DS0204 -

New operational strategies to overcome technical barriers in anaerobic digestion and extend its fields of application by using meta-omic approaches – DIGESTOMIC

New operational strategies to overcome technical barriers in anaerobic digestion and extend its fields of application by using meta-omic approaches

Anaerobic digestion is a process of organic matter degradation which produces renewable energy. Experience has shown that digester operation relies on the know-how of the developer. Such a situation is mainly due to the limitations of microbial-based management of anaerobic reactors as the microbiome still remains largely unknown. Using meta-omics approaches it should be possible to propose new operational strategies to overcome technical barriers in anaerobic digestion.

Main issues raised & general objectives

The main scientific blockage to the development of operational strategies is the lack of knowledge concerning the functioning of the microbial communities responsible for anaerobic digestion. In this project we want to use an approach leading to a deeper characterization of the reactions of the microbial ecosystem during anaerobic digestion in the case of perturbations. The studied perturbations will be: (i) high ammonia concentration, (ii) co-substrate composition variation and (iii) temperature modification. The innovative approach we propose is based on the one hand on the use of meta-omics methodologies, to obtain information on the microorganisms present, the functions expressed and the metabolites produced, but also the variations of these indicators as a function of the operating conditions; and on the other hand on the application of powerful statistical methods of data integration. <br />More precisely, the industrial goals are: <br />i. Proposal of microbial management strategies for anaerobic digesters to overcome the studied perturbations (i.e., ammonia concentration, co-substrate variation, temperature modification); <br />ii. Definition of microbial indicators of optimal performance and early warning indicators of anaerobic digestion process failure; <br />iii. Definition of possible microbial management strategies to produce biofuels or synthons for use in green chemistry. <br />Scientific goals are: <br />i. Development of a meta-omics approach to make a functional microbiome diagnostic; <br />ii. Development of biostatistical data mining methods for the fusion of meta-omics data and process parameters in order to extract key functional information from the huge amount of data generated by omics approaches; <br />iii. Evaluation of the importance of microbial ecological parameters such as microbial community diversity, homogeneity of microbial community structure and microbial community dynamics over time to maintain functional stability and robustness of bioprocesses.

The effect of high ammonia concentrations will be studied as it is the most common cause of failure, due to the low C/N ratio of sewage sludge. In a second stage, we will focus on co-digestion that can be an interesting solution to overcome this mono substrate drawback. In particular, more knowledge is needed on how the microbiome is affected by co-substrate composition variations that can lead to digester instability. This could be used as an operational lever to adapt to the variation of loading rates. Low temperature could be used when sewage sludge availability is low, in order to reduce the operational costs, and would be increased only when needed to treat high loading rates.
The adopted experimental approach will combine laboratory experiment in batch reactors, laboratory experiment in semi-continuous reactors, and full-scale pilot experiments (temperature modification). In the three tasks, the omics microbial ecology methodologies (non-targeted metabolomics, metagenomics, metatranscriptomics) will be applied to characterize in detail the reactions of the microbiome during the variation of operating parameters and to derive structure-function relationships, that is, correlations between the microbial community structure and dynamics and the reactor performances. More precisely, these data, along with performance data, will be analyzed by fusion (multi-blocks approaches) to extract the relevant correlations to favor the identification of the causes of the variations of performance and propose strategies for successful management. Through the fusion of different omics data, the goal is also to identify possible inhibitors of anaerobic digestion and to determine which biochemical reactions this metabolic regulator can influence in order to limit its action when methane production is wanted, or to amplify its inhibitory effects if it leads to the production of a high value product.

