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This book represents a synthesis of knowledge and best practice in the field of DNA-based biomonitoring at the time of writing. It has been written with end-users of molecular tools in mind, as well as those who are new to the field in research settings and are looking to gain an overall grounding in the subject area. For each of the main types of sample (water, soil / sediment, bulk invertebrates and diatoms), and for each stage of the field and laboratory processes, we outline key considerations, decisions that need to be made, factors that might influence those decisions, and trade-offs inherent in the choices made. We hope that this will help users, practitioners, and those commissioning DNA-based monitoring programmes to navigate this large field and critically evaluate the strengths and weaknesses of different analysis workflows based on context, project aims and available resources. DNA-based methods for species detection and identification have revolutionised our ability to assess biodiversity in terrestrial, freshwater and marine ecosystems. Starting from the seminal study that used eDNA to detect invasive american bullfrogs in France (Ficetola et al. 2008), research conducted over the last decade has demonstrated the power of these approaches for surveying a wide range of species and groups. Early applications included the use of eDNA to monitor Asian Carp in the USA (Jerde et al. 2013). Following heavy scrutiny, the method was eventually adopted, and is still employed today by the United States Geological Survey (USGS). A flurry of research followed, with tests designed for many threatened and invasive species including New zealand mudsnails (Goldberg et al. 2013), american crayfish (Geerts et al. 2018), gammarids (Blackman et al. 2017), and great crested newts (Biggs et al. 2015). The great crested newt eDNA test has been employed for regulatory monitoring in the UK since 2014. During the same time period, there was a proliferation of research studies that used high-throughput sequencing approaches to describe whole communities of organisms from mixed species and environmental samples, using an approach termed DNA metabarcoding (Taberlet et al. 2012c). As the field developed fast and the approaches were applied to a wide range of research and monitoring objectives, a high level of methodological variation was introduced at all stages of the workflow (Seymour 2019). Thus, while a significant level of consensus on scientific best-practice now exists in many areas, this may not be readily discerned from the now-extensive body of research literature. As environmental practitioners and policy makers are now increasingly starting to integrate DNAbased methods into routine monitoring applications including protected species licensing1, statutory monitoring2 (Hänfling et al. 2016) and environmental impact assessment3, various national and international efforts have been undertaken to standardise methods and integrate them into monitoring frameworks (Pilliod et al. 2019, Loeza-Quintana et al. 2020, Minamoto et al. 2021, Pawlowski et al. 2020a4). In Europe, the EU COST Action DNAqua-Net (Leese et al. 2018) has been working towards incorporating molecular monitoring tools for Biological Quality Elements (BQEs, e.g., fish, macroinvertebrates and phytoplankton- benthos) into the Water Framework Directive (WFD, 2000/60/EC)5 and the Marine Strategy Framework Directive (MSFD, 2008/56/EC)6. Thus, emphasis now shifts from fundamental research to robust and efficient application of DNAbased methods for operational use at large scales. This requires that scientific robustness is balanced with consideration of the practical realities faced by environmental managers. Moreover, there is increased need for strong quality assurance in a setting where non-expert field samplers and commercial laboratories are involved with the generation of data that non-specialist decision-makers then rely on to inform potentially costly action (or non-action). This places increased emphasis on robustness, replicability, traceability and ease-of-use, which may not always be the central focus of studies carried out in the academic research environment. This document aims to summarise the scientific consensus relating to every step of the field and laboratory workflows involved in the most common types of samples and analyses. We do not go into great detail regarding bioinformatics (computational processing of sequence data) and data analysis since these are extensive topics in their own right. We uniquely set the field and lab steps in the context of the practical and logistical constraints faced by environmental managers in terms of cost, logistics, safety, ease-of-use, and quality assurance, highlighting key decisions to be made and the inherent trade-offs associated with the various options. We hope that this will support non-experts, and those new to the field, to navigate the key considerations associated with planning or evaluating monitoring programmes using DNA-based monitoring methods. Additionally, it will aid decision-makers in writing and evaluating tenders and proposals, ensuring that the methods used for a given project are fit-for-purpose and that results are correctly interpreted. Alongside the many areas of emerging consensus, there remain some areas where further research is still required to balance scientific best-practice with the constraints and priorities of end-users. We hope that by shining a light on the importance of these issues, the research community will be encouraged to address them. More generally, we hope to inspire researchers in this now highly-applied scientific field to consider end-user constraints when designing and implementing research projects. This will help to accelerate uptake by users and maximise the impact of research. DNA-based bioassessment methods continue to evolve, and there are several emerging technologies that show exciting promise to move beyond even what is possible today. Examples include in-field sequencing using the MinION device from Oxford Nanopore Technologies (Pomerantz et al. 2018, Davidov et al. 2020, Hatfield et al. 2020), PCR-free metagenomic approaches (Bista et al. 2018, Giebner et al. 2020) and CRISPR for rapid detection of species, which is particularly relevant for invasive an non-native species monitoring (Williams et al. 2019, 2020). We recognise the potential of these methods, but do not consider them in detail here, since they are not yet far enough developed for routine application. EU COST Action DNAqua-Net (CA 15219 a COST (European Cooperation in Science and Technology). DNAqua-Net, European Union Horizon 2020 Published Refereed Current 14.a Mature Multi-organisational International Genetic differentiation Species distributions Method

