/análisisRESUMEN
The history of colour is fascinating from a social and artistic viewpoint because it shows the way; use; and importance acquired. The use of colours date back to the Stone Age (the first news of cave paintings); colour has contributed to the social and symbolic development of civilizations. Colour has been associated with hierarchy; power and leadership in some of them. The advent of synthetic dyes has revolutionized the colour industry; and due to their low cost; their use has spread to different industrial sectors. Although the percentage of coloured wastewater discharged by the textile; food; pharmaceutical; cosmetic; and paper industries; among other productive areas; are unknown; the toxic effect and ecological implications of this discharged into water bodies are harmful. This review briefly shows the social and artistic history surrounding the discovery and use of natural and synthetic dyes. We summarise the environmental impact caused by the discharge of untreated or poorly treated coloured wastewater to water bodies; which has led to physical; chemical and biological treatments to reduce the colour units so as important physicochemical parameters. We also focus on laccase utility (EC 1.10.3.2), for discolouration enzymatic treatment of coloured wastewater, before its discharge into water bodies. Laccases (p-diphenol: oxidoreductase dioxide) are multicopper oxidoreductase enzymes widely distributed in plants, insects, bacteria, and fungi. Fungal laccases have employed for wastewater colour removal due to their high redox potential. This review includes an analysis of the stability of laccases, the factors that influence production at high scales to achieve discolouration of high volumes of contaminated wastewater, the biotechnological impact of laccases, and the degradation routes that some dyes may follow when using the laccase for colour removal.
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Colorantes/química , Proteínas Fúngicas/química , Lacasa/química , Aguas Residuales/química , Purificación del Agua , Biodegradación Ambiental , ColorRESUMEN
BACKGROUND: The co-transformation of solid waste of natural and anthropogenic origin can be carried out through solid-state-fermentation systems to obtain bio-products with higher added value and lower environmental impact. METHODS: To evaluate the effect of Pleurotus ostreatus on co-transformation of oxo-degradable low-density polyethylene (LDPEoxo) sheets and lignocellulosic biomass (LCB), were assembled two 0.75 L microcosm systems in vertical (VMS) and horizontal (HMS) position. The pre-treated sheets with luminescent O2 plasma discharges were mixed with pine bark, hydrolyzed brewer's yeast and paper napkin fragments and incubated for 135 days at 20 ± 1.0 °C in the presence of the fungus. With the co-transformation residues, biochar (BC) was produced at 300 ± 1.0 °C (BC300) for 1 h, then used to carry out adsorption studies, using the malachite green dye (MG) at pH 4.0, 7.0 and 9.0 ± 0.2. Finally, the biochar was the substrate for the germination of carnation seeds (Dianthus caryophyllus) and Ray-grass (Lolium sp.) in vitro. RESULTS: For HMS, the decrease in static contact angle (SCA) was 63.63% (p = 0.00824) and for VMS 74.45% (p = 0.00219), concerning the pristine. Plastic roughness in VMS was higher (26%) concerning the control. Throughout the 135 days, there were fungal growth and consequently laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) activities. During the first 75 days, CO2 production increased to 4.78 ± 0.01 and 4.98 ± 0.01 mg g-1 for HMS and VMS, respectively. In MG adsorption studies, the highest amount of the colourant adsorbed at both pH 4.0 and 7.0 ± 0.2. CONCLUSIONS: Finally, the biochar or the biochar enriched with low concentrations of plant growth-promoting microorganisms and inorganic fertilizer favours the germination of Dianthus caryophyllus and Lolium sp., seeds.
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BACKGROUND: Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others. METHODS: A real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K. RESULTS: In the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t1/2 values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an Ed value of 41.40 KJ mol- 1, a low variation of KM and Vmax, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K. CONCLUSIONS: Laccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t1/2 of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.
