Name of the scientific representative of the project s coordinator, Title and Organisation:

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1 BONUS Blueprint PROJECT (1 January April 2018) The final publishable summary report Project title: Biological lenses using gene prints Name of the scientific representative of the project s coordinator, Title and Organisation: Prof. Lasse Riemann, University of Copenhagen, Denmark Phone: (0045) (office) (0045) (mobile) lriemann@bio.ku.dk Project website address: The BONUS BLUEPRINT project has received funding from BONUS (Art 185), funded jointly by the EU and This work resulted from the BONUS Blueprint project supported by BONUS (Art 185), funded jointly by the EU and the Danish Council for Independent Research, Swedish Research Council FORMAS, the German Federal Ministry of Education and Research, Academy of Finland, and the Estonian Research Council. Helsingør, Denmark, August 2018 Deniz Bombar (secretary). Lasse Riemann (project coordinator) 1

2 1. Project outline of goals and results envisaged at the beginning of the project cycle The countless planktonic microbes are the principal drivers of carbon and nutrient cycles in the Baltic Sea. Yet, they are not included among natural indicators used to describe the environmental status of the Baltic Sea as part of the EU water framework directive. This flaw has been highlighted by HELCOM and OSPAR in their work to coordinate the development of indicators and determining the good environmental status in the Baltic and North Sea areas. Through tremendous technological advancements in the last decade, we now have the methodological capacities to retrieve and process the genetic information from these microbes in a cost-efficient manner. Thereby we can gain mechanistic understanding of how the microbes drive food-web processes and how they are affected by the environment. Based on such knowledge, the BONUS BLUEPRINT project aimed to develop a framework that allows us to determine the ecological status of the Baltic Sea based on microbial genetic indicators. The expected final results are a set of indicators reflecting the biodiversity and genetic functional profiles of microbes in a given seawater sample. 2. Work carried out in the project The main challenges of BONUS BLUEPRINT were i) to describe and understand the links between environmental parameters and the genetic ii) iii) information carried by microorganisms to establish easily reproducible and automated methods of obtaining and analyzing this genetic information, and to use the genetic functional profiles to improve ecosystem models, which are needed to predict future scenarios of the Baltic Sea. Point i): BONUS BLUEPRINT included elaborate field sampling as well as experimental manipulations with natural samples and laboratory model microorganisms. We have obtained roughly 1400 nucleic acid samples and corresponding metadata, covering all different subsystems of the Baltic Sea and representing the responses of natural microbial communities and laboratory cultures to experimental additions of wellknown stressors such as eutrophication, dissolved organic matter, hypoxia, or selected pollutants. This sample set was used to assess how the microbial genetic profiles vary in genome sequenced laboratory microbes as well as along environmental gradients in the field, and in response to experimentally manipulated conditions that represent deviations from good environmental status. Point ii): It involved the development of standardized sampling procedures and bioinformatics pipelines for processing the wealth of genetic sequence data. Since 2014 we have developed and extensively tested an automatic water in-situ fixation sampler in collaboration with the BONUS Innovation project BONUS AFISMON. This sampler is optimized for obtaining undisturbed gene expression (transcriptional) profiles of the microbes. Further, we have finalized the development of the bioinformatics pipeline necessary for handling the extensive sequence datasets. This includes the elaborate and computationally extensive steps of assembling and annotating obtained sequences (what genes & functions are encoded?), generating the Baltic Sea Reference Metagenome (BARM) which functions as the sequence backbone against which all future sequence datasets can be mapped, and designing a database where the processed metagenome data are stored and can be accessed via a graphical user interface. Point iii): Understanding the biogeochemistry of the Baltic Sea is an important basis for sustainable management of this environment. Thus, any steps that may improve the predictive power of biogeochemical models used to foresee the results of environmental initiatives such as the Baltic Sea Action Plan ( are of great value. Therefore, the existing biogeochemical model BALTSEM (BAltic Sea Long-Term large Scale Eutrophication Model) has been modified to include a more comprehensive role of the bacterial community, in particular in the surface 2

