Flow cytometry: a tool for understanding the behaviour of polyhydroxyalkanoate accumulators.

The use of mixed microbial cultures (MMCs) is seen as an attractive strategy for polyhydroxyalkanoate (PHA) production. In order to optimize the MMC-PHA production process, tools are required to improve our understanding of the physiological state of the PHA-storing microorganisms within the MMC. In the present study, we explored the use of flow cytometry to analyse the metabolic state and polyhydroxybutyrate (PHB) content of the microorganisms from an MMC-PHA production process. A sequencing batch reactor under a feast and famine regime was used to enrich an MMC with PHB-storing microorganisms. Interestingly, once the PHB-storing microorganisms are selected, the level of PHB accumulation depends largely on the metabolic state of these microorganisms and not exclusively on the consortium composition. These results demonstrate that flow cytometry is a powerful tool to help to understand the PHA storage response of an MMC-PHA production process. KEY POINTS: • Flow cytometry allows to measure PHB content and metabolic activity over time. • Microorganisms showing high PHB content also have high metabolic activity. • PHB producers with low metabolic activity show low PHB content.

Behavior of 4 types of paper with printed QR codes for evaluating denture marking in conditions of extreme heat.

STATEMENT OF PROBLEM: Quick response (QR) codes are a fast and efficient technology for linking and accessing identifying information, and their use has been proposed in forensics. The heat resistance and esthetics of denture marking methods (DMMs) have been recommended by the American Dental Association (ADA), but studies on these aspects of printed QR codes are lacking. PURPOSE: The purpose of this study was to determine the optimal printed material with QR codes for implementation as a DMM adjusted to the recommendations of the ADA. MATERIAL AND METHODS: The behavior of 4 types of paper, bond paper, fiberglass filter paper, ultralong hydroxyapatite nanowire paper, and polyolefin and silica paper with printed QR codes was analyzed. They were exposed to temperatures between 100 °C and 1000 °C in a heat muffle for 1 hour. Each specimen was subjected to both a morphological and a thermogravimetric analysis (TGA) and scanned by using 3 different smartphones. RESULTS: The scans were positive for bond paper (33.3%), fiberglass fiber paper (50%), ultralong hydroxyapatite nanowire paper (100%), and polyolefin and silica paper (70.4%). The TGA revealed continuous decomposition curves (average 16.5 minutes at 624 °C). CONCLUSIONS: Printed QR codes on ultralong hydroxyapatite nanowire paper appear to be suitable as information reservoirs, even surviving incineration, and may be implemented as a DMM conforming to the ADA recommendations.

Biological Approaches in Polyhydroxyalkanoates Recovery.

Polyhydroxyalkanoates (PHA) are bio-based polymers with the potential of replace petrochemical plastics. Nevertheless, PHA commercialization is still low, due to the high production cost associated with industrial-scale development. The most cost/efficient PHA recovery strategies use organochlorine compounds or harsh reagents implying a high environmental impact. Therefore, the importance of developing an economical and efficient recovery strategy cannot be overestimated. Thus, new approaches have been reported that look for creating a sustainable production process, such as biological recovery, PHA secretion or predator bacteria. Moreover, if bioplastics would become the plastics of the future, it must be necessary to replace the traditional PHA extraction methods by environmentally friendly options. Hence, the aim of this review is to analyze trends in the development of efficient technologies for the sustainable recovery of polyhydroxyalkanoates (PHA) produced by microorganisms.

Enzymatic esterification of oleic acid by Candida rugosa lipase immobilized onto biochar.

The immobilization of Candida rugosa lipase (CRL) onto biochar was studied in a series of batch experiments. CRL sorption behavior was evaluated as a function of pH, enzyme concentration, temperature and ionic strength. As the immobilized lipase was used for the catalytic esterification of oleic acid, its resistance to solvents and thermal stability were evaluated. CRL adsorption increased by increasing temperature, and with higher pH, reaching a maximum at pH 7.0. Immobilization increased lipase stability at 40 °C by more than 80% when compared to the free enzyme. Moreover, immobilized CRL showed high stability in the presence of tert-butanol, which prevents premature deactivation of the enzyme caused by alcohols during the reaction. Immobilization of CRL increased the oleic acid conversion rate. Our results suggest that biochar is a highly promising material for the immobilization of CRL lipase for the catalytic production of esters.

