IUAP MRM network

Inter-universitary attraction poles programme on Microbial Resource Management in engineered and natural ecosystems

The Interuniversity Attraction Poles (IAP) Program aimed to provide support and build research networks within Belgian and international universities is an initiative of the Belgian Science Policy. This IAP-Phase VII (P7/25) focussed on the concept of Microbial Resource Management (MRM) in synthetic and natural ecosystems. Microbial communities represent functional biological entities with diverse metabolic capacities, and can be considered to be an infinite, yet natural resource. The strategy was to optimize the management of microbial resources to tailor to the needs of different applications. This is known as MRM. To achieve this, ecological theory (e.g. population dynamics, invasion, microbial interactions) was developed using synthetic ecosystems, alongside environmental studies using state-of-the-art molecular and proteomic tools.

Research groups

Partner 1: Ghent University (UGent)

At UGent the coordinating partner is the Center for Microbial Ecology and Technology (CMET) lead by Prof. dr. ir. Nico Boon. Within the CMET dr. Ramiro Vilchez, dr. Massimo Marzorati, Ir. Karen De Roy, Mrs. Elham Ehsani, Ir. Charlotte De Rudder, Ir. Ruben Props, Ir. Benjamin Buysschaert, Mrs. Nicole Hahn, Mrs. Christine Graveel, Mrs. Regine Haspeslagh, Mr.Tom Bellon and Ir. Frederiek-Maarten Kerckhof are or have been associated with scientific and administrative support for the MRM IAP project. Furthermore, at UGent a second research team, the research unit Knowledge Based Systems (KERMIT) lead by Prof. dr. Bernard de Baets actively contributes to the MRM IAP network. Within KERMIT dr. Jan Baetens, Mcs. Aisling Daly, Ir. Wai-Kit Tsang and Ir. Tim Depraetere are or have been associated with scientific and administrative support for the MRM IAP project in the reported period.

Partner 2: Université Catholique de Louvain (UCL)

At UCL Prof. dr. ir. Spiros Agathos, Dr. Ben A. Stenuit, Mrs Hélène Dailly, Mrs. Emna Bouhajja, Mr. Antonio Ramirez-Guanche, Mrs. Enrica Santolini, Mr. Guilherme Inocencio Matos, Mrs. Floriana Augelletti and Mr. Theocharis Efthymiopoulos are or have been associated with scientific and administrative support for the MRM IAP project in the reported period.

Partner 3: Katholieke Universiteit Leuven (KUL)

At KUL Prof. dr. ir. Dirk Springael, Dr. Benjamin Horemans, Dr. Pierre-Joseph Vaysse, Dr. Basak Ozturk, Mr. Pieter Albers, Mr. Dries Grauwels, Mr. Jeroen T’Syen, Mr. Bart Raes, Mrs. Aswini Sekhar and Mrs. Johanna Vandermaesen are or have been associated with scientific and administrative support for the MRM IAP project in the reported period.

Partner 4: Université de Mons (UMons)

At UMons Prof. dr. Ruddy Wattiez, Prof Dr. David C. Gillan, Dr. Mélanie Beraud, Dr. Stéphanie Roosa, Mr. Benoït Kunath, Mr. Guiseppe Giambarresi, Mr. Florian Liénard, Mrs. Valentine Cyriaque, Mrs. Marta Brodzik are or have been associated with scientific and administrative support for the MRM IAP project in the reported period.

Int. Partner 1: Swiss Federal Institute for Aquatic Science and Technology (EAWAG)

At EAWAG dr. Frederik Hammes is associated with scientific and administrative support for the MRM IAP project in the reported period.

Int. Partner 2: Technical University of Denmark (DTU)

At DTU Prof. dr. Barth Smets and dr. Arnaud Dechesne are or have been associated with scientific and administrative support for the MRM IAP project in the reported period.

Int. Partner 3: University of Copenhagen

At KU Prof. dr. Søren J. Sørensen, Dr. Martin Asser Hansen, Dr. Peter Holmsgaard and dr. Tim Evison are or have been associated with scientific and administrative support for the MRM IAP project in the reported period.

1. Summary of the general objectives of the research project

Microbial communities are primary drivers of element cycles on a planetary level and are used in many sustainable biotechnological applications. These communities are often complex consisting of several populations which can interact with each other by antagonistic or cooperative processes. The availability of tools and models for behaviour prediction and the proper management of such complex microbial communities, commonly referred to as Microbial Resource Management (MRM), was the main objectives of this project. The understanding of the interactions within such complex microbial communities is essential for developing MRM tools and models. The last few decades have seen a tremendous development in disparate disciplines such as molecular and evolutionary ecology together with quantum leaps in massively efficient technology platforms (genomics, proteomics, metabolomics and the corresponding meta-omics techniques for the molecular analysis of biodiversity) which allow to disentangle the interactions with high resolution. While generating vast amounts of data is currently not an issue, the theoretical framework for proper interpretation and hypothesis-driven research is still lacking, resulting in descriptive rather than hypothesis-driven studies being published (e.g. “overselling the microbiome”). Therefore, this project’s focus lies with developing knowledge to generate research hypotheses for MRM, which will enable further improvements in sustainable biotechnological applications of complex microbial communities.

The challenge for microbial ecologists and engineers is the optimal management of the microbial resources in order to guarantee stable and functional ecosystems. Therefore, the biodiversity-stability relationship and the effect of biodiversity on ecosystem functioning have become major foci in microbial ecology. The majority of studies of biodiversity-stability theory have been approached in a more phenomenological way, e.g. describing microbial ecosystems by listing species names. Hence, this project aimed at developing new ecological theories for microbial ecology as advances in (the application of) microbial ecology are limited by a lack of these conceptual and theoretical approaches. In this project, both in vitro and in silico “synthetic ecosystems” were employed to develop new microbial ecological hypotheses. By means of this unique coupling between mathematical, experimental ecological theory development and the µ-manager portfolio should allow to validate or disprove basic ecological theories on microbial communities, which so far have been blindly adopted from macro-ecology. In this project, the objective was to couple (i) invasion (ii) community dynamics (temporal aspects of community interaction), (iii) community structure (richness, evenness), (iv) community architecture (spatial structure), (v) cell to cell interactions, (vii) genetic interactions, (vi) metabolic networking, etc. to ecosystem performance.

The findings of this IAP project can have important implications and offer new opportunities for scientists working in domains such as applied and fundamental environmental sciences, food science and even medical microbiology. A combination of new ecological approaches with molecular techniques and mathematical guidance could be used to predict ecosystem function failure and hence manage robust biotechnological applications with microbial communities. To obtain this goal, 4 work packages (WP) with increasing ecosystem complexity and interactions were investigated in parallel with the construction of a modelling framework (WP5) to allow for both in vitro and in silico MRM theory development (Figure 1 1).

Figure 1 1. Overview of the division of the project in different WPs.
WP1’s main objective was to design simple but high throughput approaches to test the effect of microbial community composition (richness, evenness, diversity, architecture, species characteristics…) on certain ecological phenomena, such as invasion, horizontal gene transfer, different trophic strategies … The experiments are therefore performed in small volume systems (designated as microcosms) that allow high throughput analysis of hundreds to thousands of experiments simultaneously. Experimental design and statistical analysis of these complex setups is crucial and therefore this WP is intricately linked with WP5. WP2 further uses these high throughput methodologies with as objective to assess the influence of environmental stressors and/or stimuli on these model microbial communities in terms of resilience and robustness. In WP 3, the model microbial communities will be inoculated in their natural, but sterile habitats. The objective of this WP was to assess how specific abiotic properties of the studied ecosystems such as the occurrence of shear stress, heterogeneity, gradients, … affect the synthetic ecosystems. The goal of WP4, was to validate the theories developed in the first three WPs with existing microbial ecosystems and their natural endogenous microbial communities. Through all WPs integration with WP5 is essential. The objective of WP5 was to implement the data generated in the other work packages in models of in silico synthetic ecosystems to address (and form) ecological hypotheses.

2. Scientific achievements

2.1. WP1. Microbial interactions in synthetic communities

2.1.1.Involved partners

All Belgian partners (UGent, UCL, KUL, UMons) were involved with WP1. In WP1 robust synthetic communities are developed to serve further for experimentation in WP2 through 4. P1 (UGent, CMET) was the WP leader.

2.1.2.Summary description of the objectives

WP1’s main objective was to use simple but high throughput approaches to test the effect of microbial community composition (richness, evenness, diversity, architecture, species characteristics…) on certain ecological phenomena, such as invasion, horizontal gene transfer, different trophic strategies …

2.1.3.Summary of the scientific activities and results per partner.

P1: UGent (CMET)

Objectives

Task 1.1. Sand filter ecosystems:

Task 1.2. Soil ecosystems:

Scientific activities and results

Task 1.1. Sand filter ecosystems:

Synthetic communities were generated with different evenness but with constant richness (10 species). Synthetic communities were subsequently incubated and transferred five times in fresh LB medium (Miller) at 28°C. Each cycle lasted 48h. The total cell count was assessed by flow cytometry (Accuri C6, BD) in all time points and relative quantification of the different members of the synthetic communities was followed by amplicon sequencing of the 16S rRNA gene (Illumina MiSeq) for two time points: time zero and five.

Similarities between the time zero and time five (with 3 different evenness levels (low, medium and high)) were analyzed by means of multiple factor analyses. The result showed that there was no correlation between the community structure of the two time points but, as expected, high correlation between three different evenness was observed (Figure 2‑1).

Using a least squares means comparison (SAS PROC LSMEANS) it was observed that the evenness changed during the incubation time and the significant effects could be observed in the different time points (tpt), the interaction effect evenness*tpt and the different evenness groups with different indices like the Pielou and Shannon diversity indices (Table 2‑1). Negative correlation was found between some species which could be attributed to a decrease in total species over time.

Table 2‑1. Results of least squares means analysis.
Figure 2‑1. Result of multiple factor analysis. (A) No correlation between two time points. Blue color for time point 1 and Pink color for time point 5. (B) Correlation between three different evenness. Pink color for Low, green color for Medium and blue color for High evenness.

Overall, the community structures converged over time, irrespective of the initial evenness level:

Figure 2‑2. The beta diversity plot showed that the majority of communities evolved toward the same structure. Blue color time 0 and red color time 5.

Using SYBR-green based flow cytometry and a multi-dimensional diversity assessment based upon flow-cytometric scatter (Props et al. , in press.). The community diversity could be assessed for each separate time-point. The result of flow cytometry showed the decreasing trend in total cell counts over time when the (initial) evenness was low (and, to a lesser extent, when evenness was medium). Diversity also decreased over time specially in synthetic communities with high evenness and showed that communities with high evenness were more influenced by prolonged co-cultivation than the other communities with low or medium evenness (Figure 2‑3). Furthermore, we observed that the variability in total count over time was greatly reduced in the case of low and high initial evenness while this was not the case for medium evenness. Exact mechanistic interpretation of the data is currently under investigation.

