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Voorstel stage Bio-informatica (2017-18):

Invloed van zandwinning op de bacteriële gemeenschap in de zeebodem langsheen een verstoringsgradiënt.

Op het Belgisch deel van de Noordzee wordt jaarlijks rond de 3 miljoen m³ zand gewonnen voor zowel de bouwindustrie als voor het ophogen van onze stranden ter bescherming van de kustzone. Door zandwinning wordt de zeebodem en de biologische gemeenschappen die er leven echter onvermijdelijk verstoord. Verschillende studies tonen een duidelijk effect op het macrobenthos (organismen > 1mm) in de meest intensief ontgonnen zones maar over de invloed op de bacteriële gemeenschappen is tot nog toe weinig tot niets gekend. Nochtans zijn micro-organismen onmisbaar in een gezond ecosysteem en spelen ze een heel belangrijke rol in de biochemische processen van de zeebodem. Daarom werd in het najaar van 2015, gelijktijdig met het bemonsteren van het macrobenthos, de bacteriële gemeenschap bemonsterd in intensief ontgonnen, gematigd ontgonnen en nooit ontgonnen gebieden.De bacteriële gemeenschap van deze locaties werd bepaald door amplicon sequenering van het V3-V4 fragment van het 16S rRNA door middel van Illumina technologie. De verwerking van de ruwe data set zou antwoord moeten bieden op de volgende vragen: 1/ Beïnvloedt zandwinning de bacteriële gemeenschap en welke groepen bacteriën worden vnl. beïnvloedt? 2/ Kan het gebruik van bacteriële gemeenschappen een ‘quick scan’ methode zijn om het effect van verstoring door zandwinning te bepalen?

Abstract 1 advanced bachelor of bioinformatics 2019-2020: Nanopore sequencing of ribosomal DNA to characterize diversity in marine sediments

Bacterial and meiofaunal communities from marine sediments can be described by analyzing 16S and 18S rRNA gene sequences using a DNA metabarcoding approach with the Illumina Miseq platform. However, the PCR step in this approach suffers from a number of limitations such as PCR bias, primer mismatch, chimera formation and polymerase errors. Primer free methods could greatly improve the characterization of these communities. The long read sequencing technology of Oxford Nanopore may combat the limitations that Miseq has, but the development for biodiversity assessment is still in its infancy. The goal of this internship was to develop a bioinformatic pipeline to analyze long read minion data from environmental samples, and compare the community composition between Minion and Miseq methods to evaluate whether primer free methods are a suitable and better alternative then DNA metabarcoding.

Samples from marine sediments with different intensities of sand extraction in the North Sea have been sampled and sequenced using the Miseq and Minion. Minion data is provided as FAST5 output, this file contains the raw signal data. To process this information, base calling is required. This was done using the tool Guppy. Guppy converts the electric signal to nucleotides. FAST5 files are converted to a FASTQ file, containing Qscore, sequence length and barcodes. A summary file with data from the minion run is also generated. Demultiplexing of samples using nanopore barcodes was done using guppy_barcoder. Filtlong was used to filter out the sequences that had a read length lower than 500 base pairs. The cutoff is set to 500 bp because reads that have less base pairs will be difficult to assign a taxonomy. A total of 1676 sequences were kept, which means that 41.9% of all reads had a read length longer than 500bp. To check the amount of reads that were removed and to analyze overall read quality, the tool Nanoplot was used. This tool generates plots and text files that nicely visualize the read data.

The samples contain DNA originating from a mix of bacterial and small metazoan species. The goal was to find a way to combine the sequences from each species together. Various clustering tools (ONTtrack, vsearch and isONclust) were tried out to form clusters from the sequences that in theory should come from the same species. Our data was too divergent and contained too little reads to form a decent amount of clusters with an acceptable identity grade. Another method to group the same sequences together was required.

A local Blastn was performed against the SILVA 138 SSU Ref NR 99 (Bacteria, Archaea and Eukarya 18S/16S rRNA) database. The e-value cutoff was set at 0.05, this yielded a total of 1022 hits. This blast result generates taxonomic information for each hit. The taxonomic information in the SILVA database is not normalized in terms of taxonomic ranks, some results contain subtaxa while others do not. This makes analyzing the taxonomic levels very difficult. An R script was developed to normalize all taxonomic levels. In total 448 sequences are classified as archeae and bacteria. 505 Sequences are classified as eukaryota, 69 sequences had not been assigned a taxonomy.