Toward the use of mixed microbial cultures for the biological production of adipic and levulinic acid.

Biological synthesis of high added-value compounds like adipic acid (AA), levulinic acid (LA), or polyhydroxybutyrate (PHB) using pure culture has been separately reported. However, pure culture requires sterile conditions and the use of specific carbon sources resulting in high operating costs. Different alternatives based on the use of mixed microbial cultures (MMC) have been explored to resolve this problem. MMC have been widely reported for the production of PHB, but scarcely reported for LA production and never for AA synthesis. This work presents a novel strategy for the co-production of AA LA, and PHB using MMC. The strategy consists in selecting an MMC producer of AA, LA and PHB from an inoculum obtained from a wastewater treatment plant, which is then subjected to the feast and famine culture strategy in a sequential batch reactor, coupled with a batch reactor step to enhance the accumulation of AA and LA. The results showed that the MMC could produce a 16 ± 2, 23 ± 1 and 5 ± %1 (g compound/g volatile solids) of AA, LA and PHB, respectively, using a non-fermented residual biomass rich in pentose, namely synthetic hemicellulose hydrolysate (SHH) as the carbon source. These results contribute to generating future research to better understand and optimise the biosynthesis of these compounds by MMC.

Development and thermochemical characterization of an antioxidant material based on polyhydroxybutyrate electrospun microfibers.

The use of antioxidants such as curcumin (Cur) or quercetin (Que) in biomedical and biotechnological applications has been studied owing to their capability to prevent oxidative stress and inhibit free radicals. Using polyhydroxybutyrate (PHB) electrospun fibers is presented as a proper option to encapsulate curcumin and quercetin due to its biocompatibility and biodegradability characteristics. Electrospun fibers were obtained dissolving commercial PHB in chloroform:N,N-dimethylformamide (DMF) (4:1) at 7% m/V, and adding two different concentrations of antioxidant (Cur, and Que) 1%m/m, and 7% m/m. These polymeric solutions were electrospun at different conditions and the obtained fibers were characterized by scanning electron microscopy (SEM), thermogravimetric (TGA) analysis, and Fourier transform infrared spectroscopy (FT-IR). The curcumin and quercetin releases into phosphate buffer saline (PBS) at pH 7.4 were obtained in vitro and measured by spectrophotometry. Antioxidant activities were measured by spectrophotometry in a microplate reader using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Fibers obtained with different formulations presented a chemical composition in accordance with PHB according to FTIR spectra, the diameters fluctuate between 0.761 ± 0.123 and 1.803 ± 0.557 µm, with qualities over 0.95 according to their morphology, and the melting temperature resulted near 178 °C according to the bibliography. The crystallinity of fibers decreases while curcumin or quercetin concentration increases for the studied interval, indeed, quercetin showed a higher impact on the relative crystallinity of fibers. Antioxidant activity of active compounds is maintained after encapsulation in PHB electrospun fibers, and quercetin resulted in near four times antioxidant activity compared to curcumin according to DPPH analysis.

Applications of Electrospun Nanofibers with Antioxidant Properties: A Review.

Antioxidants can be encapsulated to enhance their solubility or bioavailability or to protect them from external factors. Electrospinning has proven to be an excellent option for applications in nanotechnology, as electrospun nanofibers can provide the necessary environment for antioxidant encapsulation. Forty-nine papers related to antioxidants loaded onto electrospun nanofibers were categorized and reviewed to identify applications and new trends. Medical and food fields were commonly proposed for the newly obtained composites. Among the polymers used as a matrix for the electrospinning process, synthetic poly (lactic acid) and polycaprolactone were the most widely used. In addition, natural compounds and extracts were identified as antioxidants that help to inhibit free radical and oxidative damage in tissues and foods. The most recurrent active compounds used were tannic acid (polyphenol), quercetin (flavonoid), curcumin (polyphenol), and vitamin B6 (pyridoxine). The incorporation of active compounds in nanofibers often improves their bioavailability, giving them increased stability, changing the mechanical properties of polymers, enhancing nanofiber biocompatibility, and offering novel properties for the required field. Although most of the polymers used were synthetic, natural polymers such as silk fibroin, chitosan, cellulose, pullulan, polyhydroxybutyrate, and zein have proven to be proper matrices for this purpose.