3 layer of the Baltic Sea and the redoxcline of its deep basins, with the goal of identifying metagenomic sequence indicators of good environmental status. 3. Main results achieved during the project With the aim to reveal effects of environmental perturbation on the microbial genetic information, field sampling and manipulation experiments were carried out. These included experiments testing the response of microbes to changes in salinity, oxygen, dissolved organic matter, pollutants, and other environmental stressors indicative of certain environmental statuses. These efforts included a large scale mesocosm experiment with natural plankton communities (Fig. 1) as well as experiments with laboratory cultures of selected microbes of which the genomes are already sequenced. Such differing approaches are necessary because only via the genome-sequenced laboratory cultures can details be revealed regarding how organisms react to different stressors at the genome level, information which later aids in the interpretation of the much more complex sequence datasets derived from field sampling or from the experiments with mixed natural communities. Figure 1: Mesocosm experiment at Linneaus University (LNU) in Sweden, testing the effects of environmental perturbations on the microbial genetic information. Empty tanks to be filled with seawater (upper left); Experiments ready to roll after adding nutrients and setting up several different perturbation treatments (upper right); a young team of PhD students from the host institution monitors irradiance levels (bottom left); Prof. Mathias Middelboe (UCPH) setting up bacterial activity measurements (bottom right). Sequencing efforts included multiple field- and experimental samples (metagenomes, metatranscriptomes, and marker-genes to assess the overall plankton diversity across the salinity gradient of the Baltic Sea). The bioinformatics processing of such sequence data involves many steps, including the annotation (i.e. definition) of different gene functions by mapping the sequences against the Baltic Sea reference metagenome (BARM; Moreover, a user-friendly web-portal for predicting environmental conditions in samples provided by the user, using a machine-learned predictor, is available at The developed bioinformatics tools allowed for quick processing and interpretation of accumulating genetic field data by the BONUS BLUEPRINT scientists and these tools are publicly available for the future. These pipelines can be compared to the generation of a fixed and extremely detailed background picture (BARM) onto which investigators can relatively easily post and assemble new sets of puzzle pieces (new metagenomes/transcriptomes) that match parts of the background. 3

4 A principal aim of the BONUS BLUEPRINT project was to demonstrate that environmental status/condition and dominant biogeochemical pathways can be deduced from the biodiversity and genetic functional profiles of microbes, the blueprint, in a seawater sample. Gene sequences obtained from cruises and experiments in the Baltic Sea were indeed correlated with environmental conditions, documenting a coupling and the proof of concept that environmental conditions can be predicted from microbial genes. A central product of the project is the Blueprint Competence Centre (BCC), which can be found at At this site sampling and laboratory protocols can be extracted where users can decide what kind of sample they want to process and what kind of information that is suitable to answer their specific question. Also links to bioinformatics tools are provided along with synthesis documents reporting the output in a scientific as well as a more popular manner. We matched the BONUS BLUEPRINT genetic data to biogeochemical model analogues. In particular we found that genes coding for elements in membrane transporter aggregates, and thus responsible for the uptake of specific substrates, are related to turnover of mineral and organic nutrients. This finding constituted the basis for two parallel model formulations. Introducing population dynamics in the modelling strategy created diverse behavior by multispecies components in the model. In turn this allowed a diverse set of virtual molecular markers, in particular genes coding for subunits of transporter aggregates, to be included in the model simulating organic and mineral nutrient uptake. A parallel strategy was to control the model-formulation by a stoichiometric relationship between organism growth and substrate consumption. Also in this case the model was translating a genomic topology forcing a more accurate response into biogeochemical model parameterizations. This latter strategy was a major input for a proposed modification of the BALTSEM model formulation. In addition to fulfilling the principal project aims BONUS BLUEPRINT has generated a wealth of novel insights into the diversity, composition and ecology of bacterioplankton in the Baltic Sea. These insights have been communicated through >23 peer reviewed publications in international research journals and contribute to an improved understanding of the role of bacteria in Baltic Sea nutrient cycling. Insights span a range of microbial processes, like carbon utilization, N 2 fixation, nitrification, break-down of pollutants, effects of riverine inflow in the face of climate change, influence of vitamins etc., with particular emphasis on effects of the vertical and horizontal chemical gradients present. A list of peer-reviewed publications is provided on the project homepage at 4. The continuity plan of the project A main aim of the BONUS BLUEPRINT project was a demonstration of the proof-of-concept that microbial genes can be used as indicators in environmental assessments. The project has forcefully demonstrated this proof-of-principle and this has been communicated to stakeholders involved in marine monitoring. Indicators used to assess the state of the environment under marine policies, however, require that a threshold value is set that represents the boundary between good and not good status. Since the metaomics approach developed within the BONUS BLUEPRINT project is relatively new there was no existing dataset that could be used as a reference point for establishing threshold values at the start of the project. The project has contributed a significant expansion of the available data from both time-series and transect studies. It is proposed to use this meta-omic information, contained in the BalticMicrobeDB database, to establish a baseline, including natural variation, against which future changes can be assessed. The publicly available database will be accessible also in the future. To allow for further development towards operational indicators, the BONUS BLUEPRINT project also proposes that a minimum of 5 years metagenomics sampling is carried out beyond the project based on: - Continued monitoring at a coastal site in the northern and southern Baltic Sea respectively, to continue capturing important variations on a temporal scale. Coastal areas have the highest impact from human activities and are also likely to indicate early changes in ecosystem functions as a 4

5 response to climate change, such as temperature and the effect of anticipated increase of riverine input. - Continued monitoring in a depth profile at a strategic location in the southern Baltic Sea with deep suboxic bottom waters. This proposal is based on the fact that biogeochemical important transformations take place in suboxic and anoxic conditions. With such monitoring effort as a starting point, the meta-omic approach could be further explored and the data retrieved put into practice in environmental status assessments. 5