Figure 2‑3. Results of flow-cytometric analysis (A) Changes in total cell counts of synthetic communities with low, medium and high evenness and (B) Diversity over time. Blue color Low, yellow color medium and red color high evenness.

Task 1.2. Soil ecosystems:

Main achievements in respect to initial WP objectives

P1: UGent (KERMIT) – supporting role (to P3)

Objectives

Task 1.1: To investigate how the diversity of the endogenous community affects the invasion of pesticide degrading inoculants in sand filter ecosystems.

Scientific activities and results
Main achievements in respect to initial WP objectives

Task 1.1:

P2: UCL

Objectives

Task 1.2, Soil ecosystems

In this task polycyclic aromatic hydrocarbons (PAH) biodegradation using synthetic bacterial communities (consisting of Proteobacteria and Acidobacteria, described in previous reports) was investigated.

Task 1.3, Marine Ecosystems

As an objective related to marine ecosystems, dimethylsulfoniopropionate (DMSP) biotransformation and sulfur cycling using bacterial communities constructed with different marine bacterial strains was investigated (see previous reports).

Additional Task: Wastewater ecosystems

As an additional objective related to wastewater ecosystems, treatment of synthetic wastewater at 4 °C using artificial, psychrophilic bacterial communities was investigated.

Scientific activities and results

Task 1.2, Soil ecosystems

Figure 2‑4. Comparison of PAHs removal (a mixture of fluorene, phenanthrene and anthracene, ca. 50 mg L-1 each) in the presence of mono-cultures, co-cultures and tri-cultures after an incubation time of 12.8 d. Total, initial cell concentrations were set to 2.0 * 107 cell mL-1. Substitutive design (replacement series) was used in which the total, initial cell concentration of the community is maintained constant (i.e., the initial cell concentration for each individual population decreased with an increase of species richness).

Task 1.3, Marine Ecosystems

Main achievements in respect to initial WP objectives

Task 1.2, Soil ecosystems

PAH biodegradation by synthetic bacterial communities and coupling of biodiversity and multifunctionality (biodegradation of different PAHs and biomass productivity, Figure 2‑5).

Figure 2‑5. Comparison of OD600 values after 4 days and 13 days of incubation in the presence of a mixture of fluorene, phenanthrene and anthracene, ca. 50 mg L-1 each.

Task 1.3, Marine Ecosystems

DMSP biodegradation by synthetic bacterial communities and coupling of biodiversity and functionality. The net biodiversity effect (NBE) measures the deviation of the mixture yield as compared to the expected value coming from monoculture yields and the relative abundance of species in the mixture. It’s different components are the dominance effect (DE), the trait-dependent and trait-independent complementarity effects (TDC and TIC) as explained in previous reports. When a synthetic consortium was grown on a mixture of carbon sources, the dominance effect strongly increased with a concomitant decrease in TIC and TDC with increasing phylogenetic diversity. Whereas if a medium containing only DMSP as a carbon source leads to dominance of TIC in the NBE (Figure 2‑6).

Figure 2‑6. Biodiversity effects (PD) obtained through Fox equation applied on the data resulting from qPCR reactions on the different synthetic communities grown in the presence of 4 different carbon sources. TIC, p= 0.0059; TDC, p = 0.0118; DE, p < 0.0001; NBE, P = 0.007. DE: dominance effect, TDC: trait-dependent complementarity effect, TIC: trait-independent complementarity effect.

Additional task, wastewater ecosystems

P3: KUL

Objectives

Task 1.1:

Task 1.2:

Scientific activities and results

Task 1.1:

Figure 2‑7. Results of competition experiment based upon covering array design for increasing richness (DIVEXP7) showing the decrease in variability with increasing richness as well as the impairment of the BAM mineralization with decreased MSH1 survival as well as a logarithmic relationship between MSH1 cell number in presence and absence of SFI.
Figure 2‑8. Illustration of reduced MSH1 survival with increased SFI richness
Figure 2‑9. Specific effects of SFI identity on MSH1 survival and modelled parameters of BAM removal curves.

Task 1.2:

Main achievements in respect to initial WP objectives

Fundamental knowledge was generated with respect to how the diversity of the endogenous community influences invasion of strain MSH1 of a sand filter community. Besides species richness, species identity plays an important role. Different approaches were developed to identify “interactive genes” in Aminobacter sp. MSH1 when in contact with sand filter isolates under conditions of oligotrophy. Bacterial species degrading the same organic pollutant can affect each other growth and activity.

Deviations from initial WP objectives

P4: UMons

Objectives

Task 1.3, Marine Ecosystems

The main objective of WP1 was to evaluate invasion of microbial communities. For that, the UMons partner developed two model synthetic communities were established in the laboratory, one in a marine and another one in a freshwater environment (see previous reports). The impact of microbial diversity and the role of HGT (horizontal gene transfer) were also studied. A large part of this WP was devoted to design the tools used in the research project: the synthetic microbial communities as such, as well as accurate methods to analyse their composition and function.

Scientific activities and results

Task 1.3, Marine Ecosystems

Two major advances have been made :

1) For (meta)proteomics, we have implemented a State-of-the-Art mass spectrometry (MS) acquisition approach, the SWATH-MS (Sequential Window Acquisition of all THeoretical fragment ions). SWATH-MS was then used to analyse a synthetic community elaborated for marine sediments (a manuscript will be submitted soon on this point). It represents a major improvement in the field of microbial ecology. The marine synthetic community was composed of 9 bacteria: Shewanella baltica OS155, Shewanella frigidimarina ACAM591, Burkholderia glumae LMG2196, Burkholderia xenovorans LB400, Mycobacterium vanbaalenii PYR-1, Methylibium petroleiphilum PM1, Cupriavidus metallidurans CH34, Escherichia coli BL21-DE3 and Pseudomonas putida SP902. These strains were selected based upon differential abundance of different genera with freshwater sediments (as determined by metaproteomics) in North Sea marine sediments at Station 130 (see the 2012-2013 reporting period). These bacteria have been cultured together for one month in SSM8 medium. At day 4 and 28, DNA extractions and qPCR experiments have been conducted in order to assess the composition of the community. At the end of incubation, the community was mainly composed of Pseudomonas putida and Shewanella baltica. At 4 and 28 days as well, hydrophilic proteins were prepared using the bead beater technology and subjected to trypsin digestion. SWATH-MS was then used. In parallel, single bacterial cultures have been prepared for each strain in the same medium and conditions, and hydrophilic proteins have been prepared according to the same protocol. Basic DDA acquisition (Data Dependent Acquisition, i.e. not the SWATH approach) was then used. Using a library composed of 9 strains we prepared a spectral library for 3 of the strains (a spectral library is a library composed of all the possible transitions obtained in one condition for one strain in particular). We then matched this library against the SWATH-MS data of the whole synthetic community. Using this approach, bacteria that were previously invisible became visible: indeed, we could detect 85 proteins from C. metallidurans as well as 76 proteins from E. coli (it was shown before by qPCR analysis that these bacteria were present in very low abundances in the synthetic community). Previous mass spectrometry approaches were not able to detect these bacteria (see previous reporting session). Thanks to the SWATH-MS approach we may suggest that E. coli is no more viable in the community, contrary to C. metallidurans that is totally functional. This conclusion may be drawn because the excreted proteome is visible (little proteins used as sensors, to interact and certainly adapt to the environment; see further details in WP2). The SWATH-MS approach proved very useful to investigate the metabolism of rare bacteria in a complex community. This may be highly relevant as investigations into the metabolism of the rare members of the microbiome have so far been precluded due to the low abundance of these members. Although very promising in its present state, the SWATH-MS approach may still be improved, for instance for the quality and peak quantity of the spectral library. A first manuscript presenting the SWATH-MS approach as a major improvement in the field of environmental microbiology (Beraud et al., in preparation) will be submitted soon. A second manuscript focused on the functionality of the synthetic community will follow.

 2) A river synthetic community was also successfully prepared on the basis of the natural community located in the Scarpe river in Férin (Northern France; Gillan et al. 2015). It is composed of 9 strains : Azoarcus communis LMG22127, Burkholderia glumae LMG2196, Burkholderia xenovorans LB400, Delftia acidovorans SPH-1, Leptothrix cholodnii SP-6, Mycobacterium vanbaalenii PYR-1, Rubrivivax gelatinosus IL144, Thauera phenylacetica B4P and Pseudomonas putida SP902. These bacterial strains were mixed together, with or without P. putida. The community was grown for 2 weeks at 16°C, 90 rpm in a threefold-diluted Luria Bertani broth. Half of the broth was refreshed after a week with fresh medium. After 2 weeks, DNA was extracted and quantitative PCR analyses (qPCR) have been performed using specific primers targeting the 16S rRNA gene for each bacterial strain. Each of the primer pairs have been tested for specificity (see previous reporting periods) and only amplify their respective target DNA. qPCR measurements are in progress and will be presented in WP2.

Main achievements in respect to initial WP objectives

We successfully designed 2 synthetic communities and were able to analyse their composition and their functionality using new proteomic approaches. The diversity of the communities was calculated using the Shannon Wiener index. However, this index was not useful as only one bacterium dominated in the community. Thanks to recent results, we are now able to thoroughly describe the function of a community. Previous experiments made on diversity and invasion (see previous reports) will now be reproduced at a larger scale to annotate the already strong conclusions we obtained: One bacterium was always dominating the community and invasion depended on the invader strength.

 
Deviations from initial WP objectives

Initially, synthetic communities were cultivated in 200 µL wells of a microtiter plate, in order to improve statistics and data volume for the WP5. Using this approach, we encountered many contamination and evaporation problems. Therefore, we decided to grow synthetic communities in flasks. We subsequently increased the time scale of the analysis to 1 month. Renewal of the medium, which is the main contributor to contamination problems, has also been reduced to only once a week (for the river community). A novel flow-through system will be soon implemented in the lab. Growth of the community in biofilms will also be investigated as described in WP3.

Synthetic communities with more than 12 species were originally planned. However it was difficult in the laboratory because prices and time of Q-PCR analysis increased with the number of species.

At last, Horizontal Gene Transfer experiments were planned for the WP1. It was decided to start this work directly in WP3, i.e. on biofilms placed in a flow-through system (see WP3).

INT1: EAWAG

Objectives

To assist MRM researchers with the establishment of advanced FCM methods for bacterial analysis.

Scientific activities and results

EAWAG hosted UGent researchers in 2013, 2014 and 2016 and collaborated in these visits on several FCM-related projects. (a) We established operational protocols for high-throughput FCM. (b) We tested and demonstrated fully-automated real-time flow cytometry in conjunction with advanced FCM fingerprinting methods.