Adsorption behavior of 2,4-dichlorophenol and pentachlorophenol in an allophanic soil.

The adsorption of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) by a variable-charge soil from southern Chile was studied in a series of batch equilibration experiments. 2,4-DCP and PCP adsorption behavior was evaluated as a function of pH (pH values of 4.5, 6.0 and 7.5) in a 0.1M KCl (25 degrees C) background solution for soil material collected at three different depths (0-20 cm, 20-40 cm, and 40-60 cm). 2,4-DCP and PCP adsorption decreased with increasing soil pH, suggesting that the undissociated species were adsorbed more readily and that electrostatic repulsion may inhibit partitioning as pH increases. The PCP adsorption was greater than observed for 2,4-DCP and decreased with soil depth. Multiple regression analysis between K(d) and various soil properties indicated that the soil organic carbon content is a strong indicator of chlorophenol adsorption, and in addition to organic carbon, the soil pH is an important property controlling adsorption behavior.

Describing chlorophenol sorption on variable-charge soil using the triple-layer model.

The sorption of 2,4-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol by a variable-charge soil from southern Chile was studied in a series of batch experiments. The chlorophenol sorption behavior was evaluated as a function of pH (pH range 4-8) at two different ionic strengths, 0.01 and 0.1 M KCl (25 degrees C). Chlorophenol sorption depended on pH and a downward shift in the soil PZC was observed with increasing chlorophenol concentration. Chlorophenol sorption decreased with increasing pH, suggesting that the undissociated species is sorbed more readily and that electrostatic repulsion may inhibit partitioning as pH increases. Data from the sorption experiments were fitted by the triple-layer model, in which monodentate outer- and inner-sphere complexes were formed between deprotonated organic molecules and active sites on the variable-charge soil.

In situ biodiesel production from greasy sewage sludge using acid and enzymatic catalysts.

This study proposes to select the most appropriate sewage sludge (greasy, primary and secondary) for in situ transesterification and to compare the technical, economic and energetic performance of an enzymatic catalyst (Novozym®435) with sulfuric acid. Greasy sludge was selected as feedstock for biodiesel production due to its high lipid content (44.4%) and low unsaponifiable matter. Maximum methyl esters yield (61%) was reached when processing the wet sludge using sulfuric acid as catalyst and n-hexane, followed by dried-greasy sludge catalyzed by Novozym®435 (57% methyl esters). Considering the economic point of view, the process using acid catalyst was more favorable compared to Novozym®435 catalyst due to the high cost of lipase. In general, greasy sludge (wet or dried) showed high potential to produce biodiesel. However, further technical adjustments are needed to make biodiesel production by in situ transesterification using acid and enzymatic catalyst feasible.

Description of mutual interactions between silicon and phosphorus in Andisols by mathematical and mechanistic models.

The Freundlich model and the Constant Capacitance Model (CCM) were used to describe silicon (Si) and phosphorus (P) sorption, both individually and for binary P-Si systems, on two Andisols with different chemical properties: Freire soil (FS) and Piedras Negras soil (PNS). Silicon sorption kinetics were examined through the Elovich equation, revealing that the initial sorption rate was 16 times greater in PNS. The Freundlich equation provides a good fit to the sorption data for both Andisols. When compared with FS, larger Si sorption capacity and lower Si affinity for the surface sites were observed in PNS; nevertheless, Si sorption decreased in both soils as P sorption increased. Slight reductions in P sorption capacity due to the presence of Si were found, whereas there was no apparent effect on P bonding intensity. The CCM was able to describe Si adsorption, and potentiometric titrations support that Si seems to be specifically sorbed mainly onto sites of negative charge. Comparable log KSiint values were obtained for both soils, indicating that Si was bound on similar sites. Phosphorus sorption was well described by the CCM, and log KPint denoted strong interactions of P with the surface sites. For binary systems, log KPint did not vary with increasing Si concentration; comparatively, log KSiint scarcely decreased with increasing P concentration in PNS, but a 28% reduction was found in FS at the highest initial P concentration.

