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.

Bulking agent in dry anaerobic digestion as a key factor for the enhancement of biogas production.

Dry anaerobic digestion (dry-AD) is an attractive process for solid wastes such as agri-food waste. However, some limitations mainly associated to lack of effective mixing, can hinder the methane production capacity of the systems. Bulking agent (BA) has been proposed as a solution to the compaction issues in systems without mechanical agitation, such as leaching bed reactors. However, effects of BA are still not clear, and, thus, the factors to consider for its dose has not been optimized yet. This work studies the effect of BA in dry-AD. Two substrates with different characteristics were proposed as models, bean peel as a lignocellulosic substrate and a mixture of food waste as a readily biodegradable substrate. Inert plastic rings were used as BA at different BA:S ratios. Assessed BA:S ratio did not affect the performance of methane production for the lignocellulosic waste, but it did significantly affect to the easily biodegradable substrate, showing up to a 28% of methane production increase. This result could be due to the presence of lignocellulosic compounds in the bean peel, behaving like a natural BA. In assays with an increased bed height, the compaction of the system was more severe, resulting in the rapid acidification of the processes. At these conditions, the positive effect of BA addition was more marked, allowing methane production and no acidification of the system. Thus, the addition of BA is a suitable strategy for improving methane production or stability in dry-AD systems without requiring the stirring of the systems.

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.

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.

On-Line Control of Feast/Famine Cycles to Improve PHB Accumulation during Cultivation of Mixed Microbial Cultures in Sequential Batch Reactors.

Production of polyhydroxyalkanoates (PHA) has generated great interest as building blocks for bioplastic production. Their production using mixed microbial cultures represents an interesting alternative, since it enables the use of organic wastes as a carbon source. Feast/famine strategy is a common way to promote selection of microorganisms with PHA accumulation capacity. However, when using waste sources, changes in substrate concentration are expected, that may affect performance and efficiency of the process. This study showed how the dissolved oxygen level can be used for online control of the cycle time, ensuring that the desired feast/famine ratio is effectively applied. An operation strategy is presented and validated, using sequential batch reactors fed with acetate as the carbon source. Production of polyhydroxybutyrate (PHB) was studied, which is the expected type of PHA to be synthetized when using acetate as substrate. Two reactors were operated by applying the proposed control strategy, to provide F/F ratios of 0.2 and 0.6, respectively. A third reactor was operated with a fixed cycle time, for comparison purposes. Results showed that the reactor that operated at an F/F ratio of 0.6 promoted higher biomass productivity and PHB content, as a result of a better use of available time, preventing unnecessary long famine times. The application of the tested strategy is a simple a reliable way to promote a better performance of feast/famine-based bioreactors involving mixed microbial cultures for PHB production.