Task 1:
In 6 lab-scale digesters ammonia was added at different rates to create different inhibition patterns. Degradation performances were monitored (gas production, degradation intermediates…), as well as the identity and activity of the microorganisms responsible for the degradation (16S metabarcoding of 140 RNA and DNA samples). In a second experiment, strategies for mitigation of ammonia inhibition were tested. Among them, the addition of support material seemed very promising to favor interactions and syntrophy between microorganisms. The effect of these support media was evaluated in the long term in six semi-continuous digesters. Results are under investigation.
Task 2:
A first experiment was set-up in batch digesters to assess the influence of feeding composition on the stability of the digesters. Fish waste, grass and sewage sludge were mixed by pairs in different proportions. Microbial dynamics were followed by 16S metabarcoding and degradation pathways were monitored by metabolomics. Different statistical methods were used to identify the most discriminant microorganisms and metabolites for the substrates. We observed that the feeding composition had a strong influence on microbial dynamics. It gives highly relevant indications for feeding strategies to overcome dysfunction during co-digestion that will be tested in semi-continuous digesters.
Task 3:
A full-scale experiment was carried out on two full-scale digesters operated in parallel and fed in exactly the same way. Digester A was used as a mesophilic anaerobic digestion (35°C) reference; while the temperature of digester B was modified. Modifications of specific biogas production were observed in the test reactor. Temperature modification showed an influence on both activity and presence of different microorganisms. Archaea were particularly sensitive to temperature modification. Degradation pathway dynamics will be monitored by metabolomics. Eight samples will be studied by metagenomics.

This project aims at developing a meta-omics approach to make a functional microbiome diagnostic. In particular it involves the development of biostatistics data mining methods for the fusion of meta-omics data and process parameters in order to extract key functional information. It will allow to extract the relevant correlations between the various types of data to favor the identification of the causes of the variations in performance and to propose strategies for successful management. Our project will give a general framework to apply these methods to other subjects of interest.
In the face of current economic, environmental and societal challenges, the waste management sector needs to gain in efficiency and attain a higher level of sustainability of the operations. New technologies and practices can help to reduce the growing quantity of residual materials as well as reduce the impacts of the processing operations. In this context, the envisaged project aims at proposing a new tool and optimized methods for the management of anaerobic digestion bioprocesses. Besides, from the point of view of sustainable development, it will contribute to increase the reliability and productivity of anaerobic digestion processes related to the re-utilization of used materials, the generation of renewable power, and the production of molecules with high added value for green chemistry.
A better management of the anaerobic bioprocesses will improve the production of biogas, by making waste a resource. In particular, this work will propose new modes of management of anaerobic digestion. The information obtained in the cases of situations of dysfunctions will be particularly interesting because it is in these situations that metabolites of interest such as ethanol, and lactic acid, can accumulate. These results could lead to developing strategies targeted to enlarge the fields of application of the process in the production of biofuels or synthons for use in green chemistry.

Conference
15th IWA World Conference on Anaerobic Digestion (17-20 Octobre 2017, Beijing, China)
Low temperature sewage sludge anaerobic digestion: full-scale proof of interest and study of microbial adaptation.
D. Conteau, O. Franchi Morales, O. Chapleur , G. Gaval, G. Traba Lago, L. Mazéas, P. Araya Kroff, B. Barillon

3th International Congress of Microbiology in Biogas (1-3 mai 2017, Wageningen, Pays-Bas)
Understanding the effect of ammonia on anaerobic microbiota during biowaste anaerobic digestion
L. Cardona, S. Poirier, C. Madigou, T. Bouchez, L. Mazéas, O. Chapleur

2nd international conference on anaerobic digestion technology (4-7 Juin 2018 , Chiang Mai, Thailand)
Sustainable alternative bioenergy for a stable life: Impact of anaerobic co-digestion on microbial community and associated degradation pathways.
L. Cardona, C. Madigou, C. Bureau, L. Rouillac, L. Mazeas and O. Chapleur

ISME conference 2018 (Aout 2018, Leipzig)
Effect of ammonia on the dynamics of anaerobic digestion microbiome: omics data integration in a time course context. O. Chapleur, S. Poirier, K-A. Lê Cao