Biosorption of pentachlorophenol by Anthracophyllum discolor in the form of live fungal pellets.

Pentachlorophenol (PCP) is an extremely dangerous pollutant for every ecosystem. In this study we have detected how PCP concentration and pH levels can influence PCP adsorption by Anthracophyllum discolor in the form of live fungal pellets. PCP adsorption was evaluated after 24 hours in KCl 0.1 M electrolyte solution with initial PCP concentrations of 5 and 10 mg L (-1) and with pH values between 4 and 9 (at intervals of 0.5). Fourier Transform Infrared Spectroscopy (FTIR) was used to identify functional groups of fungal biomass that can interact with PCP. The amount of PCP that was adsorbed by A. discolor was >80% at pH values between 5 and 5.5, whatever the concentration tested. PCP adsorption significantly decreased in liquid medium of pH > 6.0. FTIR results showed that amides, alkanes, carboxylates, carboxyl and hydroxyl groups may be important to the PCP adsorption for pellets of A. discolor. Live fungal pellets of A. discolor may be used as a natural biosorbent for liquid solutions contaminated by PCP.

Screening transesterifiable lipid accumulating bacteria from sewage sludge for biodiesel production.

Sewage sludge was evaluated as high available and low cost microbial oils feedstock for biodiesel production. Samples from four different wastewater treatment plants from La Araucanía Region in Southern Chile presented total lipids content ranging between 7.7 and 12.6%, being Vilcún sewage sludge that with the highest transesterifiable lipids content of about 50% of the total extracted lipids. The most relevant identified bacteria present in sludge samples were Acinetobacter, Pseudomonas and Bacillus, being Bacillus sp. V10 the strain with the highest transesterfiable lipids content of 7.4%. Bacillus sp. V10 was cultured using urban wastewater supplemented with glucose to achieve nitrogen depleted medium and using milk processing wastewater as a low-cost carbon source. Bacillus sp. V10 lipid profile indicates that low degree unsaturated long chain fatty acids such as C18:1 may account for approximately 50% of the lipids content, indicating its suitability to be used as raw material for biodiesel production.

Performance of an enzymatic extract in Botrycoccus braunii cell wall disruption.

Microalgae can produce and contain lipids, proteins and carbohydrates, which can be extracted and marketed as potential novel added-value bio-products. However, microalgae cell wall disruption is one of the most important challenges involved while processing this type of biomass. In this context, white-rot fungi, responsible for the biodegradation of lignin present in wood due to non-specific extracellular enzymes, could be applied for promoting microalgae cell wall degradation. Therefore, the aim of this study was to evaluate the use of an enzymatic extract produced by the white-rot fungi Anthracophyllum discolor as a biotechnological tool for Botryococcus braunii cell wall disruption. The fungus was inoculated in wheat grains and manganese peroxidase (MnP) activity was monitored while obtaining the enzymatic extract. Then, cell wall disruption trials with different MnP activity were evaluated by the biochemical methane potential (BMP). In relation to cell wall disruption, it was observed that the optimal value was obtained with enzymatic concentration of 1000 U/L with a BMP of 521 mL CH4/g VS. Under these conditions almost 90% of biomass biodegradability was observed, increasing in 62% compared to the microalgae without treatment. Therefore, the results indicate that enzymes secreted by A. discolor promoted the attack of the different cell wall components finally weakening it. Therefore, the application of this treatment could be a promissory biotechnological approach to decrease the energetic input required for the cell wall disruption step.