Optimisation of the biological production of levulinic acid in a mixed microbial culture fed with synthetic grape pomace.

Levulinic acid (LA) is a polymer with a vast industrial application range and can be co-produced as a minor by-product during the biological production of polyhydroxyalkanoates (PHA). However, the influence of key parameters as tools for favouring the production of LA over PHA is still unclear. In this study, we investigated how several critical operational conditions, i.e., carbon-nitrogen ratio (C/N), organic loading rate (OLR) and airflow, can be optimised to favour LA accumulation over PHA production by a mixed microbial culture (MMC), using synthetic grape pomace (GP) hydrolysate as the substrate. The results showed that it was possible to direct the MMC towards LA accumulation instead of PHA. The maximum LA yield was 2.7 ± 0.2 g LA/(L·d) using a C/N of 35, an airflow of 5 L/min and an OLR of 4 g sCOD/(L·d). The OLR and, to a lesser extent, the C/N ratio were the main factors significantly and positively correlated with the biological synthesis of LA.

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.

Antioxidant Activity as an Indicator of the Efficiency of Plant Extract-Mediated Synthesis of Zinc Oxide Nanoparticles.

The green synthesis of zinc oxide nanoparticles (ZnO NPs) using a diverse range of plant species has been extensively reported. Despite the success achieved by biogenic synthesis, there are problems with the control and prediction of the properties of ZnO NPs, due to phytochemical diversity between plant species. In this sense, the main objective of our work was to investigate the effect of the antioxidant activity (AA) of plant extracts on the physicochemical characteristics of ZnO NPs (production yield, chemical composition, polydispersity index (PDI), surface charge (ζ-potential) and average particle size). In order to accomplish this objective, four plant extract with different antioxidant activities were used: Galega officinalis, Buddleja globosa, Eucalyptus globulus, and Aristotelia chilensis. Phytochemical screening, quantitative analysis of phenolic compounds and antioxidant activity determination of the different extracts were carried out. Chemical species such as catechin, malvidin, quercetin, caffeic acid, and ellagic acid were the dominant components, found in the extracts studied. The A. chilensis extract showed the highest value of total phenolic compounds (TPC) and AA, followed by E. globulus, B. globosa and G. officinalis. Zetasizer, Fourier-transform infrared (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA) data show that plant extracts with lower AA leads to a decrease in the yield of ZnO NPs and an increase in the amount of residual organic extract that remains on the particles. The latter caused an increase in the average particle size, PDI and ζ-potential as a consequence of agglomeration and particle coarsening. Our result suggest that it is possible to use the AA as an indicator of the potential reducing capacity of plant extracts. In this way it is possible to guarantee the reproducibility of the synthesis process as well as ensure the formation of ZnO NPs with desired characteristics.

Use of Anthracophyllum discolor and Stereum hirsutum as a Suitable Strategy for Delignification and Phenolic Removal of Olive Mill Solid Waste.

This study evaluated the use of the white-rot fungi (WRF) Anthracophyllum discolor and Stereum hirsutum as a biological pretreatment for olive mill solid mill waste (OMSW). The WRF strains proposed were added directly to OMSW. The assays consisted of determining the need to add supplementary nutrients, an exogenous carbon source or use agitation systems, and evaluating WRF growth, enzyme activity, phenolic compound removal and lignin degradation. The highest ligninolytic enzyme activity was found at day 10, reaching 176.7 U/L of manganese-independent peroxidase (MniP) produced by A. discolor, and the highest phenolic removal (more than 80% with both strains) was reached after 24 days of incubation. The confocal laser scanning microscopy analysis (CLSM) confirmed lignin degradation through the drop in lignin relative fluorescence units (RFU) from 3967 for untreated OMSW to 235 and 221 RFU, showing a lignin relative degradation of 94.1% and 94.4% after 24 days of treatment by A. discolor and S. hirsutum, respectively. The results demonstrate for the first time that A. discolor and S. hirsutum were able to degrade lignin and remove phenolic compounds from OMSW using this as the sole substrate without adding other nutrients or using agitation systems. This work indicates that it could be possible to design an in situ pretreatment of the valorization of OMSW, avoiding complex systems or transportation. In this sense, future research under non-sterile conditions is needed to evaluate the competition of WRF with other microorganisms present in the OMSW. The main drawbacks of this work are associated with both the low reaction time and the water addition. However, OMSW is seasonal waste produced in one season per year, being stored for a long time. In terms of water addition, the necessary optimization will be addressed in future research.

Application of microbe-induced carbonate precipitation for copper removal from copper-enriched waters: Challenges to future industrial application.