ISME conference 2018 (Aout 2018, Leipzig)
Multi-omics data integration to decipher the impact of feeding composition on the microbiota of anaerobic digestion.
L. Cardona, K-A. Lê Cao, C. Madigou, C. Bureau, L. Rouillac, L. Mazeas and O. Chapleur

Chimiométrie XX – 2019 (january 2019 Montpellier)
ComDim-ICA : A procedure to perform Multiblock Independent Components Analysis
D. N. Rutledge, L. Schmidtke

Chimiométrie XX – 2019 (january 2019 Montpellier)
Unraveling the microbial community interactions in anaerobic digesters with Common Components Analysis
F. Puig-Castellví, L. Cardona, O. Chapleur, C. B. Y. Cordella, D. Jouan-Rimbaud Bouveresse, L. Mazeas and D. N. Rutledge

Anaerobic digestion is a process of degradation of the organic matter which, when it goes to its term, produces biogas rich in methane that can be valued in the form of electrical and thermal energy. In a context of environmental protection and research for increasing energy efficiency, methanization arouses a renewed interest because it allows at the same time to convert waste in an energy resource and an organic amendment that can be used for manure spreading on soil. The number of organic matter deposits is ceaselessly growing, making anaerobic digestion an important potential source of energy.

Experience feedbacks show that digesters remain little instrumented and that their operation relies on the know-how of the developer. Furthermore, the dynamics of the populations and the industries cause sudden and unpredictable changes of quantity and composition of waste to treat. Methanization is still considered not being well enough mastered to face these changes and present the desired flexibility.

Such situation is mainly due to the limitation of microbial-based management of anaerobic reactors as the microbiome which is the key player of the AD process still remains largely unknown. Nowadays omic microbial ecology methodologies (metagenomic, metatranscriptomic, metaproteomic, metabolomic) allow to perform functional microbiome diagnostics which could help (i) to build real microbial management strategy of anaerobic digestion and (ii) to set microbial indicators of optimal performance and warning indicators of process failure.

In order to achieve these goals it is important not only to study the microbial community functioning during steady state but also their reaction to operational and environmental disturbance. In the DIGESTOMIC project we want to apply the omic microbial ecology methodologies to different key known perturbations that could lead to some process failure during sewage sludge anaerobic digestion. The analysis by fusion of these omics data will allow extracting the relevant correlations between the various types of data to favour the identification of the causes of the variations of performance and proposing strategies of successful management.

In a first step, the effect of high ammonia concentration will be studied as it is the most common failure cause due to the low C/N ratio of sewage sludge. The introduction of additional wastes to conduct the co-digestion could improve significantly plant's economic feasibility by enhancing biogas production and limit the ammonia inhibition risk. However, this practice is facing one major challenge on an operational point of view due to the difficulties caused by the co-substrate composition variation that can lead to digester instability. More knowledge is needed about how microbial population is affected by the addition of a co-substrate, in terms of diversity, stability and yields. The co-substrate introduction will then be the second perturbation that will be addressed during this project. If no co-substrate is easily available it could be interesting to use temperature as an operational trigger in function of the sewage sludge availability. Temperature modification will then be the third perturbation studied during the project.

Besides, the information obtained in the cases of situations of dysfunctions will be particularly interesting because it is in these situations that metabolites of interest such as ethanol and lactic acid can accumulate. These results could allow developing strategies targeted to widen the fields of application of the process in the production of biofuels or synthons used in green chemistry. Metaomic diagnostic should be helpful to build some new approaches to produce high value products.

Project coordinator

Monsieur Laurent Mazéas (Institut de recherche en science et technologie pour l'environnement et l'agriculture)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IRSTEA Institut de recherche en science et technologie pour l'environnement et l'agriculture
AgroParisTech Institut des sciences et industries du vivant et de l'environnement
Suez Groupe SUEZ GROUPE

Help of the ANR 446,565 euros
Beginning and duration of the scientific project: October 2016 - 42 Months

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