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Proteínas Fúngicas/química , Lacasa/química , Pichia/enzimología , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Lacasa/genética , Lacasa/metabolismo , Simulación de Dinámica Molecular , Pichia/química , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Liquid waste from biological stains is considered non-domestic wastewater difficult to treat, generating high environmental impact. Therefore, the objective of this work was to carry out secondary and tertiary treatment of these effluents at a pilot scale, using a fungal/bacterial consortium followed by Chorella sp., for 15 days. In addition, to obtain an adsorbent material for Malachite Green dye removal, sludge generated in the plant and pine bark co-pyrolysis was performed. For microalgae isolation and selection of the Chlorophyceae class, Chlorococcales order, and Chorella sp. genus Winogradsky columns were employed. After 15 days of pilot plant treatment, removal percentages of 91 ± 2%, 90 ± 4% and 17 ± 2% were obtained for Colour Units, Chemical Oxygen Demand and Nitrates, respectively. Two types of class II biochar (BC500 and BC700) and one of class III (BC300) were produced. The highest value for Fixed carbon (FC) was obtained at 300 °C (27.3 ± 3%), decreasing as the temperature increased by 25.9 ± 5% and 24.8 ± 2%, for BC500 and BC700, respectively. Biochar yield was 62.1 ± 3%, 46.3 ± 4% and 31.6 ± 3% for BC300, BC500 and BC700, respectively. Finally, BC500 and BC700 biochar efficiently adsorbed Malachite Green obtaining qe values of 0.290 ± 0.032, 0.281 ± 0.015, 0.186 ± 0.009 and 0.191 ± 0.012 mg g-1 at pH values of 4.0 and 8.0 ± 0.2, respectively. Pseudo-second order model demonstrated a chemical adsorption took place, which was influenced by pH. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02780-1.
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Low-density polyethylene (LDPE) sheets (3.0 ± 0.1 cm) received sequential treatment, first by the action of direct-current low-pressure plasma (DC-LPP) with a 100% oxygen partial pressure, 3.0 × 10-2 mbar pressure, 600 V DC tension, 5.6 cm distance, 6-min treatment. Then, sheets were submitted to TiO2 photocatalysis at UV radiation at 254 nm (TiO2/UV) with a pH value of 4.5 ± 0.2 and a TiO2 concentration of 1 gL-1. We achieved a complementary effect on the transformation of LDPE films. With the first treatment, ablation was generated, which increased hydrophilicity. With the second treatment, the cavities appeared. The changes in the LDPE sheets' hydrophobicity were measured using the static contact angle (SCA) technique. The photocatalytic degradation curve at 400 h revealed that the DC-LPP photocatalysis sequential process decreased SCA by 82°. This was achieved by the incorporation of polar groups, which increased hydrophilicity, roughness, and rigidity by 12 and 38%, respectively. These sequential processes could be employed for LDPE and other material biodegradation pretreatment.
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Different genus of bacteria has been reported with the capacity to solubilize phosphorus from phosphate rock (PR). Pseudomonas sp., (A18) and Serratia sp., (C7) isolated from soils at the "Departamento de Boyacá" Colombia, where Allium cepa is cultivated. Bacteria were cultured in MT11B media and evaluated as a bio-fertilizer for A. cepa germination and growth during two months at greenhouse scale. Pseudomonas sp., and Serratia sp., cultured at 30 °C, 48 h in SMRS1 agar modified with PR, (as an inorganic source of phosphorus), presented a phosphate solubilization index (SI) of 2.1 ± 0.2 and 2.0 ± 0.3 mm, respectively. During interaction assays no inhibition halos were observed, demonstrating there was no antagonism between them. In MT11B media growth curve (12 h) demonstrated that co-culture can grow in the presence of PR and glucose concentrations 7.5-fold, lower than in SMRS1 media and brewer's yeast hydrolysate; producing phosphatase enzymes with a volumetric activity of 1.3 ± 0.03 PU at 6 h of culture and 0.8 ± 0.04 PU at 12 h. Moreover, co-culture released soluble phosphorus at a rate of 58.1 ± 0.28 mg L-1 at 8 h and 88.1 ± 0.32 mg L-1 at 12 h. After five days of evaluation it was observed that germination percentage was greater than 90 % of total evaluated seeds, when placing them in contact with the co-culture in a concentration of 1 × 108 CFU mL-1. Furthermore, it was demonstrated that co-culture application (10 mL per experimental unit to complete 160 mL in two months) at 8.0 Log10 CFU mL-1 twice a week for two months increased A. cepa total dry weight (69 ± 13 mg) compared with total dry weight (38 ± 5.0 mg) obtained with the control with water.