Current research activity with respect to microbial invasion in GAC filters is still on-going.

INT2: DTU

The comments below are for WP1 to 4 as DTU contributed to all in the same way.

Objectives

Support the analysis of the spatial structure of microbial communities

Scientific activities and results
Main achievements in respect to initial WP objectives

The invasion framework was published in a high impact journal, where we provide rigorous descriptions of the invasion process and definitions of the constitutive elements as well as theoretical foundation to interpret (and possibly control) this process

Deviations from initial WP objectives

The research of the network focused less than planned on the role of spatial structure in microbial communities hence the decision of focusing on contributing to firming up the theory behind MRM

2.2. WP2. Microbial interactions in synthetic communities under selective stress.

2.2.1.Involved partners

All Belgian partners (UGent, UCL, KUL, UMons) were involved with WP2. In WP2 the synthetic ecosystems from WP1 were subjected to selective stress. P2 (UCL) was the WP leader.

2.2.2.Summary description of the objectives

The main objective of WP2 to assess the influence of environmental stressors and/or stimuli on the model microbial communities developed in WP1 in terms of resilience and robustness.

2.2.3.Summary of the scientific activities and results per partner

P1: UGent (KERMIT) – supporting role (to P3)

Objectives

Task 2.1: To investigate how the diversity of the endogenous community affects the invasion of pesticide degrading inoculants in sand filter ecosystems under selective conditions

Scientific activities and results
Main achievements in respect to initial WP objectives

Task 2.1:

P2: UCL

Objectives

Task 2.2, Soil ecosystems:

Polycyclic aromatic hydrocarbons (PAHs) biodegradation using synthetic bacterial communities under selective pressure.

Task 2.3, Marine ecosystems:

Additional objective related to marine ecosystems: Dimethylsulfoniopropionate (DMSP) biotransformation using bacterial communities under specific relevant selective conditions

Additional task, Wastewater Ecosystems

Additional objective related to wastewater ecosystems: Treatment of synthetic wastewater at 4 °C using artificial, psychrophilic bacterial communities under selective pressure.

Scientific activities and results

Task 2.2, Soil ecosystems:

Figure 2‑10. Biodegradation kinetics of fluorene, phenanthrene and anthracene by pure cultures of strain LB126, LH128 and LB501T, respectively. Initial PAH concentrations were set to 150 mg L-1. A. Biodegradation in the absence of heavy metals (no stress). B. Biodegradation in the presence of heavy metals (a mixture of Cd, Cu and Zn, 0.8 mM each).

Task 2.3, Marine ecosystems:

Figure 2‑11. Comparison of DMSO production (used as a proxy for DMSP cleavage to DMS) under different salinity levels (30 ppt and 5.85 ppt) for the strainDSS-3 grown on DMSP 2 mM after 6 days of incubation.

WP2, Additional task, Wastewater Ecosystems

Main achievements in respect to initial WP objectives

WP2, Task 2.2, Soil ecosystems

Coupling of biodiversity, functionality and robustness (resistance and resilience) of PAH-degrading mixed cultures.

P3: KUL

Objectives

WP2, Task 2.1, Sand filter ecosystems

Determine the invasion of MSH1 in biofilms of synthetic communities in oligotrophic conditions and effect of Assimilable Organic Carbon (AOC) content and study the occurrence of HGT of catabolic genes for BAM degradation.

Scientific activities and results

WP2, Task 2.1, Sand filter ecosystems

Main achievements in respect to initial WP objectives

P4: UMons

Objectives

Task 1.3, Marine Ecosystems

It is generally recognized that stress and/or stimuli shape microbial communities. The marine sediments that were selected to serve as a model in the present study are polluted by numerous metals (Gillan et al. 2015) and some of the strains selected in WP1 present various metal-resistance systems. After consideration, we decided to use zinc as a stressor.

The UMons objectives within WP2 were therefore to analyze the effect of zinc 0.5 mM on the diversity and the functionality of the community prepared in WP1.

Scientific activities and results

Task 1.3, Marine Ecosystems

At first, the Minimum Inhibitory Concentration (MIC) for zinc was determined for each strain. Most of the strains were resistant to 0.5 mM zinc after 48h of growth in SSM8 (16°C, 90 rpm) (e.g., P. putida), while others were not able to grow even at 0.5 mM Zinc in SSM8 (e.g., Shewanella baltica). Cupriavidus metallidurans, known to be resistant to various metal stresses (MIC zinc 5 mM in rich medium, data not shown) was surprisingly sensitive to zinc (MIC = 1.5 mM after more than 50 hours). This may be an effect of the SSM8 growth medium in which Cupriavidus has difficulties to grow.

Figure 2‑12. a-d : Pie charts representing the abundances of theoretical CELLS for each species inside the community after 4 days (a, b) or 28 days (c, d) in SSM8 (a, c) or SSM8 with zinc 0.5 mM (b, d) at 16°C, 90 rpm. 16S DNA composition was assessed by Q-PCR and transformed into theoretical number of cells. Legend and data table are given in (e). RALME, ECOBD, PSEPK, SHEB5, SHEFN, BURGL, BURXL, METPP and MYCVP stand for Cupriavidus metallidurans, Escherichia coli, Pseudomonas putida, Shewanella baltica, Shewanella frigidimarina, Burkholderia glumae, Burkholderia xenovorans, Methylibium petroleiphilum and Mycobacterium vanbaalenii respectively. a #CELLSSPECIES/CELLSALL has been calculated from Q-PCR 16S DNA composition, 16S DNA/cell per species and resulting total number of cells. b Shannon-Wiener diversity index.

According to the literature, a zinc concentration of 0.5 mM in a minimal medium is sufficient to activate zinc resistance in Cupriavidus as well as in Pseudomonas. It was decided to conduct an experiment with a complete synthetic community and a zinc concentration of 0.5 mM (sensitive bacteria such as S. baltica, might survive the stress with the help of the other strains).

A 2-weeks old community composed of the 9 strains used in WP1 was grown in triplicates in SSM8 (16°C, 90 rpm) before being divided in two parts: one half was maintained in SSM8 medium (control), the other in SSM8 + zinc 0.5 mM. After 4 and 28 days of exposure to Zn, quantitative PCR (qPCR) was used to determine the relative abundance of the strains in the community (Figure 2‑12).

As expected, the presence of Zn significantly affected the structure of the synthetic community over time. S. baltica totally disappeared after 28 days of exposure to Zn. This was expected according to its weak resistance to Zn (MIC= 0.5 mM). Particularly interesting were the abundances of Escherichia coli and Cupriavidus metallidurans. According to qPCR, the proportion of E. coli was higher after 4 days of exposure when zinc was present. However, after 28 days, E. coli disappeared in all conditions (Zn-amended and controls). Proportions of C. metallidurans on the other hand were higher in the presence of Zn (qPCR, Figure 2‑12) after 4 and 28 days. Because of their low proportions in the communities (up to 2% at the best for C. metallidurans) and their specific pattern according to the conditions, these 2 species have been targeted first during the SWATH-MS experiments.

Hydrophilic proteins have been prepared from the same samples after 4 and 28 days. After trypsin digestion, the proteins have been analyzed by DIA (Mass spectrometry using SWATH acquisition, see WP1). Thanks to this method, it was possible to quantify the abundance of proteins from specific species inside the whole proteome of the community. This is the first time that the State-of-the-Art SWATH acquisition method (Gillet et al., 2012) was used in a synthetic community. A technical short communication will be submitted in the coming month (Beraud et al., in preparation). Another manuscript is expected in the coming year after reproduction of the results. This manuscript should gather the results of WP1 and WP2, and conclusions about influence of Zn 0.5 mM on community function. First results are presented here.

Two quantitative parameters will be used to study the influence of Zn 0.5 mM on the community:

The Total Area for a sample gives an idea of the abundance of each protein in the community. When summing the total area of the proteins according to species, we can have an overall idea of the functional presence of the bacteria inside the community (Figure 2‑13).

So far we only analyzed 3 bacteria: E. coli (ECOBD), C. metallidurans (RALME) and P. putida (PSEPK). When comparing the Total Area obtained for each species, results are quite similar compared to what have been observed by qPCR: PSEPK is by far more represented than ECOBD and RALME. Furthermore, ECOBD is less present after 28 days than after 4 days.

From these analysis, 585 proteins from Pseudomonas putida as well as 85 proteins from C. metallidurans and 76 proteins from E. coli were identified (745 proteins in total). This was the first time during the project that we could see the proteins from rare species using mass spectrometry.

Figure 2‑13. Total Area of proteins detected using SWATH-MS per species. 4 assays have been tested: Communities have been cultured in triplicates at 16°C, 90 rpm in SSM8 (“N4”, “N28”) or SSM8 + zn 0.5 mM (“N28”, “Z28”) for 4- (“Z4”, “N4”) or 28- days (“Z28”,”N28”). Hydrophilic proteins have been prepared, then digested using trypsin. Resulted peptides have been analysed using SWATH- MS and a Spectral library composed of 3 species (Pseudomonas putida, Escherichia coli (“ECOBD”) and Cupriavidus metallidurans (“RALME”). Total Area has been computed after normalization and resulting Log2 (Ratio between the conditions) are given as well as p-value from Student T-test.

Glycolysis proteins, ribosomal proteins, amino acids biosynthesis and GroEL protein from E. coli were disappearing with time in controls (4 to 8-fold less abundant) as well as at Day_4 in the presence of zinc. This confirms what was already obvious with the qPCR (Figure 2‑12) and Total Area (Figure 2‑13) : E. coli disappears and become inactive between Day_4 and Day_28. It cannot sustain the Zn stress either. The presence of some proteins involved in oxidative stress repair such as the Thioredoxin or the Peptide methionine sulfoxide reductase increase in the presence of zinc at both time points. Oxidative stress proteins are known to be expressed after a metal stress (Gillan, 2016).

For C. metallidurans, the proteomic profile in the community confirms its low abundance. After 28 days, ribosomal proteins suggest a higher activity in the controls. But Lipid biosynthesis, and a couple of induced transport systems suggest that inside the community, Cupriavidus is still active and interacting with its environment. When C. metallidurans is analysed in single bacterial cultures using Data-dependent Acquisition Mass Spectrometry (DDA-MS), some Czc, Cop and Cup proteins (involved in metal resistance) are upregulated in the Zn-contaminated SSM8 medium. Particularly, CzcB increased 5-fold (data not shown). None of these metal-resistance proteins were detected by SWATH-MS in the synthetic community. But other proteins, such as glutathione transporter (Q1LNI5), and phosphate/phosphonate metabolism systems (Q1LLB2, Q1LJ09), or oxidative stress response proteins (Q1LEU4) have already been proposed to react to the presence of metal stressors .