Copper contamination in watercourses is a recent issue in countries where mining operations are prevalent. In this study, the application of copper precipitation through microbe-induced carbonate precipitation (MICP) was analyzed using urea hydrolysis by bacteria to evaluate precipitated copper carbonates. This article demonstrates the application of a copper precipitation assay involving Sporosarcina pasteurii (in 0.5 mM Cu2+ and 333 mM urea) and analyzes the resultant low removal (10%). The analysis indicates that the low removal was a consequence of Cu2+ complexation with the ammonia resulting from the hydrolysis of urea. However, the results indicate that there should be a positive correlation between the initial urea concentration and the bacterial tolerance to copper. This identifies a challenge in the industrial application of the process, wherein a minimum consumption of urea represents an economic advantage. Therefore, it is necessary to design a sequential process that decouples bacterial growth and copper precipitation, thereby decreasing the urea requirement.

Effects of limonene, n-decane and n-decanol on growth and membrane fatty acid composition of the microalga Botryococcus braunii.

Botryococcus braunii is a promising microalga for the production of biofuels and other chemicals because of its high content of internal lipids and external hydrocarbons. However, due to the very thick cell wall of B. braunii, traditional chemical/physical downstream processing very often is not as effective as expected and requires high amounts of energy. In this cases, the application of two-phase aqueous-organic solvent systems could be an alternative to cultivate microalgae allowing for a simultaneous extraction of the valuable compounds without significant negative effects on cell growth. Two-phase systems have been applied before, however, there are no studies so far on the mechanisms used by microalgae to survive in contact with solvents present as a second-phase. In this study, the effects of the solvents limonene, n-decane and n-decanol on growth of the microalga B. braunii as well as the adaptive cell response in terms of their phospholipid fatty acid contents were analized. A concentration-dependent negative effect of all three solvents on cell growth was observed. Effects were accompanied by changes of the membrane fatty acid composition of the alga as manifested by a decrease of the unsaturation . In addition, an association was found between the solvent hydrophobicity (given as log octanol-water partition coefficient ([Formula: see text]) values) and their toxic effects, whereby n-decanol and n-decane emerged as the most and least toxic solvent respectively. Among the tested solvents, the latter promises to be the most suitable for a two-phase extraction system.

Evaluation of different solvent mixtures in esterifiable lipids extraction from microalgae Botryococcus braunii for biodiesel production.

Non-polar and polar solvents as well as their mixtures were tested for the extraction of microalgae lipids and thus, to evaluate their effect on total and esterifiable lipids extraction yields with potential to be converted to biodiesel. The obtained results show an increase in lipids and esterifiable lipids extraction yields when non-polar and polar solvent mixtures were used. The higher esterifiable lipids extraction yield was 19.2%wt (based on dry biomass) using a chloroform-methanol mixture (75%v/v of methanol), corresponding to a 98.9%wt esterifiable lipids extraction. In addition, esterifiable lipids extraction yield of 18.9%wt (based on dry biomass) was obtained when a petroleum ether-methanol mixture (75%v/v of methanol) was used, corresponding to a 96.9%wt esterifiable lipids extraction.

Biodiesel synthesis by direct transesterification of microalga Botryococcus braunii with continuous methanol reflux.

Direct transesterification of Botryococcus braunii with continuous acyl acceptor reflux was evaluated. This method combines in one step lipid extraction and esterification/transesterification. Fatty acid methyl esters (FAME) synthesis by direct conversion of microalgal biomass was carried out using sulfuric acid as catalyst and methanol as acyl acceptor. In this system, once lipids are extracted, they are contacted with the catalyst and methanol reaching 82%wt of FAME yield. To optimize the reaction conditions, a factorial design using surface response methodology was applied. The effects of catalyst concentration and co-solvent concentration were studied. Hexane was used as co-solvent for increasing lipid extraction performance. The incorporation of hexane in the reaction provoked an increase in FAME yield from 82% (pure methanol) to 95% when a 47%v/v of hexane was incorporated in the reaction. However, the selectivity towards non-saponifiable lipids such as sterols was increased, negatively affecting biodiesel quality.

Enzymatic biodiesel production kinetics using co-solvent and an anhydrous medium: a strategy to improve lipase performance in a semi-continuous reactor.