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Laccases (E.C. 1.10.3.2) are multicopper oxidases of great importance in the industry due to their non-specificity and high oxidative potential. Laccases are useful to bleach synthetic dyes, oxidize phenolic compounds and degrade pesticides, among others. Hence, the objective of this work was to optimize low cost culture media for recombinant (rPOXA 1B) laccase production from Pleurotus ostreatus in Pichia pastoris. To this end, low cost nitrogen sources were studied, such as malt extract, isolated soy protein and milk serum. Following, two central composite designs (CCD) were performed. In CCD-1 different concentrations of glucose USP (0-13.35 gL-1), protein isolated soy protein (5-25 gL-1), malt extract (3.5-17.5 gL-1) and (NH4)2SO4 (1.3-6.5 gL-1) were evaluated. In CCD-2 only different concentrations of glucose USP (7.9-22 gL-1) and isolated soy protein (15.9-44.9 gL-1) were evaluated. CCD-2 results led to a One Factor Experimental design (OFED) to evaluate higher isolated soy protein (20-80 gL-1) concentrations. In all designs, (CCD-1, CCD-2 and OFED) CuSO4 (0.16 gL-1) and chloramphenicol (0.1 gL-1) concentrations remained unchanged. For the OFED after sequential statistical optimization, an enzyme activity of 12,877.3 ± 481.2 UL-1 at 168 h was observed. rPOXA 1B activity increased 30.54 % in comparison with CCD-2 results. Final composition of optimized media was: 20 gL-1 glucose USP, 50 gL-1 isolated soy protein 90 % (w/w), 11.74 gL-1 malt extract, and 4.91 gL-1 (NH4)2SO4. With this culture media, it was possible to reduce culture media costs by 89.84 % in comparison with improved culture media previously described by our group.
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A simultaneous treatment of lignocellulosic biomass (LCB) and low density oxodegradable polyethylene (LDPEoxo) was carried-out using Pleurotus ostreatus at microcosm scale to obtain biotransformed plastic and oxidized lignocellulosic biomass. This product was used as raw matter (RM) to produce biochar enriched with phosphate solubilizing bacteria (PSB). Biochar potential as biofertilizer was evaluated in Allium cepa culture at greenhouse scale. Experiments including lignocellulosic mix and LDPEoxo were performed for 75 days in microcosm. Biotransformation progress was performed by monitoring total organic carbon (TOC), CO2 production, laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) enzymatic activities. Physical LDPEoxo changes were assessed by atomic force microscopy (AFM), scanning electron microscopy (SEM) and static contact angle (SCA) and chemical changes by Fourier transform infrared spectroscopy (FTIR). Results revealed P. ostreatus was capable of LCB and LDPEoxo biotransformation, obtaining 41% total organic carbon (TOC) removal with CO2 production of 2,323 mg Kg-1 and enzyme activities of 169,438 UKg-1, 5,535 UKg-1 and 5,267 UKg-1 for LiP, MnP and Lac, respectively. Regarding LDPEoxo, SCA was decreased by 84%, with an increase in signals at 1,076 cm-1 and 3,271 cm-1, corresponding to C-O and CO-H bonds. A decrease in signals was observed related to material degradation at 2,928 cm-1, 2,848 cm-1, agreeing with CH2 asymmetrical and symmetrical stretching, respectively. PSB enriched biochar favored A. cepa plant growth during the five-week evaluation period. To the best of our knowledge, this is the first report of an in vitro circular production model, where P. ostreatus was employed at a microcosmos level to bioconvert LCB and LDPEoxo residues from the agroindustrial sector, followed by thermoconversion to produce an enriched biochar with PSB to be used as a biofertilizer to grow A. cepa at greenhouse scale.