For the invasion experiments in microtiter plates in the presence of Zn, the invasion pattern of a community was assessed by qPCR using Pseudomonas putida, Cupriavidus metallidurans and Escherichia coli as invaders. No clear effects of Zn for C. metallidurans invasion was found after 5-days. Conversely, Zn at 0.5, 1.0 and 1.5 mM increased the capacity of P. putida and E. coli to invade a community composed of 5 bacteria. Although the qPCR data needs to be confirmed by proteomic approaches, it is certain that Zn triggers a stress-response in P. putida and E. coli. These responses may then increase their ability to invade the community. In other words, invasion would be facilitated by stress.

At last, for the river sediment synthetic community (WP1) MIC for various metals of each strain have been described at 16°C, 90 rpm. To mimic the stress encountered by this community in natural environment, Zn at a concentration of 0.5 mM final or Pb at a concentration of 1.5 mM final have been added to the medium. In parallel, 3 assays have been kept metal-free (LB).

After 14 days, 1.5 to 5 mL of the cultures have been pelleted and DNA was extracted. Similar to previous experiments (made on the sea sediments community), qPCR primers have been designed to specifically detect the 16S rRNA gene of each of the strain. Quality of the primers, sensibility and sensitivity of the amplification have been assessed.

So far, only qPCR for Pseudomonas putida, Thauera phenylacetica, Azoarcus communis and Delftia acidovorans were performed. The preliminary graphs can be found in Figure 2‑14.

Figure 2‑14. qPCR analysis of river sediment communities. Average Relative Intensity of the 16S DNA of 4 species (Pseudomonas putida, Thauera phenylacetica, Azoarcus communis and Delftia acidovorans) is represented using bar graph on left Y- axis. Communities have been made in 3-fold diluted LB (biological replicates “LB1”, ”LB2”, “LB3”), added with zn 0.5 mM (biological replicates “Zn1”, ”Zn2”, “Zn3”) or Pb 1.5 mM (biological replicates “Pb1”, ”Pb2”, “Pb3”). Averages and standard deviation for each condition is given (red bar, “LB”, “Zn”, “Pb”). Number of biological and/or technical replicates used to compute previous averages are represented on the right Y- Axis (circle).

Results need to be confirmed and statistical analyses must be completed. But regarding these first results, it is clear that Pseudomonas putida and Delftia acidovorans are both quite abundant in the community compared to Azoarcus communis and Thauera phenylactetica. Furthermore, P. putida seems to be less abundant with zinc, which is consistent with the sensitivity of the strain to zinc. On the contrary, T. phenylacetica and D. acidovorans, both resistant to concentration > 1.5 mM Zn were more abundant in the presence of zinc. Communities elaborated without P. putida as well as functional analysis using SWATH-MS analysis will be used to further explore these communities

Main achievements in respect to initial WP objectives

We have analysed the final diversity and functionality of a marine community in the presence of a zinc stress. Thanks to qPCR experiments, shifts in the composition of the community, due to metal addition, have been visualized. In almost every conditions, P. putida was dominating the community. So far, functional analysis seems to confirm the positive role of Zn on C. metallidurans. It is more abundant with Zn than without. Further proteomic analyses are needed to conclude : is it because other strains such as P. putida and S. baltica are disappearing or because C. metallidurans is favored?

Furthermore, the river sediment community, composed of a new set of bacteria present the same pattern. Q-PCR analysis and proteomics experiments from the coming month should give clues about the link between diversity and functionality.

INT1: EAWAG

Objectives

To advise and assist MRM researchers with biodegradation experimental set-ups in low nutrient environments.

Scientific activities and results

EAWAG is hosting and training a KUL researcher in the use of FCM and working in AOC free environments (on-going at time of writing). Current research activity with respect to microbial invasion in GAC filters is still on-going.

2.3. WP3. Synthetic communities in a sterile environmental matrix

2.3.1.Involved partners

All Belgian partners (UGent, UCL, KUL, UMons) were involved with WP2. P3 (KUL) was the WP leader.

2.3.2.Summary description of the objectives.

Ecological hypotheses will be evaluated using the model microbial communities from WP1, in their natural, but sterile habitats.

2.3.3.Summary of the scientific activities and results per partner.

P1: UGent (KERMIT) - Supporting role (to P3).

Objectives

Task 3.1: To investigate how the diversity of the endogenous community affects the invasion of pesticide degrading inoculants in sand filter ecosystems in a sterile environmental matrix

Scientific activities and results
Main achievements in respect to initial WP objectives

The results of the statistical analyses are being used to help determine how the diversity (richness) of the endogenous community affected the degradation of BAM by MSH1 in sand filter ecosystems in a sterile environmental matrix

P2: UCL

Objectives

Task 3.2, Soil ecosystems:

Polycyclic aromatic hydrocarbons (PAHs) biodegradation by artificial bacterial communities within a sterile, soil background matrix.

Task 3.3, Marine ecosystems:

Additional objective related to marine ecosystems: Dimethylsulfoniopropionate (DMSP) biotransformation by bacterial communities within sterile, marine water.

Additional Task, Wastewater ecosystems

Additional objective related to wastewater ecosystems: Treatment of sterile, real wastewater at 4 °C using artificial, psychrophilic bacterial communities.

Scientific activities and results

Test of Sterilization protocol: autoclaving soil three times (121 °C, 45 min) over 3 consecutive days, at 24 h intervals. Intermediate incubation periods of 24  h at room temperature will be carried out to kill sporulating microorganisms. To inhibit fungal growth, cycloheximide (actidione, 50 µg mL-1) will also be added in sterile systems. Other experiments and tasks within this WP are still on-going.

Main achievements in respect to initial WP objectives

At UCL the work on WP3 is still in development hence no achievements with respect to the initial WP objectives can be reported yet.

Deviations from initial WP objectives

At UCL the work on WP3 is still in development hence no deviations with respect to the initial WP objectives can be reported.

P3: KUL

Objectives

Task 3.1, Sand filter ecosystems

Determine invasion of MSH1 in a sterile sand filter matrix with and without a synthetic community

Scientific activities and results

Invasive behavior of MSH1 in sterile lab scale sand filters: Sand filters continuously fed with sterile water in the absence or presence of a synthetic community showed that no apparent effect of the community was observed in the initial invasive phase.

Main achievements in respect to initial WP objectives

At KUL the work on WP3 is still in development hence no achievements with respect to the initial WP objectives can be reported yet.

Deviations from initial WP objectives

At KUL the work on WP3 is still in development hence no deviations with respect to the initial WP objectives can be reported.

P4: UMons

Objectives

Task 1.3, Marine Ecosystems

The aim of UMons within WP3 was to use fluorescent mobile DNA to follow genetic transfers inside a synthetic community. For this, bacterial strains have been genetically modified and tools have been designed (sterilised environmental matrices and glassware).

Scientific activities and results

Task 1.3, Marine Ecosystems

Several studies report on the importance of mineral particles for bacterial communities. Mineral particles, such as sand are a surface for bacterial attachment and biofilm formation. It was reported that biofilms are important for formation and maintenance of a bacterial community as well as for resistance to various stresses (including invasion). Some of the bacteria used in the community (like Escherichia coli) were shown to survive in marine environment only due to the presence of sediments. Furthermore, natural seawater contains numerous carbon sources that may influence the composition of the synthetic community.

We designed an efficient protocol to sterilize environmental water and sediments, i.e. to remove living organisms while keeping the physico-chemical composition of the medium unaffected. Three different sterilization procedures have been tested so far on fresh river water harvested the day before. Unfortunately, these results were unsuccessful: autoclaving was the most efficient sterilisation method but failed to inactivate all bacterial spores. In addition, autoclaving affected the chemical composition of the water (its pH). No further experiments have been conducted so far on this point. In parallel, glass beads (diameter of 104 µm) have been selected to elaborate an artificial sediment on which bacterial strain will be able to elaborate a biofilm. First biofilm test experiments have been started (data not shown).

Two mobile genetic elements (a plasmid and a genomic island), featuring Pb resistance systems, will be tagged with fluorescent proteins in order to be able to track their movements in the synthetic communities. This part of the work is in progress (Valentine Cyriaque, PhD student, in Prof. S. Sørensen laboratory). For that, the strain Pseudomonas putida KT2440 with the fluorescent plasmid pKJK5-pA10403-gfpmut3-Km (Bahl et al., 2009) is used. This strain contains the fluorescent protein GFP that is repressed in the host strain (P. putida). The GFP on the plasmid is only expressed when the plasmid is transferred to a receiver strain. Plasmid transfer is thus easily detected via fluorescent microscopy. A low-copy number version of the plasmid, pLENT, containing pbrUTRABCD, involved in lead resistance in C. metallidurans was constructed using MuA-transposition in E. coli. The construct will be transferred into P. putida.

Mobile genetic elements known as ICE (Integrative Conjugative Elements) are among the most abundant conjugative elements in the prokaryotic world and may subsequently be very important in shaping communities (Guglielmini et al., 2011). As a consequence, we decided to also tag an ICE in Delftia acidovorans SPH-1with GFP. The pbr system must also be inserted in the strain. The HGT experiments (tagged plasmid and ICE) will be done in simulated river sediment communities designed earlier (by a Master student, Marta Brodzik; see WP1, WP2). The tagged P. putida donor will also be used on the synthetic marine community (see WP1 and 2).

Finally, in a manuscript soon to be published (Cyriaque et al., in preparation) the plasmidome of a natural sediment bacterial community in freshwater, held in microcosms for 6 months, was examined. Some of the microcosms were treated with Zn, others were left untreated. The 16S rRNA composition of the community is currently being analyzed by Illumina sequencing.

Main achievements in respect to initial WP objectives

On the date of this ex-post evaluation UMons only started working on WP3 a few months ago. So far, the glassware has been selected and the genetic manipulations (tagged plasmid with GFP) are almost finished. We expect first results in the coming year. If successful, the experiments with tagged ICE elements will be the first in synthetic communities.

Deviations from initial WP objectives

So far, the sterilization process for the natural matrices has been unsuccessful. We are now using synthetic medium to mimic natural sediments. Experiments made so far have shown no big differences between river water and MilliQ water, as long as carbon sources are added.

2.4. WP4. Validation in autochthonous communities

2.4.1.  Involved partners

All Belgian partners (UGent, UCL, KUL, UMons) were involved with WP4. P4 (UMons) was the WP leader.

2.4.2.  Summary description of the objectives

In WP4 the results obtained in the previous work packages were validated for natural ecosystems.

2.4.3.  Summary of the scientific activities and results per partner

P2: UCL

Objectives

Task 4.2, Soil ecosystems

Polycyclic aromatic hydrocarbons (PAHs) biodegradation by artificial bacterial communities within a non-sterile, soil background matrix.

Task 4.3, Marine ecosystems

Additional objective related to marine ecosystems: Dimethylsulfoniopropionate (DMSP) biotransformation by bacterial communities within non-sterile, marine water.

