Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics.

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.

Impact of Antibiotics as Waste, Physical, Chemical, and Enzymatical Degradation: Use of Laccases.

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.

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study.

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.

A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases.

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.

LDPE Transformation by Exposure to Sequential Low-Pressure Plasma and TiO2/UV Photocatalysis.

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.

Wastewater treatment from a science faculty during the COVID-19 pandemic by using ammonium-oxidising and heterotrophic bacteria.

During and after the pandemic caused by the SARS-CoV-2 virus, the use of personal care products and disinfectants increased in universities worldwide. Among these, quaternary ammonium-based products stand out; these compounds and their intermediates caused substantial changes in the chemical composition of the wastewater produced by these institutions. For this reason, improvements and environmentally sustainable biological alternatives were introduced in the existing treatment systems so that these institutions could continue their research and teaching activities. For this reason, the objective of this study was to develop an improved culture medium to cultivate ammonium oxidising bacteria (AOB) to increase the biomass and use them in the treatment of wastewater produced in a faculty of sciences in Bogotá, D.C., Colombia. A Plackett Burman Experimental Design (PBED) and growth curves served for oligotrophic culture medium, and production conditions improved for the AOB. Finally, these bacteria were used with total heterotrophic bacteria (THB) for wastewater treatment in a pilot plant. Modification of base ammonium broth and culture conditions (6607 mg L-1 of (NH4)2SO4, 84 mg L-1 CaCO3, 40 mg L-1 MgSO4·7H2O, 40 mg L-1 CaCl2·2H2O and 200 mg L-1 KH2PO4, 10% (w/v) inoculum, no copper addition, pH 7.0 ± 0.2, 200 r.p.m., 30 days) favoured the growth of Nitrosomonas europea, Nitrosococcus oceani, and Nitrosospira multiformis with values of 8.23 ± 1.9, 7.56 ± 0.7 and 4.2 ± 0.4 Log10 CFU mL-1, respectively. NO2- production was 0.396 ± 0.0264, 0.247 ± 0.013 and 0.185 ± 0.003 mg L-1 for Nitrosomonas europea, Nitrosococcus oceani and Nitrosospira multiformis. After the 5-day wastewater treatment (WW) by co-inoculating the three studied bacteria in the wastewater (with their self-microorganisms), the concentrations of AOB and THB were 5.92 and 9.3 Log10 CFU mL-1, respectively. These values were related to the oxidative decrease of Chemical Oxygen Demand (COD), (39.5 mg L-1), Ammonium ion (NH4+), (6.5 mg L-1) Nitrite (NO2-), (2.0 mg L-1) and Nitrate (NO3-), (1.5 mg L-1), respectively in the five days of treatment. It was concluded, with the improvement of a culture medium and production conditions for three AOB through biotechnological strategies at the laboratory scale, being a promising alternative to bio-augment of the biomass of the studied bacteria under controlled conditions that allow the aerobic removal of COD and nitrogen cycle intermediates present in the studied wastewater. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03961-4.

Use of biochar and a post-coagulation effluent as an adsorbent of malachite green, beneficial bacteria carrier, and seedling substrate for plants belonging to the poaceae family.

Wastewater treatment plants produce solid and semi-solid sludge, which treatment minimises secondary environmental pollution because of wastewater treatment and obtaining new bioproducts. For this reason, in this paper, the co-pyrolysis of biogenic biomasses recovered from a biological reactor with immobilised fungal and bacterial biomass and a tertiary reactor with Chlorella sp. used for dye-contaminated wastewater treatment was carried out. Biogenic biomasses mixed with pine bark allowed the production and characterisation of two types of biochar. The raw material and biochar were on the "in vitro" germination of Lolium sp. seeds, followed by adsorption studies for malachite green (MG) dye using the raw material and the biochar. Results showed that using 60 mg L-1 of a cationic coagulant at pH 6.5 allowed for the recovery of more than 90% of the microalgae after 50 min of processing. Two biochar resulted: BC300, at pH 5.08 ± 0.08 and BC500, at pH 6.78 ± 0.01. The raw material and both biochars were co-inoculated with growth-promoting bacteria; their viabilities ranged from 1.7 × 106 ± 1.0 × 101 to 7.5 × 108 ± 6.0 × 102 CFU g-1 for total heterotrophic, nitrogen-fixing and phosphate-solubilising bacteria. Re-use tests on Lolium sp. seed germination showed that with the post-coagulation effluent, the germination was 100%, while with the biochar, with and without beneficial bacteria, the germination was 98 and 99%, respectively. Finally, BC500 adsorbed the highest percentage of malachite green at pH 4.0, obtaining qecal values of 0.5249 mg g-1 (R2: 0.9875) with the pseudo-second-order model. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03766-x.