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Bacterias/metabolismo , Carbón Orgánico/metabolismo , Lignina/metabolismo , Cebollas/crecimiento & desarrollo , Fosfatos/metabolismo , Pleurotus/metabolismo , Polietileno/química , Agricultura , Biodegradación Ambiental , Biomasa , Fermentación , Cebollas/metabolismo , Cebollas/microbiología , Pleurotus/crecimiento & desarrolloRESUMEN
Low-density polyethylene (LDPE) waste generates an environmental impact. To achieve the most suitable option for their degradation, it is necessary to implement chemical, physical and biological treatments, as well as combining procedures. Best treatment was prognosticated by Plackett-Burman Experimental Design (PB), evaluating five factors with two levels (0.25 mM or 1.0 gL-1 glucose, 1.0 or 2.0 mM CuSO4, 0.1 or 0.2 mM ABTS [2, 20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)], pH 4.5 ± 0.2 or 7.0 ± 0.2 and 30 or 90 day incubation), which was reproduced for 150 days. Therefore, PB identified a sequential treatment (plasma followed by fungus) for partial LDPE biodeterioration. Sheets were pretreated with glow discharge plasma (O2, 3.0 x 10(-2) mbar, 600 V, 6 min.), followed by Pleurotus ostreatus biodeterioration. Fungus growth, colonization percentage, and pigment generation followed. Laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) activities were appraised. Additionally, contact angle (CA), functional group presence and changes and carbonyl and vinyl indices (Fourier transformed infrared spectroscopy) were evaluated. LDPE surface changes were assessed by Young's modulus, yield strength, scanning electronic microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Plasma discharge increased hydrophilicity, decreasing CA by 76.57% and increasing surface roughness by 99.81%. P. ostreatus colonization was 88.72% in 150 days in comparison with untreated LDPE (45.55%). After this treatment carbonyl groups (C = O), C = C insaturations, high hydrophilicity CA (16 ± 4) °, and low surface roughness (7 ± 2) nm were observed. However, the highest Lac and LiP activities were detected after 30 days (Lac: 2.817 U Lac g-1 and LiP: 70.755 U LiP g-1). In addition, highest MnP activity was observed at day 120 (1.097 U MnP g-1) only for P. ostreatus treated LDPE. Plasma favored P. ostreatus adsorption, adherence, growth and colonization (88.72%), as well as partial LDPE biodeterioration, supported by increased hydrophilicity and presence of specific functional chemical groups. The approximate 27% changes in LDPE physical properties support its biodeterioration.
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Biodegradación Ambiental , Gases em Plasma , Pleurotus , Polietileno , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Oxidación-Reducción , Pleurotus/crecimiento & desarrollo , Pleurotus/metabolismo , Polietileno/química , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Factores de Tiempo , Sustancias Viscoelásticas/químicaRESUMEN
Cellulose-pulping requires chemicals such as Cl2, ClO2, H2O2, and O2. The black liquor (BL) generated exhibits a high chemical oxygen demand (COD), five-day biochemical oxygen demand (BOD5), and chlorophenol content, along with an augmented colour and increased pH. BL is often discharged into water bodies, where it has a negative impact on the environment. Towards that end, laccases are of great interest for bioremediation, since they can degrade aromatic and non-aromatic compounds while reducing O2 to water instead of H2O2. As such, we evaluated Pleurotus ostreatus and Pichia pastoris (which produces rPOXA 1B laccase) in the treatment of synthetic BL (SBL) in an "in vitro" modified Kraft process followed by CuO/TiO2/visible light photocatalysis. Treating SBL with P. ostreatus viable biomass (VB) followed by CuO/TiO2/visible light photocatalysis resulted in 80.3% COD removal and 70.6% decolourisation. Toxic compounds such as 2-methylphenol, 4-methylphenol, and 2-methoxyphenol were eliminated. Post-treated SBL exhibited low phytotoxicity, as evidenced by a Lactuca sativa L seed germination index (GI) > 50%. Likewise, SBL treatment with P. pastoris followed by VB/CuO/TiO2/visible light photocatalysis resulted in 63.7% COD removal and 46% decolourisation. Moreover, this treatment resulted in the elimination of most unwanted compounds, with the exception of 4-chlorophenol. The Lactuca sativa L seed GI of the post-treated SBL was 40%, indicating moderate phytotoxicity.
