Distribution ofListeria spp., andListeria monocytogenesin micro- and small-scale meat product processing plants.

Listeriosis is a disease caused by L. monocytogenes, a relevant microorganism as a causative agent of foodborne diseases - FBD. This study aimed to evaluate the distribution of Listeria spp., and L. monocytogenes in different production areas in two small plants (A and B) and two micro-food processing plants (C and D) producing meat derivatives, located in different cities of Colombia. The methodology implemented was i. The analysis of sampling points is based on a harmonised tool. ii. Four samplings in each production plant between 2019 and 2020. iii. Isolation and identification of microorganisms through conventional microbiology, a semi-automated system, molecular serotyping and clonal characterisation by ERIC-PCR. L. monocytogenes frequency in the production plants belonging to the study ranged between 5.9 and 28.6 %; for Listeria spp., plants A and D had isolated, plant A had the highest proportion, while for L. monocytogenes geno-serotypes found were: 1/2a, 1/2c, 4a-4c, 4b, 4d - 4e, with geno-serotype 4b as the most frequent. Furthermore, possible persistent isolates were detected in plant C as the feasible sources of contamination, based on failures in flow management, raw material contaminated with L. monocytogenes, lack of standardised cooking processes and transfer of the microorganism through equipment and surfaces. Finally, in three of the four production plants assayed, L. monocytogenes or Listeria spp. were present in the packaging area in some of the samples taken during the study, which calls for increased and frequent monitoring, as well as constant technical support for the control of L. monocytogenes in micro and small-scale production plants.

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.

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.

Analysis of the assessment of antimicrobial susceptibility. Non-typhoid Salmonella in meat and meat products as model (systematic review).

BACKGROUND: The scientific publications of antimicrobial susceptibilities and resistance must be precise, with interpretations adjusted to the standard. In this frame, knowledge of antimicrobial resistance is fundamental in pathogenic microorganisms such as Salmonella spp., known for many annual deaths worldwide. The objective of this work was to compare the interpretation of standards, the concentrations, and the breakpoints, to study antimicrobial resistance in Non-Typhoidal Salmonella (NTS) isolated from beef, pork, and chicken meat, meat products, and propose additional considerations that improve the use and usefulness of published results. RESULTS: After refining the search based on meeting the inclusion and exclusion criteria, 48 papers were selected. In 33 (68.8%) of them, the disc diffusion method was used, in 11 (22.9%) the MIC determination method, and in 4 (8.33%) were used both. In 24 (50%) of the articles, the selection of a different (correct) standard could have had an impact on the interpretation of antimicrobial susceptibility, which observed when considering three scenarios, i) comparison between the year of the isolation versus the implemented standard, ii) comparison between the year of submission versus implemented standard and iii) comparison between the year of publication versus implemented standard. CONCLUSIONS: The most frequent scenario was the inadequate selection of standards, indicating that some studies had not ensured that applied standards kept in line with the date of isolation, date of publication and interpretation of susceptibilities. We proposed 2 years for standards use for resistance and multi-resistance interpretations. On the other hand, we invite researchers to publish their results in the shortest possible time, and editors and reviewers of scientific journals to prioritise these types of studies and verify the correspondence between the standard cited and the one used and the one to be taken into account.

Antimicrobial Resistance of Non-Typhoid Salmonella in Meat and Meat Products.

Salmonella enterica serovars are associated with numerous annual deaths worldwide and are responsible for a large number of foodborne diseases. Within this frame of reference, knowledge of antimicrobial susceptibility represents the fundamental approach of most Salmonella treatments. Therefore, scientific publications of antimicrobial susceptibilities and resistance must be precise, with interpretations adjusted to a particular standard. Hence, the three objectives in this study were: (i) to describe the frequency of antimicrobial-resistant isolates of Non-Typhoidal Salmonella (NTS) isolated from beef, pork, chicken meat, and other meat products; (ii) to describe the distribution of serovars and their multi-resistance to antibiotics for clinical use (veterinary and human) between 1996 and 2019; and (iii) to propose additional considerations that could improve the use and usefulness of the published results. Our results determined that the predominant isolates came from poultry. Enteritidis and Typhimurium were the most reported serovars by MIC (with both having the highest resistance to TET) while the lowest resistance was to CIP and CRO for Enteritidis and Typhimurium, respectively. The multi-resistance pattern AMP AMC CEP GEN KAN STR TET was the most frequently observed pattern by MIC in Montevideo and Seftenberg, while, for disc diffusion, the pattern AMP STR TET was the most frequent in the Bredeney serotype. In conclusion, researchers should carry out homogeneous sampling procedures, identify the types of the samples, use standard identification methods, and employ appropriate standards for antimicrobial susceptibility interpretation. Additionally, there is also a need for all WHO members to comply with the WHA 73.5 resolution. Our final recommendation is for all producers to reduce antibiotic prophylactic use.

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.

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.

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.

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.

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.

Management of swine mortalities through the use of a mixed composting-accelerating bio-inoculant.

A composting-accelerating bio-inoculant (Bacillus subtilis, Talaromyces sayulitensis (HC1), Steinernema sp., and Heterorhabditis sp.) was evaluated in a composting process made up of a different mix of wood chips, pig manure, urine, and swine mortality (raw material RM). Three different treatments (T1, T2, and T3) were assessed, and physicochemical, microbiological, and entomological evaluations were carried out at 0 and 45 days of the composting process. The highest organic nitrogen (1.34 %) concentration was detected in swine mortality, whereas the highest total oxidizable organic carbon (39.1 %) concentration was observed in wood chips. Salmonella spp., was not identified in any of the raw materials. Clostridium spp., count was 5.5, 2.0, and 1.0 Log10 unit, for pig manure, wood chips, and swine mortality, respectively. Pig manure, swine mortality, and wood chip total coliform count was 6.21, 5.32, and 1 Log10 unit, respectively. Helminth eggs were not detected in any of the RM and Cryptosporidium spp., oocysts were occasionally found in pig manure and wood chips. Several types of flies were identified, Musca domestica, Muscina stabulans, Stomoxys calcitrans, Fannia canicularis, Sarcophaga sp., and Calliphora sp. Treatment 3 (45.11 % swine mortality, 33.33 % wood chips, and 21.55 %, urine and bio-inoculant) had the greatest total oxidizable organic carbon availability, the highest carbon/nitrogen (C/N) ratio (20.67, p < 0.05), and the lowest dipterous larvae count. Moreover, Salmonella sp., was not observed and had only low Clostridium spp., and fecal coliform count. The bio-inoculant's effect on C/N ratio, cation exchange capacity, and electrical conductivity were beneficial, and resulted in production of a fertilizer complying with EPA 600/1-87-014, EPA 40 CFR Part 258, and NTC5167/11 norms. According to the characterization protocols used in this study the compost was apparently free from bacterial and parasitic pathogens and minimal dipteran counts. Last, maturation time was 15 days shorter compared with control (C4).

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.

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.

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.

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.

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.

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.

Production and characterization of a human lysosomal recombinant iduronate-2-sulfatase produced in Pichia pastoris.

Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg-1 H-1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.

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.