Additional Task, Wastewater ecosystems

Additional objective related to wastewater ecosystems: Treatment of real wastewater (non-sterile) at 4 °C using artificial, psychrophilic bacterial communities.

Scientific activities and results

The work on WP4 at UCL is still preliminary, no results can be reported yet.

P3: KUL

Objectives

Task 4.1, Sand filter ecosystems

 

Determine invasion of MSH1 in pilot scale sand filters in DWTP

Scientific activities and results

Task 4.1, Sand filter ecosystems

Invasive behavior of MSH1 in sterile and non-sterile lab scale sand filters fed with non-sterile groundwater

Invasion of MSH1 in sand filters continuously fed with groundwater containing the residing groundwater bacteria was assessed for sterile sand filter columns and for sand filter columns previously primed with a sand filter community coming from DWTPs. The continuous supply of groundwater bacteria led to reduction in BAM removal both in the absence or presence of a synthetic community after a period of time. No apparent effect of the sand filter community was observed in the initial invasive phase. However, the continuous supply of bacteria probably led to a loss of MSH1 from the sand filters and as a consequence also BAM-degrading activity was lost. Important to note was that the priming of the sand filter with extracted communities from sand filter in DWTPs extended the period before loss of BAM-degrading activity occurred. As such the occurrence of other species can have a positive effect on survival of the invader at a later stage.

Invasive behavior of MSH1 in pilot scale sand filters continuously fed with BAM-contaminated groundwater of a DWTP

The bioaugmentation of MSH1 in pilot scale sand filters was assessed under different strategies of cell delivery (suspended or carrier embedded cells). Species richness and evenness of the system and how this is impacted by bioaugmentation will be determined.

Main achievements in respect to initial WP objectives

At KUL the work on WP4 is still in development hence no achievements with respect to the initial WP objectives can be reported yet.

Deviations from initial WP objectives

At KUL the work on WP4 is still in development hence no deviations with respect to the initial WP objectives can be reported.

P4: UMons

Objectives

Task 4.3, Marine ecosystems

The aim of WP4 is to reach the higher complexity and use the results obtained during the first three WP.

Scientific activities and results

Task 4.3, Marine ecosystems

Sediments have been harvested from two sites along the same river. The first site, MetalEurop, is located close to a former foundry closed since 2003 and still contaminated with various metals. The second site, Férin, situated upstream, presents the same physicochemical properties than MetalEurop except for the metal contamination. In a manuscript (Cyriaque et al., in prep) we have compared the plasmidome of the two communities and have performed microcosm experiments.

For that, 50 g of sediments from Férin have been harvested and transferred into the microcosms. Sediments were kept at 15°C, 70 rpm and overlaying water was changed periodically (autoclaved water from Férin), every week. After a week of equilibration, gradual additions of Zn (to reach a final concentration of 2870 mg/kg; "Zn samples") or Cd, Cu, Pb & Zn (to reach 36.8 mg/kg, 86.3 mg/kg, 902.2 mg/kg and 2870 mg/kg; “M” or “M+” samples) have been performed over an 8 weeks period. The final theoretical concentration of each metals correspond to the concentration published for sediments in MetalEurop. Bioavailable metal concentrations have been checked. For each condition, sediments have been made in triplicate, including the controls (no metals added). Every 2 months, viable counts as well as Denaturing Gradient Gel Electrophoresis (DGGE) analysis have been performed (using a fragment of the 16S rRNA gene) (Figure 2‑16)

Figure 2‑16. DGGE profile evolution of 16S DNA in each microcosm conditions. Natural sediments have been transferred in microcosm and kept for 6 months at 15°C, 70 rpm. After 1 week, metal have been gradually added in the microcosms over a period of 8 weeks. Samples were:  “Control” (no addition); “M+” (cadmium (36.8 mg/kg theoretical concentration), copper (86.3 mg/kg theoretical concentration), lead (902.2 mg/kg theoretical concentration) and zinc (2870 mg/kg theoretical concentration)); “Zn” (zinc 2870 mg/kg theoretical concentration). DGGE analysis of the resulting sediments communities have been done every 2 months.

Only 1 band was clearly visible in the DGGE profile of M+ samples after 6 months. Consistent with the results from WP2 on the synthetic sea water community (section 2.2.3), metal addition reduces the evenness of the community of natural river sediments. This band has been sequenced and was shown to represent Sphingomonadaceae, i.e. α-Proteobacteria known to degrade aromatic compounds.

The plasmidome was analysed using shotgun metagenomics after 6 months and compared to natural sediments (“T0”). Plasmid host taxonomic composition are reported in Figure 4.2.

Microcosm incubation had an influence on plasmid host taxonomic composition (Figure 2‑17). Numerically, E. coli plasmids dominate in all microcosms despite the fact that the bacterium was not far detected abundant with DGGE analysis. This suggests that the microcosm experiment promoted the transfer of E. coli plasmids and related functions without particularly favoring the abundance of the bacterium in itself. E. coli could be an important vector of horizontal gene transfer in freshwater.

Figure 2‑17. Heatmap of plasmid host genus diversity from the mobilome of Férin (T0) and each microcosm condition (control, Zn, M) including the 10 most abundant genera for all of them. The colour intensity is related to the abundance (%) of each genus (annotation was performed using M5NR database)

Another interesting result is that metal additions have led to an increase in the abundance of α-Proteobacterial plasmids. This was consistent with observations made in Férin and MetalEurop by metagenomics (Cyriaque et al., in preparation). α-Proteobacteria were also more abundant in contaminated microcosms M+ after 6 months (Figure 2‑17). In this particular case, plasmid abundance increases with host abundance.

Functional annotation of the plasmidome revealed that metal resistance systems are significantly more abundant in microcosms contaminated with metals. Levels of the cobalt-zinc-cadmium resistance system (Czc) increased 2 times in the Metaleurop plasmidome. qPCR assays have been performed to follow czcA but problems occurred because metallic contaminants inhibited the PCR.

The next step will be to use the fluorescent strains (developed in WP3) in similar microcosms and follow the transfer of the plasmid or ICE element in natural sediments. As transfer does not mean protein expression, improvements of the SWATH-MS approach to analyse specific proteins from the sediments will be tested to see if the selected resistance mechanisms are expressed in the new hosts.

Main achievements in respect to initial WP objectives

The WP4 only started recently. So far we could produce results similar to those observed in the synthetic community regarding the diversity of the community. Selective extraction of the plasmidome proved efficient. A manuscript will be published soon (Cyriaque et al., in preparation) and new experiments will start as soon as the tools developed during WP3 are available.

Deviations from initial WP objectives

The qPCR approach was not effective for the metal-contaminated sediments placed in microcosms. However, the Illumina sequencing technology was more appropriate and finally less time consuming and less expensive.

2.5. WP5. The modelling framework (in parallel with WP1 through 4)

2.5.1.Involved partners

The UGent partner (KERMIT, Bernard De Baets) and international partner 2 (DTU, Barth Smets) as well as international partner 3 (KU, Søren Sørensen) were involved in WP5. P1 (UGent, KERMIT) was the WP leader.

2.5.2.Summary description of the objectives

In WP5 in in silico models were developed in parallel to W1 to 4 to increase (mechanistic) insights into (synthetic) ecosystems which can be tuned with parameters obtained from the in vitro work within the other WPs.

2.5.3.Summary of the scientific activities and results per partner.

P1: UGent - KERMIT

Objectives

Task 5.1: Simplified synthetic ecosystems

In this task we aimed to construct a spatiotemporal modelling framework to mechanistically explain hypotheses regarding community biodiversity and functionality (particularly in the face of competition between species).

Task 5.2: Synthetic ecosystems with selective conditions

In this task we aimed to introduce variable environmental conditions to the modelling framework, as such expressing the dependence of crucial microbial processes on such changing conditions using functions rather than parameter values.

Task 5.3: Synthetic systems and autochthonous communities on an environmental matrix

In this task we aimed to parameterize and validate our models, to permit a quantitative assessment of the essential microbial ecosystem characteristics, such as the species abundance and their spatial distribution.

Scientific activities and results

For this WP, there is some overlap between our scientific activities and results, since for example developing an IBM is an activity, with the result being the IBM itself.

A spatially explicit individual-based modelling framework and extension modules (e.g. additional species, variable community evenness, different competition structures, fixed/non-fixed competition outcomes, interaction with changing environment, resource-dependent demographic processes) were developed. Subsequently, In silico experiments using above IBMs, conducted using High Performance Computing (HPC) infrastructure at UGent were performed and followed by a Sensitivity analysis of IBM.

Deviations from initial WP objectives

Our scientific activities have proceeded according to the initial WP objectives.

INT2: DTU

Objectives

Contribute to the modelling efforts

Scientific activities and results

Review and compare modelling framework for microbial co-metabolism

Main achievements in respect to initial WP objectives

This comparison was published in a high impact journal

Deviations from initial WP objectives

The research of the network focused less than planned on the role of spatial structure in microbial communities

INT3: KU

Objectives

Development of domain specific language tools for bioinformatics analysis.

Scientific activities and results

Development of bioinformatics tools, sequencing, travel for collaborative meetings and organization for the consortium and IT. Specifically for the development of workflow pipelines for multiple step data processing in, for example, multiple FASTA entries and analysis was adapted for parallelization, validated in practical work and published online. Validation was performed during the completion of the Ph.D. study performed by P.N. Holmsgaard and scientific work detailed in publications below was based on the pipelines described here.

BioDSL (pronounced Biodiesel) is a Domain Specific Language for creating bioinformatics analysis workflows. A workflow may consist of several pipelines and each pipeline consists of a series of steps such as reading in data from a file, processing the data in some way, and writing data to a new file. BioDSL is built on the same principles as Biopieces, where data records are passed through multiple commands each with a specific task. The idea is that a command will process the data record if this contains the relevant attributes that the command can process. E.g. if a data record contains a sequence, then the command reverse_seq will reverse that sequence.

2.6. Annual and other meetings

Annually, a meeting was coordinated by the UGent partner with each of the different partners hosting the meeting. Always presentations were given by each of the partners to exchange research and get input and experimental design recommendations. Additionally an IAP business meeting was organized.

Apart from annual meetings the individual partners organized meetings as need, some of which are listed below:

3. Position of the IAP network

3.1. Cutting-edge research

3.1.1.Scientific highlights in comparison to the state of the art

In Belgium, some research groups were focussing on environmental microbiology, looking at functional stability in ecosystems. Most of the research groups were using modelling, exploration or experimentation approaches to examine these ecosystems. However since the visionary review paper of Prosser et al. five years ago, it became clear that these three “schools of microbial ecology” should be united into conceptual and theoretical approaches. Understanding the microbial collaborations and their outcome within such a complex microbial community is essential for the development of a Microbial Resource Management (MRM) framework that would allow prediction and management of the behavior of microbial communities. In the present IAP project, we united five research groups to further develop the Microbial Resource Management (MRM) concept. For biotechnological applications, pure cultures and complex microbial communities are conventionally used, while in the present project, we developed very efficient synthetic communities, with intermediate complexity and high controllability for examining the different types of ecosystems.