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Catálisis , Celulosa/química , Lacasa/química , Oxígeno/química , Análisis de la Demanda Biológica de Oxígeno , Cobre/química , Cresoles/química , Guayacol/química , Peróxido de Hidrógeno/química , Lacasa/genética , Luz , Pichia/química , Pichia/genética , Pleurotus/química , Pleurotus/genética , Titanio/químicaRESUMEN
Laccases catalyze the oxidation of various aromatic organic compounds concomitantly with molecular oxygen reduction to water. Triphenylmethane dyes are synthetic compounds widely used in diverse industries. Their removal from effluents is difficult, due to their high degree of structural complexity; hence, their high concentration in effluents cause a negative impact on the environment. In the present work, molecular docking was used to evaluate interactions between rGlLCC1 or rPOXA 1B enzymes with Crystal Violet (CV) or Malachite Green (MG) dyes. In addition, removal tests of the two dyes were performed. Van der Waals interactions were obtained for only the CV dye for both GlLCC1 and POXA 1B enzymes. Nevertheless, in the GlLCC1 model, two π-π interactions were observed. For the MG dye only, Van der Waals interactions were obtained. Moreover, amino acid composition interacting in each model with each dye was similar. It is important to highlight that by molecular docking, none of the estimated ligand configurations generated hydrogen bonds. Thus, explaining the difficulty to degrade CV and MG. Regarding CV, maximum decolorization percentage was 23.6 ± 1.0% using Ganoderma lucidum supernatant and 5.0 ± 0.5% with Pleurotus ostreatus supernatant. When using recombinant laccase enzyme concentrates, decolorization percentages were 9.9 ± 0.1 and 7.5 ± 1.0% for rGlLCC1 and rPOXA 1B, respectively. On the other hand, for the MG dye, maximum decolorization percentages were 52.1 ± 5.1 and 2.3 ± 0.2% using G. lucidum and P. ostreatus concentrates, respectively. Whereas with recombinant laccase enzymatic concentrates, values of 9.4 ± 0.8% were obtained, with rGlLCC1, and 2.1 ± 0.1% when using rPOXA 1B. These findings represent an important step in bioremediation processes improvement and efficiency of industry-generated products, using environmentally friendly alternatives.
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Proteínas Fúngicas/química , Violeta de Genciana/química , Simulación del Acoplamiento Molecular , Pleurotus/enzimología , Reishi/enzimología , Colorantes de Rosanilina/química , Proteínas Fúngicas/genética , Pleurotus/genética , Reishi/genéticaRESUMEN
The original version of this article unfortunately contained a mistake. The replacement image of Fig. 4 provided by the first corresponding author, Aura M. Pedroza-Rodríguez, is incorrect and that the originally submitted Fig. 4 should have been retained. The original article has been corrected.
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Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL-1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 × 10-5 mM s-1, with an apparent Km of 5.36 × 10-2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.
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Abstract The triphenylmethane Malachite Green (MG) and Crystal Violet (CV) dyes are cationic dyes and mix with domestic wastewater when dumped; increasing, among others, the chemical and biological oxygen demand and can cause acute toxicity at different trophic levels. Promoting the removal (decolorization) of MG and CV, and laccase activity (54.8 ± 8.9 and 30.6 ± 2.9 UL-1 respectively) by using P. ostreatus viable biomass needed parameters such as pH (4.5 and 6.0), temperature (25 to 30 °C), stirring speed (120 rpm), percentage of inoculum (2% v/v), and dye concentration (20 and 10 mg L-1). In adsorption studies, it was showed that an acidic pH favors the adsorption of both dyes and the model of pseudo-second order describes best the phenomenon of adsorption. Finally, the germination index (GI), using Lactuca sativa seeds for the initial dyes solutions, was < 50%; demonstrating its high phytotoxic effect. When dye solutions were treated with viable biomass, the GI increased, leaving open the possibility to perform future research to determine if the aqueous solutions, post-treated with P. ostreatus, could be used in treatments that generate less toxic water which could be used in processes that do not require potable water.
