Coalescence expérimentale de communautés microbiennes dans des digesteurs anaérobies

Anaerobic digestion is a biological process carried out by a complex and synergistic network of microbial communities allowing the degradation of organic matter such as agricultural waste or effluents from wastewater treatment plants, into biogas, a gas recoverable into energy. The mechanisms influencing microbial communities at the heart of this process but also in nature remain misunderstood because of a low understanding of their dynamics. The objectives of this project are therefore to develop an anaerobic digestion system to better understand the dynamics of microbial community assembly. Thus, a new continuous reactor process has been developed with automated feeding, biomass wasting and degassing functions. Automation and multiplexing of reactors allows for the continuous parallel manipulation of 30 reactors in parallel. In addition to the automation, many parameters are versatile, such as the substrate loading (once a minute up to batch conditions), the reactor volume (50 to 200 mL), the temperature (room to 55°C), but also the use of the aerobic system or the implementation of other tools such as LEDs for phototrophic cultures. Capable of accurately quantifying the performance of a methanogenic ecosystem, this system has enabled us to test the structure and the performance of five different methanogenic ecosystems that have been mixed and tested individually. By mixing different methanogenic ecosystems the Archaea diversity has increased transiently. Besides, a correlation is observed between the diversity of mixed communities and their methanogenic performance; yet the individual communities have a better functioning at the same level of diversity. Interestingly, the mixture of some communities has allowed for better methane production than individual communities, suggesting the development of specific interactions in these communities. In a novel way compared to the literature and that the majority of individual bacterial communities are found in mixed communities. Contrary to the idea of selecting a more adapted or functional community, here the majority of communities have settled. These experiments suggest that a parameter such as the functionality of a bioprocess can be improved by bioaugmentation.La digestion anaérobie est un procédé biologique effectué par un réseau complexe et synergique de communautés microbiennes permettant la dégradation de matière organiques comme les déchets agricoles ou les effluents de station d’épuration en biogaz, un gaz valorisable en énergie. Les mécanismes influençant les communautés microbiennes au cœur de ce procédé mais aussi dans la nature restent incompris du fait de la faible compréhension de leur dynamique. Les objectifs de ce projet visent à donc développer un système de digestion anaérobie permettant de mieux comprendre la dynamique de l’assemblage des communautés microbiennes. Ainsi un nouveau procédé de réacteurs en continu dont les fonctions d’alimentation de soutirage et de dégazage sont automatisées a été développé. L’automatisation et le multiplexage des réacteurs permettent la manipulation de 30 réacteurs en continu en parallèle. Outre l’automatisation ce système, de nombreux paramètres sont flexibles comme le taux de charge (une fois par minute jusqu’à une condition batch), le volume de réacteur (50 à 200mL), la température (pièce – 55°C), mais aussi l’utilisation du système en aérobie ou l’implémentation d’autres outils comme des LEDs pour les cultures phototrophes. Capable de quantifier précisément la performance d’un écosystème méthanogène, ce système nous a permis de tester la structure et la performance d’écosystèmes méthanogènes mis en mélanges et testés de façon individuelle. En mélangeant des écosystèmes méthanogènes différents, la diversité des Archées a augmenté transitoirement. Une corrélation est d’ailleurs observée entre la diversité de ces communautés mélangées et leur performance méthanogène, seulement à même diversité les communautés individuelles ont un meilleur fonctionnement. L’assemblage de certaines communautés mélangées a pourtant permis une meilleure production de méthane que les communautés individuelles, ce qui suggère le développement d’interactions spécifiques de ces communautés. De façon nouvelle par rapport à la littérature, la majorité des communautés bactériennes individuelles sont retrouvées dans les communautés mélangées. Soit contrairement à l’idée d’une sélection d’une communauté plus adaptée ou plus fonctionnelle, ici la majorité des communautés se sont implantées. Ces expériences suggèrent qu’un paramètre tel que la fonctionnalité d’un bioprocédé peut-être amélioré par bioaugmentation

Replication of experiments in anaerobic digestion: simpler, greater and easier than you had ever hoped

Reliability of results in science is done by replication of experiments, but it is often expensive and labour intensive. Indeed, operating anaerobic digesters in a continuous mode requires some experience and a lot of manpower. The number of anaerobic digesters that can be handled in parallel is therefore limited. In practice, experiments are often repeated several times, starting always with a fresh inoculum. One approach is to store and to restore the activity of the same inoculum, with no guarantee on the preservation of the functioning. Unfortunately, there is no consensus on any standardized strategy for the long-term preservation of complex ecosystems. Here, we present a multiplexed chemostat system that minimizes costs and manpower for operating several anaerobic digesters in parallel, and present a first application of the system. Up to thirty reactors may be operated by a single person. This system may unlock ecological studies as well as process optimization in the context of anaerobic digestion, where several conditions need to be tested in parallel. The vocation of this device is to make it available to the scientific community