This IAP project has been instrumental in consolidating/establishing the use of synthetic community research to understand/elucidate ecological theories and within microbial community interactions bith on the methodological and conceptual level. Moreover, before the IAP network, most work on synthetic communities contained no more than 2 or 3 species. Diversity was usually described thoroughly before inoculation or after a while using global analyses such as DGGE and metagenomics which are not always accurate. Within this IAP, we wanted to extend the size of these synthetic communities to be able to mimic better the real complexity. For example, the community of the sea sediments and the river sediments of UMons was composed of 9 bacterial species and has been thoroughly characterised using qPCR. The 194 synthetic communities developed by CMET consisted of 10 different species pooled together with differing evenness. The sand filter synthetic community by KUL consisted of 13 sand filter isolates. In this way the synthetic communities employed by the network to test and evaluate hypotheses that surpassed the state of the art.

A second main aim was to develop a toolbox to study these ecosystems. Within the network so far a plentitude of methods and tools are now available, which have a higher level of standardisation and accessibility for bioinformatics processing than what is the current state of the art. This is a necessary step in making such facilities available to a wider research and industrial audience in Europe in both scientific and high technology private sector development. For instance, metaproteomics was impaired by the data dependency of the acquisition. UMons is the first to use SWATH-MS to specifically analyse rare bacteria proteome inside synthetic communities. Furthermore, in contrast to typical microscopic modelling approaches, the spatially explicit IBMs we have developed within the framework of the IAP incorporate variable community evenness and resource dependence in demographic processes, two factors that have been shown by in vitro experiments to be key to ecosystem stability and functionality, but thus far largely overlooked by in silico approaches. By incorporating these two factors, we have brought existing models closer to reality. Similarly, KUL is the first to use techniques as TraDIS, differential (meta) omics and DFI for elucidating molecular interactions between organisms. Another important example is that CMET together with KERMIT is developing various advanced binning techniques for a cost-effective and fast tracking of diversity of (synthetic) ecosystems by means of flow cytometry. Using flow cytometry we are now able to describe individual cells in terms of fluorescence 1 and scatter signals; this gives rise to a multiparametric description of each cell. We can apply this technique in order to characterize microbial species. As flow cytometry is capable of measuring thousands of cells in a short span of time, we use machine learning techniques in order to assess to which extent microbial cells can be distinguished on an individual basis. By measuring bacterial species separately, we can aggregate data coming from these measurements to create in-silico communities. This opens up the opportunity to use supervised learning techniques such as ‘Linear Discriminant Analysis’ and ‘Random Forests’ in order to label unknown cells. Using statistical properties of in-silico communities we can show that we are able to retrieve these diversities with satisfying accuracy. This new technology is under full development while we are writing this report and will change the way we can analyze synthetic communities. Last but not least, most IAP labs (KUL, CMET, UMons, UCL) have developed high throughput systems for assessing microbial ecology theories and within community interactions using synthetic communities.

Through the different work packages and tasks, increased insight in the effects of complementarity and selection effects of biodiversity on functionality and robustness of synthetic microbial communities have been obtained. The network has gone beyond the state of the art in the invasion field by providing a rigorous conceptual framework but also novel evidence of the role of the resident community evenness and of environmental conditions. The evidence is both of experimental and modelling nature, highlighting the unique perspective provided by the current network on ecological hypothesis testing.

3.1.2.Perspectives of the network research domain for the coming 5 to 10 years

During the first the years of the project, the expertise of the different partners was shared during our scientific meetings and workshops. Based on this exchange of idea’s, collaborations inside the IAP are getting more and more intense. Within the project, we are starting to see streamlining of ideas and experimental setups. As a consequence, more similar patterns between the partner experiments can be found. Further exchange of students on the projects will results essential for statistical confirmation of the results and particularly to improve the model built in WP5. CMET and KERMIT routinely start joint master thesis projects though, within the IAP network more joint master thesis projects could be coordinated among different partners.

We anticipate that the fruitful links that were formed between the “µ-manager” partners will continue in the future, as a good working relationship was formed between them. For instance, it is anticipated that the links between CMET and KERMIT as well as KERMIT and KUL will continue, due to the many interesting possibilities for collaboration offered by the complementary research fields. Within UGent, between KERMIT and CMET, it is also foreseen that the legacy of the IAP will continue via the master’s theses completed within its framework, for instance an author of one of those is now employed as a PhD student in KERMIT, continuing to work using the models and methods developed within the IAP framework. In addition, KERMIT has applied for a postdoc position within a GOA project, based on the modelling knowledge and expertise gained during the IAP. Furthermore, tight collaborations were developed between UMons and KUL on the one hand and UMons and CMET on the other hand regarding the use of meta-proteomics in unravelling functionality of complex communities including the complex molecular mechanisms involved in the interactions between community members. The unravelling of mechanisms behind interaction (either collaborative or antagonistic) is key to future management of microbial communities and has applications in various fields of biotechnology (white, red and green biotechnology) and remains of all labs involved in the IUAP in the future. As such, the further “meta-proteomic” collaborations between UMons and the CMET and KUL labs is seen as highly important for further progress in understanding the molecular interactions within communities. As a consequence of these collaborations, between KUL, UMons and UGent, a tight collaboration has been initiated regarding the micro-managing of microbial communities in the context of space applications. Already a number of common ESA funded projects have been setup and in the future, the IAP partners will submit more joint projects. KUL and UCL consolidated/increased collaboration regarding PAH biodegradation.

As outlined above (here and in section 3.7), many new project proposals are being written with the network partners at national, bilateral and international levels with the network partners together to assure durability of the established partnerships and continuation of the work performed within the network. The network functions well as it is, however if it should be changed in the coming years it would be beneficial to include more international and small partners, especially those that would be able to apply the results of the network, and smaller research departments in diverse areas of research or associated disciplines such as health care. In addition, bio-informaticians might play an important role in future in developing and employing tools to dissect molecular networks and associated models in microbial communities. The integration of scientist involved in process modelling in order to develop mechanistic models of complex community functioning would be beneficial and needed. Several project proposals (like the H2020 EXPLOITOMICS and a national SBO-FWO project proposal mentioned above) include these items.

Finally, the first work packages and efforts delivered in the first three years of the network have focused intensively on method development and validation. Now that we have these techniques available as well as new hypotheses based upon WP1 through 3 we anticipate that in the next years will see these preliminary approaches come to full fruition, which will allow the network to consolidate its position as a major international player in microbial ecology.

3.1.3.Critical mass of the IAP network

We feel that the network is getting international recognition, based on the number of invited lectures (almost 30, see 4.2) we are also convinced that this will further expend within the next few years, as the new methodologies, applications and tested hypotheses within the first work packages will gain recognition in several peer reviewed papers which are currently being drafted. The IAP brought together top teams working all over Belgium on similar projects and lead to increased knowledge on ecosystem functionality. Based upon their involvement in the IAP network, KERMIT was invited to join a consortium of marine scientists applying for a GOA grant which demonstrates the interest from other scientific fields in the methods developed in WP5. Also other collaborations will arise based on the same kind of interest from others. This possibility is illustrated by a symposium on spatially explicit modelling, organized by Jan Baetens, which was proposed to and accepted by the European Conference on Mathematical and Theoretical Biology (Nottingham, July 2016). This symposium will include speakers from an international selection of universities, and demonstrates the wide interest in the type of methods that we have used and promoted via the IAP.

CMET and EAWAG have, through their extensive collaboration within the network gained a critical mass in the method development and implementation of flow cytometry in microbial ecology. The IAP support allowed very close collaboration and as a result, within the scope of the IAP, Dr. Hammes from EAWAG will install an online flow cytometer at UGent for high frequent single cell analysis of bioreactors. This unique equipment (not commercially available) will boost our knowledge on short term dynamics. Several new research lines are on-going and will be started in the near future to consolidate this position.

UMons has developed critical mass and expertise in method development and implementation of meta-proteomics in community understanding, both at this IAP as for example, for space research with UGent and KUL..

The IAP project further allowed to further consolidate/establish the topic of within microbial community interactions focusing on environmental microbiology and pollutant degradation in the lab of D. Springael at KUL and to contribute in establishing a critical mass to proceed on this topic in the next years. The microbiology of natural and engineered systems for treating and mitigating pesticide pollution of surface and groundwater ecosystems plays an important role at KUL. Taking into account these topics, the further collaborations with UMons (for community metaproteomics), EAWAG/DTU (drinking water microbiology) and KU (sequencing, within community interactions) and CMET (flow cytometry – KUL purchased a Accuri Flow Cytometer on the IAP) are in that sense highly important.

All these examples listed above clearly shows that this IAP allowed us to develop a critical amount knowledge and infrastructure which are all complementary for the different partners:

  • UGent-CMET: high throughput planktonic microcosms and (online) flow cytometry
  • UGent-KERMIT: modelling, neural networks and flow cytometry fingerprinting
  • KUL: high throughput biofilm based microcosms
  • UCL: high throughput chemostat reactors.
  • UMons: state-of-the-art metaproteomics

3.2. International role

A. Collaboration with international partner(s) within the network

International partners were invited during the annual meeting. They were presenting their work. UMons recently sent a PhD student in the laboratory of S. Sørensen at KU, benefiting from their facilities and expertise to create strains used for horizontal gene transfer. Similarly, KUL sent a PhD student to the lab of F. Hannes of EAWAG. The same PhD student will be visiting B. Smets of DTU later in the project. Publications should come out of these collaborations. KUL also worked together with KU for Illumina and 454 sequencing. The budget of the international partners was used mainly for training and mobility. The international partners contributed to critical discussions during the scientific meetings. The international partners allowed the network to benefit from their knowledge and international network, which is greatly beneficial to disseminate the knowledge obtained within the network and consolidate the critical mass of the network. Through joint publications and project proposals (e.g. CMET & EAWAG, KUL & KU, CMET & DTU, KUL & DTU, KUL & CMET & UMons & KU) the network as a whole could benefit from the international partners.