Resumen Los colorantes trifenilmetánicos Verde Malaquita (MG) y Crystal Violeta (CV) son catiónicos y al ser vertidos se mezclan con aguas residuales domésticas, incrementando, entre otros, la demanda química y biológica de oxígeno; pudiendo causar toxicidad aguda en diferentes niveles tróficos. En este estudio se encontró que los parámetros pH (4,5 y 6,0), temperatura (25 y 30 °C), velocidad de agitación (120 r.p.m.), porcentaje de inóculo (2 % v/v) y concentración de colorante (20 y 10 mgL-1), presentaron un efecto significativo (p < 0.05) para favorecer la remoción (decoloración) de MG y CV, así como la actividad lacasa (54,76 ± 8,91 y 30,59 ± 2,89 UL-1 respectivamente) al utilizar biomasa viable de P. ostreatus. En los estudios de adsorción se evidenció que pH ácidos favorecen la adsorción de ambos colorantes y que el modelo de Pseudo-segundo orden describe mejor el fenómeno de quimisorción. Finalmente los índices de germinación (IG) empleando semillas de Lactuca sativa, para los colorantes iniciales fueron < 50 %; demostrando su efecto fitotóxico elevado. Cuando las soluciones de colorantes fueron tratadas con biomasa viable, el IG aumentó, dejando abierta la puerta para la realización de investigaciones futuras con la intensión de determinar si las soluciones acuosas, postratadas con P ostreatus, pueden ser utilizadas en tratamientos que generen aguas menos tóxicas y que estas puedan ser empleadas en otros procesos que no requieran agua potable.
Resumen Os corantes de tipo trifenilmetano Verde Malaquita (VM) e Cristal Violeta (CV) são corantes catiônicos e se misturam com águas residuais domésticas quando descartadas; aumentando, entre outros, as demandas químicas e biológicas de oxigênio, podendo causar toxicidade aguda em diferentes níveis tróficos. Promoveu-se a remoção (descoloração) de VM e CV, e atividade da lacase (54.8 ± 8.9 e 30.6 ± 2.9 UL-1 respectivamente) utilizando como parâmetros necessários para a biomassa viável de P. ostreatus como pH (4,5 e 6,0), temperatura (25 a 30 °C), velocidade de agitação (120 RPM), porcentagem de inócuo (2 % v/v), e concentração de corante (20 e 10 mg L-1). Em estudos de absorção, se demonstrou que um pH mais ácido favorece a absorção de ambos corantes e o modelo de pseudo-segunda ordem descreve melhor o fenômeno da absorção. Finalmente, o índice de germinação (IG), utilizando sementes de Lactuca sativa para as soluções iniciais dos corantes, foi < 50 %; demonstrando assim seu alto efeito fitotóxico. Quando as soluções de corante foram tratadas com a biomassa viável, o IG aumentou, deixando em aberto a possibilidade de realizar futuras investigações para determinar se as soluções aquosas, tratadas com P. ostreatus, poderiam ser utilizadas em tratamentos que gerem águas menos tóxicas, que poderia ser utilizada em processos que não requerem água potável.
RESUMEN
Objective. To isolate biosurfactant-producing microorganisms from the rhizosphere of fique and to select the best genus to evaluate the effect of nutritional and fermentation conditions on the production of rhamnolipids. Materials and methods. Rhizospheric soil was sampled in three areas of Cauca. The best genus was selected for the experimental designs (Plackett Burman and 2² factorial) and to find the production conditions for the growth kinetics at an Erlenmeyer flask scale. Results. Isolates from the rhizosphere of fique plants were from groups (or genera) of Bacillus, Pseudomonas and Actinomycetes, being Pseudomonas the more responsive in preliminary testing for emulsification. From the results of the experimental designs and the kinetics of production, we found that rhamnose synthesis associated with rhamnolipids (3.2 g/l) and emulsification (68% EC24) was significantly favored (p <0.0001) by cultivating an inoculum of 10% v/v of Pseudomonas fluorescens in a medium composed of: soybean oil 2% (v/v), K2HPO4 0.2% (w/v), yeast extract 0.4 g/l, NH4NO3 3.7 g/l, 1 ml trace elements (CoCl3 20 mg/l, H3BO(3)30 mg/l, ZnSO(4)10 mg/l, Cu2SO(4)1 mg/l, Na2MoO(4)3 mg/l, FeSO (4)10 mg/l MnSO(4)2,6 mg/l) and pH 7.2. Conclusion. Of all the microbial genera isolated from the rhizosphere of fique, Pseudomonas fluorescens had the greatest potential in the production of biosurfactants of the rhamnolipids family.
