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Different extraction methods have been proposed to study the ingestion of microplastics by marine organisms, including enzymatic digestion. While mussels have been the focus of research, crustaceans' enzymatic digestion has received little attention. An overlooked source of information for microplastic research is analysis of long-term time-series biotic samples. These collections are invaluable for the detection and monitoring of changes in ecosystems, especially those caused by anthropogenic factors. Here, crustacean larvae collected in two periods, 1985 and 2020, in the central North Sea were used to develop and optimise an effective and gentle enzymatic digestion method suitable for microplastic research. Sequential breakdown of these chitinaceous samples via a mechanical and surfactant (Sodium Dodecyl Sulphate 1% v/v) pre-treatment, followed by proteinase K (100 mU/mL) and chitinase (50 mU/mL) digestion, efficiently removed >96% of biomass of 1985 and 2020 samples. The optimised method was effective without interfering with the identification of naturally weathered microplastics via FTIR Spectroscopy.

The utilisation of the organic fraction of municipal solid waste as feedstock for bioethanol production could reduce the need for disposal of the ever-increasing amounts of municipal solid waste, especially in developing countries, and fits with the integrated goals of climate change mitigation and transport energy security. Mixed culture fermentation represents a suitable approach to handle the complexity and variability of such waste, avoiding expensive and vulnerable closed-control operational conditions. It is widely accepted that the control of pH in these systems can direct the fermentation process toward a desired fermentation product, however, little empirical evidence has been provided in respect of lignocellulosic waste substrates and different environmental inocula sources. We evaluated ethanol production from the organic fraction of municipal solid waste using five different inocula sources where lignocellulose degradation putatively occurs, namely, compost, woodland soil, rumen, cow faeces and anaerobic granular sludge, when incubated in batch microcosms at either initially neutral or acidic pH and under initially aerobic or anaerobic conditions. Although ethanol was produced by all the inocula tested, their performance was different in response to the imposed experimental conditions. Rumen and anaerobic granular sludge produced significantly the highest ethanol concentrations (30 mM) under initially neutral and acidic pH, respectively. A mixed-source community formed by mixing rumen and sludge (R C S) was then tested over a range of initial pH. In contrast to the differences observed for the individual inocula, the maximal ethanol production of the mixed community was not significantly different at initial pH of 5.5 and 7. Consistent with this broader functionality, the microbial community analyses confirmed the R C S community enriched comprised bacterial taxa representative of both original inocula. It was demonstrated that the interaction of initial pH and inocula source dictated ethanologenic activity from the organic fraction of municipal solid waste. Furthermore, the ethanologenic mixed-source community enriched, was comprised of taxa belonging to the two original inocula sources (rumen and sludge) and had a broader functionality. This information is relevant when diverse inocula sources are combined for mix culture fermentation studies as it experimentally demonstrates the benefits of diversity and function assembled from different inocula.

Mixed Culture Fermentation is a promising route for bioethanol production from organic wastes. Yet, achieving a stable ethanologenic activity in undefined mixed cultures remains a challenge. This work aimed to retain ethanol production from organic municipal solid waste by microbial communities enriched from sheep rumen and anaerobic sludge mixtures, under low process control (initially aerobic conditions and initial pH ≤ 5.5). To find a stable operating window, sequential inoculum transfer intervals were evaluated (14 and 3-days). Soluble fermentation product profiles and changes in the prokaryotic communities were monitored. The originally inoculated batches always produced high ethanol concentrations (60 mM; 0.070 LEtOH/Kgwaste), equivalent to 1/6 of the current corn grain-based ethanol industrial production process. Fermentative activity and community richness significantly decreased in both transfer times regimes tested. However, the 3-day transfer interval led to a stable community which consistently produced ethanol (30 mM) as its main soluble fermentation product. Originally inoculated and 3-day transferred communities consistently enriched for a solventogenic Clostridium and an acid-tolerant Pseudomonas species. Ethanologenesis, as a dominant catabolic process, is an inherent property of these mixed culture fermentations, and its maintenance across successive transfers is critically dependant on the inoculum transfer time.

The utilisation of the organic fraction of municipal solid waste as feedstock for bioethanol production could reduce the need for disposal of the ever-increasing amounts of municipal solid waste, especially in developing countries, and fits with the integrated goals of climate change mitigation and transport energy security. Mixed culture fermentation represents a suitable approach to handle the complexity and variability of such waste, avoiding expensive and vulnerable closed-control operational conditions. It is widely accepted that the control of pH in these systems can direct the fermentation process toward a desired fermentation product, however, little empirical evidence has been provided in respect of lignocellulosic waste substrates and different environmental inocula sources. We evaluated ethanol production from the organic fraction of municipal solid waste using five different inocula sources where lignocellulose degradation putatively occurs, namely, compost, woodland soil, rumen, cow faeces and anaerobic granular sludge, when incubated in batch microcosms at either initially neutral or acidic pH and under initially aerobic or anaerobic conditions. Although ethanol was produced by all the inocula tested, their performance was different in response to the imposed experimental conditions. Rumen and anaerobic granular sludge produced significantly the highest ethanol concentrations (∼30 mM) under initially neutral and acidic pH, respectively. A mixed-source community formed by mixing rumen and sludge (R + S) was then tested over a range of initial pH. In contrast to the differences observed for the individual inocula, the maximal ethanol production of the mixed community was not significantly different at initial pH of 5.5 and 7. Consistent with this broader functionality, the microbial community analyses confirmed the R + S community enriched comprised bacterial taxa representative of both original inocula. It was demonstrated that the interaction of initial pH and inocula source dictated ethanologenic activity from the organic fraction of municipal solid waste. Furthermore, the ethanologenic mixed-source community enriched, was comprised of taxa belonging to the two original inocula sources (rumen and sludge) and had a broader functionality. This information is relevant when diverse inocula sources are combined for mix culture fermentation studies as it experimentally demonstrates the benefits of diversity and function assembled from different inocula.