Production of pine sawdust biochar supporting phosphate-solubilizing bacteria as an alternative bioinoculant in Allium cepa L., culture.

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.

Evaluation of two microcosm systems for co-treatment of LDPEoxo and lignocellulosic biomass for biochar production.

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.

Non-domestic wastewater treatment with fungal/bacterial consortium followed by Chlorella sp., and thermal conversion of the generated sludge.

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.

Low-cost media statistical design for laccase rPOXA 1B production in P. pastoris.

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.

Phosphate-solubilizing Pseudomonas sp., and Serratia sp., co-culture for Allium cepa L. growth promotion.

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.

Tertiary treatment (Chlorella sp.) of a mixed effluent from two secondary treatments (immobilized recombinant P. pastori and rPOXA 1B concentrate) of coloured laboratory wastewater (CLWW).

Industrial development has increased wastewater (WW) volume; generating contamination and disturbing ecosystems, because of breeching disposal parameters. In this work, Coloured Laboratory Wastewater (CLWW), (1500.00 colour units, CU) was separately submitted to two secondary treatments. For the first one CLWW was treated for three cycles C1, C2 and C3 with P. pastoris X33/pGAPZαA-LaccPost-Stop producing rPOXA 1B laccase, immobilized in calcium alginate beads. For the second-one, rPOXA 1B enzyme concentrate was used (three processes: P1, P2, and P3). Both treatments were carried out in a 15 L reactor with 10 L effective work volume (EWV) with 72 h hydraulic retention time. C1, C2, and C3 effluents were flocculated and filtered through quartzite sand, while P1, P2, and P3 effluents were only filtered through quartzite sand. The mixture of secondary effluents was submitted to a tertiary treatment with Chlorella sp. For C1, C2, C3, P1, P2, and P3, CU removal was of 99.16, 99.58, 99.53, 96.72, 97.05 and 96.47%, respectively. Discharge parameters, total organic carbon (TOC), inorganic carbon (IC), chemical oxygen demand (COD) and biological oxygen demand (BOD5) decreased, although they reached different final values. After the tertiary treatment (144 h) effluent discharge parameters were reduced to 34 ± 4 CU, TOC to 6.6 ± 0.9 mg L-1 and COD to 155 ± 4 mg L-1. It was demonstrated that secondary treatments (immobilized recombined cells or recombinant enzyme concentrate) combined with Chlorella sp., (tertiary treatment) attained a considerable removal of discharge parameters, demonstrating a promissory alternative for CLWW sequential treatment.

Simultaneous bioconversion of lignocellulosic residues and oxodegradable polyethylene by Pleurotus ostreatus for biochar production, enriched with phosphate solubilizing bacteria for agricultural use.

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.

Media improvement for 10 L bioreactor production of rPOXA 1B laccase by P. pastoris.

In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] substrate concentrations. Plackett-Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L-1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L-1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were V max of 3.163 × 10-2 mM min-1 and K m of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures.

Biodeterioration of plasma pretreated LDPE sheets by Pleurotus ostreatus.

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.

Detoxification of pulping black liquor with Pleurotus ostreatus or recombinant Pichia pastoris followed by CuO/TiO2/visible photocatalysis.

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.

Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

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.

Correction to: Malachite Green and Crystal Violet Decolorization by Ganoderma lucidum and Pleurotus ostreatus Supernatant and by rGlLCC1 and rPOXA 1B Concentrates: Molecular Docking Analysis.

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.

Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization.

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.