Objetivo. Aislar microorganismos de la rizosfera de fique capaces de producir biosurfactantes y seleccionar el mejor gé nero para evaluar el efecto de las condiciones nutricionales y de fermentación en la producción de ramnolípidos. Materiales y mé todos. Se realizaron muestreos de suelos rizosfé ricos en tres zonas del Cauca. El mejor gé nero fue seleccionado para realizar los diseños experimentales (Plackett Burman y factorial 2²) y establecer las condiciones de producción para las ciné ticas de crecimiento a escala de Erlemeyer. Resultados. Se aislaron bacterias del gé nero Bacillus, Pseudomonas y del grupo Actinomycetes, siendo Pseudomonas el grupo con mayor respuesta en las pruebas preliminares de emulsificación. A partir de los resultados obtenidos en los diseños experimentales y ciné ticas de producción, se estableció que la síntesis de ramnosa asociada con ramnolípidos (3,2 g/l) y la emulsificación (68% EC24) se favorecieron significativamente (p<0.0001) al cultivar un inoculo de 5% v/v de Pseudomonas fluorescens en un medio compuesto por: aceite de soya 2% (v/v), K2HPO4 0,2% (p/v), extracto de levadura 0,4 g/l, NH4NO(3)3,7 g/l, 1 ml de elementos traza (CoCl(3)20 mg/l, H3BO(3)30 mg/l, ZnSO(4)10 mg/l, Cu2SO4 1 mg/l, Na2MoO(4)3 mg/l, FeSO(4)10 mg/l MnSO(4)2,6 mg/l) y pH 7.2. Conclusión. Se aislaron 3 gé neros microbianos a partir de rizosfera de fique, siendo Pseudomonas fluorescens la bacteria con mayor potencial en la producción de biosurfactantes de la familia de los ramnolípidos.
Objetivo. Isolar microorganismos da rizosfera da piteira capazes de produzir biossurfactantes, selecionar o melhor gênero para avaliar o efeito das condições nutricionais e de fermentação na produção de rhamnolipídeos. Materiais e mé todos. Foram realizadas amostragem de solos rizosfé ricos em três á reas do Cauca. O melhor gênero foi selecionado para realizar desenhos experimentais (Plackett Burman e fatorial 2²) e definir as condições de produção para as ciné ticas em escala de erlenmeyer. Resultados. Foram isoladas bacté rias do gênero Bacillus, Pseudomonas e grupo de Actinomycetes. As Pseudomonas foram o grupo com maior resposta nos testes preliminares de emulsificação. A partir dos resultados obtidos nos desenhos experimentais e ciné ticas de produção foi estabelecido que a síntese de ramnose associados com rhamnolipídeos (3,2 g/l) e a emulsificação (68% EC24) foram favorecidos significativamente (p<0,0001) ao cultivar um inoculo de 5% v/ v de Pseudomonas fluorescens em um meio composto por: óleo de soja 2% (v/v), K2HPO(4)0,2% (p/v), extrato de levedura 0,4 g/l, NH4NO3 3,7 g/l, 1 ml de oligoelementos (CoCl(3)20 mg/l, H3BO(3)30 mg/l, ZnSO(4)10 mg/l, Cu2SO(4)1 mg/l, Na2MoO(4)3 mg/l, FeSO(4)10 mg/l MnSO4 2,6 mg/l) e pH 7,2. Conclusão. Três gêneros microbianos foram isolados da rizosfera da piteira, sendo Pseudomonas fluorescens a bacté ria com maior potencial na produção de biossurfactantes da família dos rhamnolipídeos.