Multiplexed chemostat system for quantification of biodiversity and ecosystem functioning in anaerobic digestion

<div><p>Continuous cultures in chemostats have proven their value in microbiology, microbial ecology, systems biology and bioprocess engineering, among others. In these systems, microbial growth and ecosystem performance can be quantified under stable and defined environmental conditions. This is essential when linking microbial diversity to ecosystem function. Here, a new system to test this link in anaerobic, methanogenic microbial communities is introduced. Rigorously replicated experiments or a suitable experimental design typically require operating several chemostats in parallel. However, this is labor intensive, especially when measuring biogas production. Commercial solutions for multiplying reactors performing continuous anaerobic digestion exist but are expensive and use comparably large reactor volumes, requiring the preparation of substantial amounts of media. Here, a flexible system of Lab-scale Automated and Multiplexed Anaerobic Chemostat system (LAMACs) with a working volume of 200 mL is introduced. Sterile feeding, biomass wasting and pressure monitoring are automated. One module containing six reactors fits the typical dimensions of a lab bench. Thanks to automation, time required for reactor operation and maintenance are reduced compared to traditional lab-scale systems. Several modules can be used together, and so far the parallel operation of 30 reactors was demonstrated. The chemostats are autoclavable. Parameters like reactor volume, flow rates and operating temperature can be freely set. The robustness of the system was tested in a two-month long experiment in which three inocula in four replicates, i.e., twelve continuous digesters were monitored. Statistically significant differences in the biogas production between inocula were observed. In anaerobic digestion, biogas production and consequently pressure development in a closed environment is a proxy for ecosystem performance. The precision of the pressure measurement is thus crucial. The measured maximum and minimum rates of gas production could be determined at the same precision. The LAMACs is a tool that enables us to put in practice the often-demanded need for replication and rigorous testing in microbial ecology as well as bioprocess engineering.</p></div

Detailed prices of major components required for the construction of one LAMACs module containing six reactors.

<p>Detailed references for these items can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193748#pone.0193748.s006" target="_blank">S1 Table</a>.</p

Biogas production of four anaerobic digesters measured by the LAMACs.

<p>A) Actual accumulated biogas over time (red curves) and linear regressions (blue lines) B) Biogas production rates derived from linear regressions.</p

Dynamics of biomass concentration for twelve anaerobic digesters over nine weeks.

<p>Biomass concentration is expressed as volatile solids. A color code was applied by inoculum origin; Light gray stands for INOC A replicates, dark-gray stands for INOC B replicates and black stands for INOC C replicates. During weeks 4 to 6, a simple substrate with halved loading rate in terms of COD was applied to the reactors.</p

Development of average weekly biogas production rates over time for anaerobic digesters with three different inocula.

<p>Four replicated anaerobic digesters were operated per condition. White boxes stand for the INOC A condition, light gray boxes for INOC B and dark-gray boxes for INOC C. The Kruskal Wallis test was significant in the first eight weeks and the Dunn post hoc test for pairwise multiple comparison displays inocula differences with ‘*’ symbols. In weeks 4 to 6, the simple substrate was used instead of the complex substrate.</p

General view of the LAMACs and of components of one chemostat in the LAMACs module.

<p>(A) 3D view of a LAMACs module, i.e., six chemostats. The electric box containing controlling cards is on the top of the module and above any source of liquids to prevent electric failure in case of leakage. A waterproof stirring plate is underneath the heating block containing the six reactor vessels. Three peristaltic pumps are aligned above each vessel. The upper range of bags is for biogas collection; the lower range serves as substrate reservoirs. The dimensions of one module are 50 cm width × 52 cm length × 100 cm height. (B) Schematic view of a chemostat detailing the use of the three-hose connector. One port is used as inlet for feeding, and two ports as outlet for biomass wasting and degassing. One port for manual sampling is sealed with a rubber stopper. A 0.45 μm pore-size filter is placed in the feedline ahead of the reactor to prevent contamination of the medium.</p

Stable temperature of the LAMACs during operation.

<p>Average temperature and standard deviation over three days of operation at three different positions in the heating block. Data points were recorded every 15 minutes, with 280 total data points.</p

Linear regression of biogas accumulation over time.

<p>Linear regression of biogas accumulation over time.</p