Enzymatic biodiesel production kinetics under previously optimized conditions were investigated. Waste frying oil (WFO) was used as the raw material, Novozym 435 as catalyst, methanol as acyl acceptor and tert-butanol as co-solvent. To investigate pure transesterification kinetics improving product properties, 3Å molecular sieves were incorporated into the reaction to provide an anhydrous medium avoiding the side reactions of hydrolysis and esterification. The effects of either WFO or methanol on the reaction rate were analyzed separately. The reaction was described by a Ping Pong mechanism and competitive inhibition by methanol. The results obtained in the kinetics study were applied in the operation of a semi-continuous reactor for biodiesel production. The operational conditions of each reaction cycle were: methanol-to-oil ratio 8/1 (mol/mol), 15% (wt) Novozym 435, 0.75% (v/v) of tert-butanol, 44.5°C, 200 rpm and 4h of reaction time. The enzymes were successively reused by remaining in the reactor during all the cycles. Under these conditions, biodiesel production yields higher than 80% over 7 reaction cycles were observed. Both the kinetics study and the reactor operation showed that Novozym 435 was not inhibited at high methanol concentrations and that the kinetics of the proposed enzymatic process could be comparable to the conventional chemical process.

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.

Lipase-catalyzed process in an anhydrous medium with enzyme reutilization to produce biodiesel with low acid value.

One major problem in the lipase-catalyzed production of biodiesel or fatty acid methyl esters (FAME) is the high acidity of the product, mainly caused by water presence, which produces parallel hydrolysis and esterification reactions instead of transesterification to FAME. Therefore, the use of reaction medium in absence of water (anhydrous medium) was investigated in a lipase-catalyzed process to improve FAME yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as raw material, methanol as acyl acceptor, and 3Å molecular sieves to extract the water. The anhydrous conditions allowed the esterification of free fatty acids (FFA) from feedstock at the initial reaction time. However, after the initial esterification process, water absence avoided the consecutives reactions of hydrolysis and esterification, producing FAME mainly by transesterification. Using this anhydrous medium, a decreasing in both the acid value and the diglycerides content in the product were observed, simultaneously improving FAME yield. Enzyme reuse in the anhydrous medium was also studied. The use of the moderate polar solvent tert-butanol as a co-solvent led to a stable catalysis using Novozym 435 even after 17 successive cycles of FAME production under anhydrous conditions. These results indicate that a lipase-catalyzed process in an anhydrous medium coupled with enzyme reuse would be suitable for biodiesel production, promoting the use of oils of different origin as raw materials.

Innovative approaches for effective selection of lipase-producing microorganisms as whole cell catalysts for biodiesel production.

The high cost of commercial lipases limits their industrial application in the production of biodiesel or fatty acid methyl esters (FAME). This disadvantage has encouraged the search for lipase-producing microorganisms (LPMs) as potential whole cell catalysts for FAME production. The aim of this study, therefore, was to evaluate innovative procedures for easy selection and testing of LPMs as a low-cost whole cell catalyst, based on catalytic performance, methanol tolerance and physico-chemical cell surface properties. The latter (in particular the cell surface hydrophobicity and charge) were analyzed because of their crucial role in microbial adhesion to surfaces and the concomitant increase in cell immobilization and bioavailability of hydrophobic substrates. Biocatalysis experiments performed in the presence of nutrient, rapeseed oil and methanol were an effective tool for studying and identifying, in just two experiments, the capacity of different LPMs as biocatalysts in organic media, as well as the methanol tolerance of the cell and the lipase. This indicates the potential for using live microorganisms for FAME production. Another finding was that the inhibitory effect of methanol is more significant for lipase activity than LPM growth, indicating that the way in which alcohol is supplied to the reaction is a crucial step in FAME production by biocatalysts. According to these results, the application of these innovative assessments should simplify the search for new strains which are able to effectively catalyze the FAME production process.

Biotechnological processes for biodiesel production using alternative oils.

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.

Improving fatty acid methyl ester production yield in a lipase-catalyzed process using waste frying oils as feedstock.

The application of waste frying oil (WFO) mixed with rapeseed oil as a feedstock for the effective production of fatty acid methyl esters (FAME) in a lipase-catalyzed process was investigated. The response surface methodology (RSM) was used to optimize the interaction of four variables: the percentage of WFO in the mixed feedstock, the methanol-to-oil ratio, the dosage of Novozym 435 as a catalyst and the temperature. Furthermore, the addition of methanol to the reaction mixture in a second step after 8 h was shown to effectively diminish enzyme inhibition. Using this technique, the model predicted the optimal conditions that would reach 100% FAME, including a methanol-to-oil molar ratio of 3.8:1, 100% (wt) WFO, 15% (wt) Novozym 435 and incubation at 44.5 degrees C for 12 h with agitation at 200 rpm, and verification experiments confirmed the validity of the model. According to the model, the addition of WFO increased FAME production yield, which is largely due to its higher contents of monoacylglycerols, diacylglycerols and free fatty acids (in comparison to rapeseed oil), which are more available substrates for the enzymatic catalysis. Therefore, the replacement of rapeseed oil with WFO in Novozym 435-catalyzed processes could diminish biodiesel production costs since it is a less expensive feedstock that increases the production yield and could be a potential alternative for FAME production on an industrial scale.