RESUMEN
Objetivo. Evaluar tres tratamientos (lagunaje, fotocatálisis con TiO2 y desinfección química) para la inactivación de coliformes totales y Escherichia coli presentes en agua residual doméstica empleada para riego agrícola. Materiales y métodos. El agua residual fue caracterizada por medio de análisis físicos, químicos y microbiológicos. Posteriormente fue sometida a un tratamiento de lagunaje facultativo (TLF), pos tratamiento fotocatalítico (PTFTiO2/UV) y pos tratamiento químico (PTQ NaClO). Valorando la capacidad desinfectante de cada uno de ellos para inactivar coliformes totales y E. coli. A continuación se procesaron tres nuevos lotes de agua residual y se utilizaron para realizar pruebas de riego a escala de laboratorio por 30 días, empleando como modelo plantas de Lactuca sativa variedad Batavia y evaluando la concentración inicial y final de los dos grupos. Resultados. El PTFTiO2/UV fue significativamente superior que TLFLF y el PTQ NaClO (p<0,0001) obteniendo 100% de inactivación para coliformes y E. coli a los 30 minutos de irradiación a escala de reactor. Respecto a las pruebas de riego de L. sativa se demostró que al utilizar el agua tratada por PTFTiO2/UV no se presentó contaminación con E. coli y coliformes a los 30 días de proceso. Por el contrario en las plantas regadas con agua tratada por TLF y PTQ NaClO se observó un incremento en las dos poblaciones generando un problema de contaminación de las hortalizas al finalizar la prueba de laboratorio. Conclusión. La fotocatálisis heterogénea TiO2 fue un método eficaz para la reducción de coliformes y E.coli en aguas residuales domésticas.
Objective. To evaluate three treatments (facultative stabilization ponds, heterogeneous photocatalysis with TiO2 and chemical disinfection with sodium hypochlorite) for the inactivation of coliforms and Escherichia coli presen in domestic wastewaters used in agricultural irrigation. Materials and methods. Wastewater was characterized by physical, chemical and microbiological analyses and was then exposed to a facultative pond treatment (FPT), post-photocatalytic treatment (PTFTiO2/UV) and post-chemical treatment (PTQNaClO) to assess the disinfecting capacity of each method in the inactivation of total coliforms and E. coli. Three new samples of wastewater were processed and used in irrigation tests on a laboratory-scale basis for 30 days, using Lactuca sativa cultivar. Batavia as a model plant and evaluating the initial and final concentrations of the two groups. Results. PTFTiO2/UV was significantly higher than FPT and PTQNaClO (p<0.0001), obtaining 100% of inactivation of coliforms and E. coli after 30 minutes of irradiation at a reactor scale. Regarding the irrigation tests with L. sativa, we showed that using water treated by PTFTiO2/UV there is no contamination with E. coli and coliforms after 30 days. On the contrary, plants irrigated with water treated by FPT and PTQNaClO showed an increase in the two populations originating a contamination problem in the vegetable by the end of the laboratory experiments. Conclusion. The heterogeneous photocatalysis with TiO2 was an effective method in the reduction of coliforms and E. coli present in domestic wastewater.
Objetivo. Avaliar três tratamentos (lagoas, fotocatálise com TiO2 e desinfecção química) para inativação de coliformes totais e Escherichia coli presentes em águas residuais domésticas utilizadas na irrigação dos cultivos. Materiais e métodos. O efluente foi caracterizado por analises físicos, químicos e microbiológicos. Posteriormente foi submetido a tratamento de lagoas facultativas (TLF), pós-tratamento fotocatalítico (PTF TiO2/UV) e pós-tratamento químico (PTQ NaClO). Avaliando a capacidade desinfetante de cada um para inativar coliformes totais e E. coli. Posteriormente, foram processados três novos lotes de águas residuais para testar a irrigação em escala de laboratório por 30 dias, utilizando-se como modelo plantas de Lactuca sativa variedade Batavia e avaliando a concentração inicial e final dos dois grupos. Resultados. O PTFTiO2/UV foi significativamente maior a TLFLF e PTQ NaClO (p<0,0001) apresentando 100% de inativação para coliformes e E. coli em 30 minutos de irradiação a escala do reator. Quanto aos testes de irrigação de L. sativa foi demonstrado que, ao utilizar a água tratada com PTFTiO2/UV não se apresentou contaminação com E. coli e coliformes a 30 dias do tratamento. Em contrapartida, em plantas irrigadas com água tratada por TLF e PTQ NaClO observou-se um aumento nas duas populações gerando um problema de contaminação de hortaliças no final do teste de laboratório. Conclusão. A fotocatálise heterogênea TiO2 foi um método eficaz para reduzirem coliformes e E. coli em águas residuais domésticas.