Additional file 1. Additional figures.

Heterotrophic bacteria play a key role in the degradation of organic matter and carbon cycling in river sediments. These bacterial communities are directly influenced by environmental variables that differ spatially and temporally in rivers. Here, we studied the longitudinal patterns of sediment bacterial community composition and dissolved organic matter utilization under base flow and drought conditions in a Mediterranean river. Our results indicated that sediment microbial communities were affected by dissolved organic matter quality and origin along the river continuum. In headwaters the potential degradation of cellulose and hemicellulose was greater (i.e., higher β-glucosidase and β-xylosidase activities), suggesting higher microbial utilization of allochthonous detritus from terrestrial origin. Conversely, the accumulation and transport of more recalcitrant compounds (i.e., decrease in the recalcitrant index) became potentially relevant downstream. Furthermore, discharge fluctuations had clear effects on bacterial community composition and dissolved organic matter use. The hydrological fragmentation of the river continuum during drought period generated sediment microhabitats dominated by gamma and delta-Proteobacteria, with a greater potential capacity to degrade a wide range of compounds, particularly nitrogen containing moieties. During base flow conditions, we observed a higher occurrence of alpha-Proteobacteria and a greater potential use of more recalcitrant carbon compounds, mostly of terrestrial origin. Overall, our findings suggest an upstream-downstream longitudinal transition of sediment microbial communities that rely on allochthonous to autochthonous dissolved organic matter, and a shift toward autochthonous dissolved organic matter reliance during drought This study was supported by the Spanish Ministry of Economy and Competitiveness through projects FLUMED-HOTSPOTS (CGL2011-30151-C02) and FUNSTREAM (CGL2014-58760-C3-R)

The Chlamydiae are a phylum of obligate intracellular bacteria comprising important human and animal pathogens, yet their occurrence in the environment, their phylogenetic diversity and their host range has been largely underestimated. We investigated the seasonality of environmental chlamydiae in a Tyrrhenian coastal lake. By catalysed reporter deposition fluorescence in situ hybridization, we quantified the small planktonic cells and detected a peak in the abundance of environmental chlamydiae in early autumn with up to 5.9x10(4) cells ml(-1). Super-resolution microscopy improved the visualization and quantification of these bacteria and enabled the detection of pleomorphic chlamydial cells in their protist host directly in an environmental sample. To isolate environmental chlamydiae together with their host, we applied a high-throughput limited dilution approach and successfully recovered a Vexillifera sp., strain harbouring chlamydiae (93% 16S rRNA sequence identity to Simkania negevensis), tentatively named CandidatusNeptunochlamydia vexilliferae'. Transmission electron microscopy in combination with fluorescence in situ hybridization was used to prove the intracellular location of these bacteria representing the first strain of marine chlamydiae stably maintained alongside with their host in a laboratory culture. Taken together, this study contributes to a better understanding of the distribution and diversity of environmental chlamydiae in previously neglected marine environments.

Amoebae play an important ecological role as predators in microbial communities. They also serve as niche for bacterial replication, harbor endosymbiotic bacteria and have contributed to the evolution of major human pathogens. Despite their high diversity, marine amoebae and their association with bacteria are poorly understood. Here we describe the isolation and characterization of two novel marine amoebae together with their bacterial endosymbionts, tentatively named 'Candidatus Occultobacter vannellae' and 'Candidatus Nucleophilum amoebae'. While one amoeba strain is related to Vannella, a genus common in marine habitats, the other represents a novel lineage in the Amoebozoa. The endosymbionts showed only low similarity to known bacteria (85-88% 16S rRNA sequence similarity) but together with other uncultured marine bacteria form a sister clade to the Coxiellaceae. Using fluorescence in situ hybridization and transmission electron microscopy, identity and intracellular location of both symbionts were confirmed; one was replicating in host-derived vacuoles, whereas the other was located in the perinuclear space of its amoeba host. This study sheds for the first time light on a so far neglected group of protists and their bacterial symbionts. The newly isolated strains represent easily maintainable model systems and pave the way for further studies on marine associations between amoebae and bacterial symbionts.

