Miljöövervakning av växtplankton i Kattegatt och Östersjön med rDNA-barcoding och mikroskopi : En jämförelse av molekylärbiologisk metodik och mikroskopi.

Växtplankton utgör en viktig del av den marina näringsväven. Därför pågår nationell och regional övervakning av växtplanktons utbredning i tid och rum, antal, biomassa och biodiversitet i haven runt Sverige. EU-direktiv och mellanstatliga konventioner influerar övervakningens utformning. Vissa algblomningar kan vara skadliga, därför finns ett visst fokus på alger som producerar gifter eller är skadliga på annat sätt. För att utföra övervakningen på ett kostnadseffektivt sätt behöver flera olika metoder kombineras. I den här rapporten presenteras resultat från en jämförelse av mikroskopbaserad analys av växtplanktonprover med resultat baserad på rDNA-metabarcoding. Provtagning skedde med hjälp av ett så kallat FerryBox-system i juli 2013 på arton platser längs en salthalts-gradient (ca 3-24 promille) från Bottenviken via Bottenhavet och Egentliga Östersjön till Kattegatt. Resultat från rDNA metabarcoding visade på en mycket större biodiversitet jämfört med data från mikroskopi. Totalt 89 olika organismer identifierades med mikroskop (Utermöhl-metoden). Bland dessa ingår samlingsgrupper som ”oidentifierade flagellater” och ”oidentifierade encelliga organismer”. Baserat på rDNA data noterades totalt ca 2000 olika organismer (exklusive heterotrofa bakterier) vilket innebär att mer än 95% av biodiversiteten förbisågs med Utermöhl-metoden. För prokaryoter (16S rDNA) noterades 36 olika Operational Taxonomic Units (OTUs) för cyanobakterier och för eukaryoter (18S) noterades 1860 olika OTU. Flera organismer som noterades med mikroskopi saknades helt i rDNA data. Det tyder på att referensdatabaser för 16S och 18S rDNA saknar sekvenser för vanligt förekommande arter i haven runt Sverige. Det kan också bero på att just sekvensdelen 18S är identisk för vissa arter/släkten, varför klassificeringen hamnade på en högre taxonomisk nivå. rDNA-barcoding ger inte samma typ av data som mikroskopi, det saknas bl.a. motsvarigheter till cellantal och biomassa. En alternativ molekylärbiologisk metod är så kallad kvantitativ PCR (qPCR). Med qPCR kan mängden DNA för enskilda organismer bestämmas, mängden DNA är relaterat till cellantal. Idag är det endast möjligt att utföra qPCR på ett fåtal organismer i ett prov.  Författarna föreslår att rDNA-barcoding av plankton införs i marina övervakningsprogram i Sverige som komplement till annan metodik. Förutom att rDNA-data ger högupplöst information om biologisk mångfald, så har rDNA barcoding ett lågt pris per prov om många prover analyseras och är inte beroende av mikroskopisters skicklighet att identifiera organismer. Införandet bör innehålla följande delmoment: 1. Pilotstudie – rDNA-data för ett helt år jämförs med mikroskopi och flödescytometri; 2: Sekvensering av vanligt förekommande arter som saknas i referensdatabaser; 3: Standardisering av provtagningsprotokoll; 4: Standardisering av metodik för sekvensering; 5: Utveckling av qPCR för utvalda skadliga arter; 6: Standardisering av dataflöden och jämförelser med internationella referensdatabaser; 7: Uppbyggnad av system för datahantering hos nationell datavärd och 8: Utveckling av bedömning av miljöstatus gällande t.ex. biodiversitet och invasiva arter baserat på rDNA data.Phytoplankton is a fundamental part of the marine food web. Therefore, a national monitoring programme that is focused on the spatial and temporal distribution of phytoplankton in the sea around Sweden is ongoing. The design of surveillance is based on EU directives and intergovernmental conventions. Since some algal blooms can be harmful, there is a special focus on the algae that produces toxins and are potentially harmful in other ways. To carry out the monitoring in a cost effective way, several methods are conducted. This report presents a comparison of the results yielded by microscope-based analysis and rDNA metabarcoding-based analysis. Sampling was done with the help of a Ferrybox system in July 2013 in eighteen locations along a salinity gradient (3 - 24‰) from the Bothnian Bay through the Bothnian Sea and the Baltic Proper to the Kattegat. Results of rDNA metabarcoding showed a much greater biodiversity compared to the result from microscopic counting (Utermöhl method). In total, only 89 organisms were identified by Utermöhl method and the rest were marked as "unidentified flagellates" and "unidentified unicellular organisms". rDNA metabarcoding recorded a total number of almost 2,000 different organisms (excluding heterotrophic bacteria), which means that more than 95% of biodiversity was overlooked by Utermöhl method. Altogether 36 Operational Taxonomic Units (OTUs) were identified as cyanobacteria from the prokaryotes data (16S rDNA) and 1860 different OTUs were found in eukaryotes data (18S rDNA). There were still several organisms missing from the list that microscopic counting yielded. This result suggests that the reference databases for the 16S and 18S rDNA sequences lack some species common in the seas surrounding Sweden. Another reason may be that the 18S rDNA is identical to other species or a genus leading to that the classification is at a higher taxonomic level. Metabarcoding provides different types of data than microscopic counting. The rDNA-based data can offer a high resolution on biodiversity but cannot offer data on cell counts and biomass as microscopic counting does. An alternative molecular biological method is known as quantitative PCR (qPCR) which can determine the amount of DNA from individual organisms so that the cell count of the organisms can be inferred. At present it is only possible to perform qPCR on a small number of organisms in a sample. The authors suggest introducing the rDNA metabarcoding approach of plankton analysis in Swedish marine monitoring programs as a complement to other methods. Besides its advantage regarding the high resolution on biodiversity, rDNA barcoding has a low price per sample when many samples are analysed in one go, and it is not dependent on the taxonomists’ skill on identifying organisms. The plan includes the following elements: 1. Pilot study - rDNA data from a full year should be compared with microscopy and flow cytometry data; 2: Sequencing of the common species in the Baltic that are missing in the reference databases; 3: Standardization of sampling protocols; 4: Standardization of sequencing method; 5: Developing qPCR method targeting selected harmful species; 6: Standardization of sequencing data and comparing it with the available reference databases; 7: Structuring the data management system for the monitoring data and 8: Development of the assessment of the environmental status regarding biodiversity and invasive species based on rDNA data