B. International activities

  • In 2017, the network will organize an international symposium on microbial resource management.
  • As shown above the IAP partners contributed European research projects.
  • Organised international workshops/conferences related to the IAP framework:
    • “2nd International Conference on Microbial Diversity 2013 (MD 2013)” 23th-25th October 2013, Torino, Italy, Member of the Scientific Committee and chairman of the oral session “Competition, dominance and evenness: how microorganisms manifest their supremacy” CMET (Nico Boon)
    • “MedRem-2014 - “Microbial Resource Management for Polluted Marine Environments and Bioremediation”. 16-18 January 2014, Hammamet, Tunisia. Member of the Scientific Committee and chairman. CMET (Nico Boon)
    • MEWE and biofilms IWA specialist conference. 4-7 September 2016, Copenhagen, Denmark. Member of the Scientific Committee - organized by Barth Smets DTU (INT2) - CMET (Nico Boon)
    • Barth Smets (DTU, INT2) organised a workshop at the University of Edinburgh “Computational methods for spatially structured microbial populations” - Attended by Jan Baetens - July 2013
  • The IAP members have been invited to give talks at many international conferences or institutes, where the work of the IAP consortium was presented:
    • “Microbial community analysis to better understand biological stability” at the “Biological Stability of Drinking Water” workshop, organised by the UNESCO‐IHE Institute for Water Education, Delft, The Netherlands, 23rd May 2012. (CMET, N. Boon)
    • “Microbial community analysis to better understand ecosystem functioning” at the “PhD course. Microbial Community Management in Aquaculture”, organized by Ghent University, 20th -22nd August 2012, Gent, Belgium (CMET, N. Boon)
    • “Flow cytometry community fingerprinting to detect quickly stress in drinking water systems” at the Annual Meeting of the German Society for Cytometry, Bonn, Germany, 10-12th October 2012. Keynote lecture. (CMET, N. Boon)
    • “The role of evenness and invading populations to preserve microbial community functionality”. Royal Netherlands Institute for Sea Research, NIOZ, Yerseke, The Netherlands, 28th February 2013. (CMET, N. Boon)
    • “Microbial Resource Management: from high throughput models to pilot reactors” at Taida College, Nankai University, Tianjin, China, 18th April 2013. Invited oral presentation. (CMET, N. Boon)
    • “µ-workshop on flow cytometry community fingerprinting” at School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 22nd April 2013. (CMET, N. Boon)
    • “Microbial Resource Management: from high throughput models to pilot reactors” at School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 24th April 2013. (CMET, N. Boon)
    • “Microbial Resource Management – new tools and explorations” at College of Environment and Resources, Jilin University, Changchun, China, 25th April 2013. (CMET, N. Boon)
    • “Developing and applying ecological theories for microbial resource management” at the 5th Congress of European Microbiologists (FEMS 2013), Leipzig, Germany, 21-25 July 2013. (CMET, N. Boon)
    • “Synthetic microbial communities: high throughput models to test new ecological hypothesis” at the 2nd International Conference on Microbial Diversity 2013 (MD 2013), Torino, Italy, 23-25 October 2013. (CMET, N. Boon)
    • Stenuit, B. and Agathos, S.N. ‘Conceptual and methodological framework to manage microbial robustness using molecular systems synecology: Applications for the biodegradation of contaminants of emerging concern’ The 2nd Thünen Symposium on Soil metagenomics. December 11-13, 2013, Braunschweig, Germany
    • Springael D., Sekhar A., Albers C.N., Horemans B., Smets B., Aamand J. Biofilms put to work : application of microbial biofilms in various processes. Eurobiofilms 2013, Ghent, Belgium.
    • Ramiro Vilchez-Vargas, Agnes Waliczek, Frederiek-Maarten Kerckhof, Belen Rodelas, Daniela J. Näther, Ruy Jauregui and Dietmar H. Pieper. Taxonomic and catabolic bacterial profiles in contaminated soils: A high resolution analysis shows the same players with different scripts. ISSM2014 - ninth international symposium on subsurface microbiology, 5-10 October, California USA. Invited Speaker.
    • “Synthetic microbial ecosystems: an exciting tool to understand and apply microbial communities” at the Summer School Molecular and Physiological regulation of medical and environmental microbial biofilms, Vaalbeek, Belgium. 15-18 september 2014. (CMET, N. Boon).
    • Stenuit, B. and Agathos, S.N. ‘Robustness and ecological resilience of microbial communities: from intrinsic natural dynamics to disturbance-driven responses’ Understanding Microbial Communities: Function, Structure and Dynamics (Programme theme, 11th August 2014 to 19th December 2014), Workshop on Engineering and Control of Natural and Synthetic Microbial Communities, The Isaac Newton Institute for Mathematical Sciences, November 26-28, 2014, Cambridge, UK
    • “Microbial community engineering: discovering the methanome” at Swiss Federal Institute for Environmental Science and Technology (EAWAG), Department of Process Engineering, Dübendorf, Switzerland, 8th March 2015. (CMET, N. Boon).
    • “Flow cytometry community fingerprinting for drinking water systems” at the 10th RME Conference Food Feed Water Analysis – innovations and breakthroughs. Noorderwijkhout, the Netherlands, 20-22 April 2015. (CMET, N. Boon).
    • “Synthetic microbial ecosystems: an exciting tool to understand and apply microbial communities” at the “13th Symposium on Bacterial Genetics and Ecology (BAGECO-13)”, Milan, Italy, 14-18 June 2015. Keynote presentation. (CMET, N. Boon).
    • Augelletti, F., Agathos, S.N., Stenuit, B. ‘Responses of synthetic microbial communities perturbed in multiplexed continuous bioreactors as an ecologically relevant proxy for microbial robustness prediction in natural and engineered ecosystems’ 13th Symposium on Bacterial Genetics and Ecology (BAGECO), June 14-18, 2015, Milan, Italy
    • Vandermaesen J., Daly A., Baetens J., De Baets B., Boon N., Springael D. (2015) Unravelling the relationship between indigenous community diversity and success of bioaugmentation using synthetic microbial ecosystems. 13th Symposium on Bacterial Genetics and Ecology (BAGECO). Conference Proceedings.
    • Stenuit, B. and Agathos, S.N. ‘Construction of chemostat arrays for the study of PAHs biodegradation by bacterial consortia under disturbances’ 6th European Bioremediation Conference (EBC-VI), June 29 - July 2, 2015, Chania, Crete, Greece
    • “Synthetic microbial ecosystems: an exciting tool to understand and apply microbial communities” at the School of Engineering, Glasgow University, Glasgow, Scotland, 19th August 2015. (CMET, N. Boon).
    • “Flow cytometry community fingerprinting for drinking water systems” at the School of Engineering, Glasgow University, Glasgow, Scotland, 21st August 2015. (CMET, N. Boon) .
    • “Synthetic microbial ecosystems: an exciting tool to understand and apply microbial communities” at the symposium “Ecology and evolution in microbial communities” of the Department of Fundamental Microbiology, Université de Lausanne, Lausanne, Switserland, 6th November 2015. (CMET, N. Boon).
    • Kerckhof, F.-M., Ho, A., De Rudder, C., Heyer, R., Benndorf, D. , Heylen, K. and Boon, N. (2016) “Happily ever after? How repeated subcultivation influences a methanotrophic marriage” - oral presentation at the session on biogeochemical cycling - KNVM Spring Meeting - 22 & 23 March 2016.
    • Springael, D., Horemans, B., Degryse J., Boonen, J., Walravens E. Lapanje A., Wittebol J., “Bioaugmentation-Based Treatment of 2,6-Dichlorobenzamide (BAM)-Contaminated Groundwater in Drinking Water Production - 10th Tenth International Conference on Remediation of Chlorinated and Recalcitrant Compounds, May 22-26 2016, Palm Springs, USA.
    • Horemans, B., Raes, B., Springael, D. (2015). Molecular markers as an application to optimize bioaugmentation in sand filters for drinking water production. Gordon Research Conference on Applied and Environmental Microbiology. Mount Holyoke, MA, USA, 12-17 July 2016.
  • Besides the interactions with the IAP international partners, collaborations were consolidated/established with other international partners such as Konny Smalla (

3.3. Durability of the IAP

As the ecological questions raised by the network have never been properly addressed and assessed before, continuation of the IAP is of great relevance to the field of environmental microbiology. The IAP bring together teams with different facilities and expertise. Discussion with national and international partners appeared really fruitful for all partners. WP4 just started and is the most biologically relevant part, hence many new hypotheses will be formed during the last year of the IAP. A continuation would allow in depth investigation and possibly application of these findings. Hence, we feel that there is certainly scope for a prolongation of the IAP, given the interesting avenues of research that have been opened by the activities already completed. There is increasing recognition from different scientific disciplines of the need for, and the opportunities offered by, modelling integration and feedback. We consider the modelling side of the network a great advantage and a unique opportunity for the field of theoretical and applied microbial ecology. This is especially true for the spatially explicit models that are developed within the IAP, because they allow for the more realistic simulation of ecosystem dynamics, while their relatively high computational demand poses less and less of a problem. Such an interdisciplinary approach is exists in the IAP network, and with some modifications offers potential for further advancement. Specifically integration of the data obtained in the final WP into predictive models would greatly benefit from additional validation through continuation of the MRM IAP network. We are convinced that the approach we take (i.e. by using synthetic communities) crucial progress can be made in the understanding of microbial communities and system microbial ecology. This fundamental research has a broad range of applications in biotechnology (white, red and green biotechnology). Many microbial community dominated processes (for ex. fermentation processes in food and feed sector, waste valorisation, production of new chemicals) are considered to be of extreme interest and prefereable over chemical or physical solutions. In addition to this, microbial consortia have several benefits compared to single individual organisms (including synthetic monocultures that are currently preferentially used in biotechnology applications, i.e., they can perform complex functions that individual species cannot and are generally more robust to perturbations. Finally, understanding inter-microbial interactions within detrimental polymicrobial communities (such as biofilms in clinical and industrial settings) opens the doors to development of more effective and sustainable antimicrobials.

Inclusion of more small projects or parts of projects derived from the scientific results gained for the evaluation of protocols but with specific aims would be a useful tool for inclusion of international partners. In reporting the partners may suggest such tasks or they could be suggested at meetings by the core partners. We observed that the MRM network, after its first collaboration in the current form within a IAP phase, has reached thrilling results, conclusions and collaborations which are worthwhile to pursue and solidify during a next IAP-phase.

4. Output

4.1. IAP publications

List the 10 most relevant publications or co-publications directly related to the achievements of the IAP project as a whole. A complete list of publications is already provided in the scientific activity reports of the first three years of the project, The complete list of the recent publications (not included in the activity reports of the first three years) is provided in Annex 1 of this review report.