The increase in traded container volumes worldwide puts pressure on the hinterland road network, leading congestion and emission problems. This leads to a requirement to develop intermodal transportation systems. In this chapter, we analyze the most important features of such container transportation systems for the hinterland supply chain. At the network design level, we review the current state of the art and we identify avenues for future research. Among others, we highlight that the coordination of container shipments across the container supply chain is a particularly relevant issue as hinterland networks involve several actors. At the operational level, we characterize the most important factors influencing the trade-off between intermodal transportation and truck-only deliveries. In addition, we provide a case study of coordination at an intermodal barge terminal in the Netherlands. We highlight that the exchange of information is the key enabler for efficient hinterland intermodal transportation and we show that a better information system can be of crucial importance.

Coastal countries have traditionally relied on the existing marine resources (e.g., fishing, food, transport, recreation, and tourism) as well as tried to support new economic endeavors (ocean energy, desalination for water supply, and seabed mining). Modern societies and lifestyle resulted in an increased demand for dietary diversity, better health and well-being, new biomedicines, natural cosmeceuticals, environmental conservation, and sustainable energy sources. These societal needs stimulated the interest of researchers on the diverse and underexplored marine environments as promising and sustainable sources of biomolecules and biomass, and they are addressed by the emerging field of marine (blue) biotechnology. Blue biotechnology provides opportunities for a wide range of initiatives of commercial interest for the pharmaceutical, biomedical, cosmetic, nutraceutical, food, feed, agricultural, and related industries. This article synthesizes the essence, opportunities, responsibilities, and challenges encountered in marine biotechnology and outlines the attainment and valorization of directly derived or bio-inspired products from marine organisms. First, the concept of bioeconomy is introduced. Then, the diversity of marine bioresources including an overview of the most prominent marine organisms and their potential for biotechnological uses are described. This is followed by introducing methodologies for exploration of these resources and the main use case scenarios in energy, food and feed, agronomy, bioremediation and climate change, cosmeceuticals, bio-inspired materials, healthcare, and well-being sectors. The key aspects in the fields of legislation and funding are provided, with the emphasis on the importance of communication and stakeholder engagement at all levels of biotechnology development. Finally, vital overarching concepts, such as the quadruple helix and Responsible Research and Innovation principle are highlighted as important to follow within the marine biotechnology field. The authors of this review are collaborating under the European Commission-funded Cooperation in Science and Technology (COST) Action Ocean4Biotech – European transdisciplinary networking platform for marine biotechnology and focus the study on the European state of affairs., This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by COST (European Cooperation in Science and Technology) program.AR, KK, and TR: the publication is part of a project that has received funding from the European Union Horizon 2020 Research and Innovation Programme under grant agreement no. 774499 – GoJelly project. AR and KK: this research was funded by the Slovenian Research Agency (research core funding P1-0245 and P1-0237). AR: this publication has been produced with financial assistance of the Interreg MED Programme, co-financed by the European Regional Development Fund (Project No. 7032, internal ref. 8MED20_4.1_SP_001) – B-Blue project. AB: acknowledges the support from the Research Council of Norway through the grant 267474 from the HAVBRUK2 program. MLC: acknowledges the Portuguese Foundation for Science and Technology (UIDB/04326/2020), the European Maritime and Fisheries Fund (MAR2020 OSTEOMAR/16-02-01-FMP-0057 and ALGASOLE/16.02.01-FMP-0058), the European Regional Development Fund (Atlantic Area BLUEHUMAN/EAPA/151/2016 and INTERREG V-A Spain-Portugal ALGARED+), and the European Commision (H2020-MSCA-ITN BIOMEDAQU/766347). MFC: wishes to acknowledge the funding from CEEC program supported by FCT/MCTES (CEECIND/02968/2017); ACTINODEEPSEA project (POCI-01-0145-FEDER-031045) co-financed by COMPETE 2020, Portugal 2020, ERDF and FCT; Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by FCT and ERDF. MC: financial support from the Programme of the Institute of Oceanology, PAS (grant no. II.3) and National Science Centre in Poland (project number NCN 2016/21/B/NZ9/02304). MCu: acknowledges the funding from the Ministerio de Ciencia e Innovación of Spain (SAF2009-0839 and RTA 2015-00010-C03-02) and INTERREG-MAC2/1.1b/279 (AHIDAGRO). AD-M: acknowledge financial support from INTERREG-MAC/1.1b/042 (BIOTRANSFER2) and Agustín de Betancourt Programme (Cabildo de Tenerife and Universidad de La Laguna). AD: work has been supported by the ERDF Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1, Operational Programme “Growth and Employment” (No. 1.1.1.2/VIAA/1/16/048). RJF: funding for this research was provided under the Marine Research Programme 2014–2020, through the Marine Institute of Ireland under grant PBA/MB/16/01 “A National Marine Biodiscovery Laboratory of Ireland (NMBLI)” and through the Food Institutional Research Measure, administered by the Department of Agriculture, Food, and the Marine, Ireland under grant issue 17/F/260 (MaraBioActive). SG: this work was supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019). SG thanks financial support provided by FCT/MCTES through grant IF/00700/2014 and OceanTresaures project PTDC/QUIQUI/119116/2010. NID: wishes to acknowledge the funding from the Croatian Science Foundation Project CELLSTRESS (IP-2018-01-5840). MMa and TD: we wish to acknowledge funding from the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI) under grant no. 239 (SPINAQUA project). AM-G: acknowledges the financial contribution from the project BYTHOS funded by the European Union’s Interreg V-A Italia-Malta Programme under project code C1-1.1-9. HM-M: financial support from National Science Centre in Poland 2016/21/B/NZ9/02304 and 2017/25/B/NZ9/00202. MMe: this work has been supported by the French Government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX-01. MMe: thanks the Canceropôle Provence-Alpes-Côte d’Azur, and the Provence-Alpes-Côte d’Azur Region for the financial support provided to the MetaboCell project. DO: supported by the Doctorate Study program in Ecology and Environmental Sciences, Marine Research Institute, Klaipėda University, Lithuania. CR: we gratefully acknowledge the Research Council of Norway, the Møre and Romsdal County Council and Møreforsking AS for their financial contributions through the PROMAC (244244; www.promac.no), the Norwegian Seaweed Biorefinery Platform (294946; http://seaweedplatform.no/), and the Blå-Grønn (55031) projects. ER: this work benefited from financial support from the PACA Canceropôle, the National Cancer Institute, the PACA Regional Council and the French Government, managed by the National Research Agency as part of the Université Côte d’AzurJEDI Investissement d’Avenir project (ANR-15-IDEX-01). JS: work was supported by the Slovenian Research Agency (P4-0127 and J4-1771). IS: financial support from Ministry of Education, Youth and Sports of the Czech Republic (project CZ.02.1.01/0.0/0.0/17_048/0007323). XT: the tool “RRI Roadmap” was developed as part of the European Horizon 2020 project MARINA “Marine Knowledge Sharing Platform for Federating Responsible Research and Innovation Communities” under the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 710566 (2016–2019). OT: his contribution is carried out with the support of the Marine Institute and is funded under the Marine Research Programme by the Irish Government (Grant-Aid Agreement No. PBA/MB/16/01).