Tribological aspect of the surface topography variations for injection cams

In automotive industry, the environmental demands of low emissions, low fuel consumptions and long lifecycles lead to higher and higher engineering constraints. For example, high pressures on injection cams are needed for an improved combustion. Therefore friction and wear are constant problems encountered in camshaft development. The contact between roller and cam is a mix of sliding and rolling which leads to a wide range of failure modes. The uniqueness of this contact is also due to variations all around the cam depending on a multitude of parameters such as radius of curvature, load, sliding velocity. A previous study described surface topography as a function of cam shape and running time. The different types of wear mechanisms are strongly linked to contact pressures which are also dependent on roughness.This paper deals with the repercussion of observed variations of surface topography on the tribological behaviour of the cam roller contact. A number of cams have been mapped. The measurements are made by a non-contact light interferometer to monitor changes in the surface roughness. The Greenwood-Williamson contact model has been developed and applied to those measurements in order to collect characteristics of the microscopic pressures distribution.The results of the study show the significant effect of topography variations on the tribological behaviour of the cam roller contact. Cam angle roughness fluctuations arestudied as well as running time dependency. The flat part of the cams with a high amount of peaks of sharp radii gives the worst tribological conditions and the running-instudy shows the beneficial effect of peak smoothening on contact pressures. Tribological characteristics such as pressure distribution are valuable results which are helping to build characteristics of a recommended surface more able to face wear problems. In that case, an interesting prospect will be to virtually screen surfacesproduced with different finishing processes and find out the best ones in order to reduce wear. The study of different roller processes will be as well of great interest

Evaluation of cam and roller surfaces and their manufacturing process by functional characterisation

Friction and wear are constant problems encountered in camshaft development. Thecontact between roller and cam is a mix of sliding and rolling which leads to a wide rangeof failure modes. The uniqueness of this contact is also due to variations all around thecam of a multitude of parameters. A previous study described surface topography as afunction of cam shape. The different types of wear mechanisms are strongly linked tocontact pressures which are also dependent on roughness. The aim of the paper is toevaluate the quality of camshafts and rollers produced with different manufacturinghistories. The evaluation utilises standard roughness parameters as well as rough contactparameters. The surfaces measurements are made by a non-contact light interferometerand a set of 3D roughness parameters is evaluated for each measure. The Greenwood-Williamson contact model has been developed and also applied to the measurements inorder to collect characteristics of the microscopic pressures. The results of the study showthe significant effect of topography variations on the tribological behaviour of the camroller contact and rank the different manufacturing processes according to functionalcharacterization. The verification of the ranking using experiments is the continuity of thisstudy

Evaluation of cam and roller surfaces by wear testing and functional characterisation

Friction and wear are constant problems encountered in camshaft development. The contact between roller and camis a mix of sliding and rolling which leads to a wide range of failure modes. The uniqueness of this contact is also dueto variations all around the cam of a multitude of parameters such as load and radius. A previous study describedsurface topography as a function of cam shape. The different types of wear mechanisms are strongly linked to contactpressures which are also dependent on roughness. The aim of the paper is to develop a rough contact model which willbe utilized as a tool to rank surfaces and their ability to face wear problems. In order to verify the tool, rough contactresults are compared to roughness parameter variations due to wear produced in a cam roller rig test. The surfacemeasurements used for this study are made by a non-contact light interferometer. The Greenwood-Williamson contactmodel has been developed in a deterministic way and the elasto-plastic behaviour of the material has been integratedto the model. The outputs of the simulation give a ranking of surfaces which is compared to their roughness variationsdue to wear. The study shows that the model developed is a reliable tool to rank and define surface quality since theresults are correlated to wear. However, the results show as well some discrepancies which could be corrected in thefuture by integrating to the model two new features: a rough to rough contact including sliding between surfaces. Thisnew model should be verified by an accurate experimentation using relocation between unworn and worn surfaces

The evolution of surface topography of injection cams

Friction and wear are constant problems encountered in camshaft development. Among a wide amount of factors, surface topography is an important feature to improve in this complex system. Four parts of different shapes can be differentiated on an injection cam profile. Therefore the machining of such component is quite particular. Considering the complex shape of an injection cam, this paper is divided in two studies: 1. Variations of surface topography around freshly produced cams in order to emphasize the particular profile of cams which leads to a complex machining. 2. Evolution of surfaces during run-in in order to discover the effects of wear process on a group of roughness parameters. The results show a variation of density of peaks along the freshly produced cam surface and highlight also the importance of roller type on the evolution of topography. Further results show that summits of the surface are rounded off during run-in. Concerning the future, results of the study are encouraging to investigate more deeply the tribological behaviour (contact mechanic, oil film thickness) of the surfaces