  1. Van Nevel, S., De Roy, K., & Boon, N. (2013). Bacterial invasion potential in water is determined by nutrient availability and the indigenous community. FEMS microbiology ecology, 85(3), 593-603.
  2. De Roy, K., Marzorati, M., Negroni, A., Thas, O., Balloi, A., Fava, F., Verstrate, W., Daffonchio, D. & Boon, N. (2013). Environmental conditions and community evenness determine the outcome of biological invasion. Nature communications, 4, 1383.
  3. Benner, J., Helbling, D. E., Kohler, H. P. E., Wittebol, J., Kaiser, E., Prasse, C., Ternes, T. A., Albers, C. N., Aamand, , Horemans, B, Springael, D., Walravens E. & Boon. N (2013). Is biological treatment a viable alternative for micropollutant removal in drinking water treatment processes?. Water research, 47(16), 5955-5976.
  4. Horemans, B., Vandermaesen, J., Breugelmans, P., Hofkens, J., Smolders, E., & Springael, D. (2014). The quantity and quality of dissolved organic matter as supplementary carbon source impacts the pesticide-degrading activity of a triple-species bacterial biofilm. Applied microbiology and biotechnology, 98(2), 931-943.
  5. Horemans, B., Hofkens, J., Smolders, E., Springael, D. (2014). Biofilm formation of a bacterial consortium on linuron at micropollutant concentrations in continuous flow chambers and the impact of dissolved organic matter. FEMS Microbiology Ecology, 88, 184-194.
  6. Ho, A., de Roy, K., Thas, O., De Neve, J., Hoefman, S., Vandamme, P., Heylen, K., & Boon, N. (2014). The more, the merrier: heterotroph richness stimulates methanotrophic activity. ISME Journal. 8: 1945-1948.
  7. Stenuit, B., & Agathos, S. N. (2015). Deciphering microbial community robustness through synthetic ecology and molecular systems synecology. Current opinion in biotechnology, 33, 305-317.
  8. Gillan, D. C., Roosa, S., Kunath, B., Billon, G., & Wattiez, R. (2015). The long‐term adaptation of bacterial communities in metal‐contaminated sediments: a metaproteogenomic study. Environmental microbiology, 17(6), 1991-2005.
  9. Daly, A. J., Baetens, J. M., & De Baets, B. (2015). The impact of initial evenness on biodiversity maintenance for a four-species in silico bacterial community. Journal of theoretical biology, 387, 189-205.
  10. Gillan, D. C. (2016). Metal resistance systems in cultivated bacteria: are they found in complex communities? Current Opinion in Biotechnology, 38, 123–130. http://doi.org/10.1016/j.copbio.2016.01.012

4.2. Outreach, dissemination and impact to society

Research conducted within the framework of the MRM project was presented as part of the Biowiskundedagen initiative, where secondary school students are invited to visit the FBE to learn about the work that goes on here. (http://www.biowiskundedagen.ugent.be/)

The BioDSL (http://maasha.github.io/BioDSL/, developed by KU) domain specific language was made publically available and source code is available on GitHub (https://github.com/maasha/BioDSL) , which will allow further development and collaboration. Public bioinformatics forums were also informed of the existence of BioDSL to increase dissemination and visibility: https://www.biostars.org/p/166470/ and http://seqanswers.com/forums/showthread.php?t=64377

KUL attended an end user meeting with De Watergroep (non-scientific stakeholder) for the use of Aminobacter sp. MSH1 in sand filters in drinking water treatment plants

Applying ecological principles from within this network prof. Boon (CMET) submitted multiple patents for applications outside of the IAP network:

  1. Boon, E. Courtens, S. Vlaeminck (2014). Nitrification process for the treatment of wastewater at high temperature. WO2014/154559
  2. Teughels, N. Boon, M. Quirynen, G. Loozen (2014). Prebiotic oral care compositions containing an alkyl glycoside. KU Leuven & University Ghent, Belgium, PCT/US2013/077915. Submitted on 27 december 2013
  3. Teughels, N. Boon, M. Quirynen (2014). Prebiotic oral care compositions containing amino acids. KU Leuven & University Ghent, Belgium, PCT/US2013/077920. Submitted on 27 december 2013
  4. Teughels, N. Boon, M. Quirynen (2014). Prebiotic oral care compositions containing carboxylic acids. KU Leuven & University Ghent, Belgium, PCT/US2013/077923. Submitted on 27 december 2013
  5. Teughels, N. Boon, M. Quirynen (2014). Prebiotic oral care methods using a saccharide. KU Leuven & University Ghent, Belgium, PCT/US2013/077925. Submitted on 27 december 2013

4.3. PhD and postdoc training

UMons: 1 post doc for the last 3 years (Dr. Mélanie Beraud) and 1 PhD student financed by a F.R.I.A project (Valentine Cyriaque) started September 2015.

UCL: 1 post-doc (Benoit Stenuit) financed by the IAP.

KU: 1 PhD student (P.N. Holmsgaard, non-IAP) finished his PhD thesis within the framework of the IAP

KUL: 3 Post-docs (Pierre-Jo Vaysse (IAP), Benjamin Horemans (non-IAP), Basak Ozturk (IAP)) and 4 PhDs: (Joke Vandermaesen (IAP) (Joint with UGent), Pieter Albers (non-IAP), Aswini Sekhar (non-IAP) and Bart Raes (non-IAP))

UGent: 3Post-docs (Ramiro Vilchez-Vargas (IAP), Massimo Marzorati (non-IAP), Jan Baetens (non-IAP)) and 9 PhDs (Sam Van Nevel (non-IAP) – delivered, Karen De Roy (non-IAP) – delivered, Benjamin Buysschaert (non-IAP), Ruben Props (non-IAP), Charlotte De Rudder (non-IAP), Nicole Hahn (non-IAP), Elham Ehsani (IAP), Frederiek-Maarten Kerckhof (IAP) - about to be delivered (public defence 13/6), Aisling Daly (IAP))

 

Annex

The complete list of the recent publications related to the IAP-project (not included in the activity reports of the first three years).

 A. Publications per team (not in activity reports of first three years)

P1. UGent (CMET)

  1. Van Nevel, S., Buysschaert, B., De Gusseme, B. & Boon, N. (2016). Flow cytometric examination of bacterial growth in a local drinking water network. Water and Environment Journal. http://dx.doi.org/10.1111/wej.12160
  2. Buysschaert, B. , Byloos, B. , Van Houdt, R., Leys, N. & Boon, N. (2016). Reevaluating multicolor flow cytometry for microbial viability. Applied Microbiology and Biotechnology, in press.
  3. Props, R., Monsieurs, P., Mysara, M. & Boon, N. (2016). Measuring the biodiversity of microbial communities by flow cytometry. Methods in Ecology and Evolution, in press.

P1. UGent (KERMIT)

  1. Daly, A., Baetens, J., De Baets, B. (2016). In silico substrate dependence increases community productivity but threatens biodiversity. Physical Review E, 93(4): 042414

P3. KUL

  1. Horemans, B., Bers, K., Romero, E.R., Juan, E.P., Dunon, V., De Mot, R., Springael, D. (2016). Functional redundancy of linuron degradation in microbial communities of agricultural soil and biopurification systems. Applied and Environmental Microbiology, 82(9): 2843-2853.
  2. Dealtry, S., Nour, E.H., Holmsgaard, P.N., Ding, G.C., Weichelt, V., Dunon, V., Heuer, H., Hansen, L.H., Sørensen, S.J., Springael, D., Smalla, K. (2016). Exploring the complex response to linuron of bacterial communities from biopurification systems by means of cultivation-independent methods. FEMS Microbiology Ecology, in press
  3. T'Syen, J., Tassoni, R., Hansen, L., Sorensen, S., Leroy, B., Sekhar, A., Wattiez, R., De Mot, R., Springael, D. (2015). Identification of the amidase BbdA that initiates biodegradation of the groundwater micropollutant 2,6-dichlorobenzamide (BAM) in Aminobacter MSH1. Environmental Science and Technology, 49(19):11703-13
  4. Fida, T., Breugelmans, P., Lavigne, R., van der Meer, J., De Mot, R., Vaysse, P., Springael, D. (2014). Identification of opsA, a gene involved in solute stress mitigation and survival in soil in the polycyclic aromatic hydrocarbon-degrading Novosphingobium LH128. Applied and Environmental Microbiology, 80 (1), 3350-3361.
  5. Horemans B, Albers P, Springael D. 2016. The biofilm concept from a bioremediation perspective. In: Biofilms in Bioremediation. pp 23-40. Ed. Gavin Lear. Caister Academic Press, UK.

P4. UMons

  1. Gillan, D. C. (2016). Metal resistance systems in cultivated bacteria: are they found in complex communities? Current Opinion in Biotechnology, 38, 123–130.

INT3. KU

  1. Holmsgaard, P. N., Sørensen, S. J., & Hansen, L. H. (2013). Simultaneous pyrosequencing of the 16S rRNA, IncP-1 trfA, and merA genes. Journal of Microbiological Methods, 95(2), 280-284. http://dx.doi.org/10.1016/j.mimet.2013.09.016
  2. Holmsgaard, P. N. (2013). Second generation sequencing for elucidating the diversity of bacteria and plasmids in soil (Unpublished Ph.D thesis). Ph.d.-afhandling. Københavns Universitet.

B.  List of co-publications

  1. UGent-CMET (P1) & EAWAG (INT1): Van Nevel S, Koetzsch S, Weilenmann H-U, Boon N, Hammes F (2013). Routine bacterial analysis with automated flow cytometry. Journal of Microbiological Methods 94: 73-76. http://dx.doi.org/10.1016/j.mimet.2013.05.007
  2. DTU (INT2), EAWAG (INT1) & UGent-CMET (P1) : Kinnunen, M., Dechesne, A., Proctor, C., Hammes, F., Johnson, D., Quintela-Baluja, M., Graham, D., Daffonchio, ., Fodelianakis, S. , Hahn, N., Boon, N., Smets, B. F. (2016). A conceptual framework for invasion in microbial communities. The ISME Journal, 1–7. http://dx.doi.org/10.1038/ismej.2016.75
  3. KUL (P3) & KU (INT2): Dealtry, S., Nour, E.H., Holmsgaard, P.N., Ding, G.C., Weichelt, V., Dunon, V., Heuer, H., Hansen, L.H., Sørensen, S.J., Springael, D., Smalla, K. (2016). Exploring the complex response to linuron of bacterial communities from biopurification systems by means of cultivation-independent methods. FEMS Microbiology Ecology, in press
  1. KUL (P3) & KU (INT3): T'Syen, J., Tassoni, R., Hansen, L., Sorensen, S., Leroy, B., Sekhar, A., Wattiez, R., De Mot, R., Springael, D. (2015). Identification of the amidase BbdA that initiates biodegradation of the groundwater micropollutant 2,6-dichlorobenzamide (BAM) in Aminobacter MSH1. Environmental Science and Technology, 49(19):11703-13
  1. KUL (P3), Ugent-CMET (P1) and UGent-KERMIT (P1) : Vandermaesen, J., Daly, A., Baetens, J., De Baets, B., Boon, N., Springael, D. (2015) Unravelling the relationship between indigenous community diversity and success of bioaugmentation using synthetic microbial ecosystems, In Proceedings of the 13th Symposium on Bacterial Genetics and Ecology, Milan, Italy, 14-18 June 2015.