Members of the bacterial phylum Planctomycetes are reported in marine water samples worldwide but quantitative information is scarce. In this thesis the abundance, distribution and phylogenetic diversity of Planctomycetes were investigated in different coastal systems, the German North Sea island Helgoland, the Tyrrhenian coastal lake Lago di Paola and the adjacent marine coastal waters. Seawater was collected in different seasons at the station "Kabeltonne" near the island Helgoland. An increase in Planctomycetes absolute numbers was registered in spring showing a positive correlation with phytoplankton blooms. The analysis of size-fractionated seawater samples and of macroaggregates showed that ~ 90% of the Planctomycetes reside in the > 3µm size fraction. At Lago di Paola Planctomycetes increased in summer - during stratification - in surface waters and mainly responded to chlorophyll a concentrations confirming their suggested role in the degradation of algal biomass. Abundances were higher in the coastal lake than in the adjacent marine waters. With both studies 224 almost full length 16S rRNA gene sequences of Planctomycetes have been added to the database and six coastal clades have been defined. At Helgoland the clade-specific counts revealed a seasonal succession which might be linked to phytoplankton blooms. At Lago di Paola, cell numbers indicated the simultaneous presence of all clades in the surface and bottom water in similar abundances. In clone libraries from Lago di Paola, 20 almost full length 16S rRNA gene sequences affiliated with Chlamydiales were retrieved, two main clusters were identified and two oligonucleotide probes were designed. It was possible to visualize high numbers of individual environmental chlamydiae as well as chlamydiae inside a host, presumably a protist.