Many species had more than one sequence. From these sequences a consensus sequence was generated at species level with SPOA v3.0.1 (, which is based on a partial order alignment (POA) algorithm. The consensus sequences were again blasted again the SILVA database and set to the correct taxonomy. The blastn is performed again because the consensus sequences should be more correct and can yield a different result than the original blast. The blast accuracy improved with 21%, the mean value of the bitscore from the original blast was 346.40 while the bitscore from the blast containing consensus sequences was 437.34. In order to analyse the E-value, the log value was taken and the 0 scores were ignored. The original blast had an average of -160.407, an average of -183.055 was calculated for the consensus blast. The lower the value the better. 

The Minion pipeline resulted in the detection of 81 unique metazoan genera and 24 unique bacterial genera. The same DNA samples were also processed with the Miseq data, which yielded a total of 568 unique metazoan genera. In total 27 genera were found in both Miseq and Minion data. A total of 249 unique bacteria was found with Miseq. Twelve of these genera were found in both sequencing techniques.


A semi-automated pipeline was developed to process and analyze long read data from mixed environmental samples. Consensus sequences have shown to be of higher quality than original raw Minion sequences. We expected to receive more genera from Minion data in comparison with Miseq because we should not have a bias from primers, but this was not the case. A reasoning behind this may be that the Minion wet-lab protocol is not fully optimized, hence the low amount of sequences that were left after filtering. 


Abstract 2 advanced bachelor of bioinformatics 2019-2020: Optimizing diversity estimates for metazoa from DNA metabarcode datasets

Biodiversity plays an important role in a healthy seabed. The goal of the experiment is to examine biodiversity into a single seabed. One way to do this is with DNA metabarcoding, where DNA is extracted directly from the sediment and a genetic marker is amplified through PCR. Depending on the genetic marker, it is possible to study bacteria (16S) or small multicellular organisms (18S). After the analysis with the Illumina Miseq. The outcome is a lot of sequences per sample. They must then be filtered and corrected. Contamination can also occur in the samples during the lab experiment, which must be removed to obtain a reliable estimate of the biodiversity using metabarcode data.

This project was aimed at investigating the biodiversity in one sandbank (Thorntonbank) in which 15 different samples were taken at 3 different sand extraction impact zones (HIGH, MED, LOW). The names of impact zones indicate the different heights were the samples were taken. It was also examined whether total DNA gave the same results as iDNA

After receiving the 16S and 18S datasets, the primers were removed be using a bash script. Several bioinformatic pipelines were performed in Rstudio. The first was DADA2. This pipeline is determining the amplicon sequence variants (ASV) and will also remove the chimeras. During this pipeline, the taxonomy was assigned using an existing RPD database to determine the different genera. The total amount of ASVs for 16S was 1653 after DADA2 and for 18S it was 3529 ASVs. In 16S they were 227 unique genera and in 18S they were 282 unique genera.

In a next step the phyloseq pipeline was performed. This provides a visual representation of the DADA2 data. One of the most important plots was the Non-metric Multi-dimensional Scaling plot (NMDS plot), from which was deduced that for most samples there was no difference between the total DNA and iDNA. For most cases there were also clusters of the samples taken at the same impact zone. In addition, the pipeline also gave a barplot with the different Genera that occurred per sample and a heatmap with the ASVs per sample. In 16S the species where filtered on phylum and there was no clear difference between the reference and the different impact zones and in 18S there was a higher percentage Opisthokonta in the reference and HIGH samples then the other samples.

The decontam pipeline was carried out after phyloseq, this removed the contaminants. We worked with 2 methods, the frequency method and the Prevalence. The frequency method takes into account the distribution of the frequency of each sequence feature as a function of the input DNA concentration is used to identify contaminants.

The Prevalence method will check which ASVs are present in the negative control and will then remove them from the other samples. When the ASV appeared as contaminant in both methods, they were removed from the dataset. In 16S two ASVs were flagged as contaminants and in 18S zero ASVs were flagged as contaminants.

The last pipeline performed was the LULU pipeline. The purpose of LULU is to reduce the number of ASVs to achieve more realistic biodiversity metrics. This is done by evaluating the co-occurrence patterns of ASVs among samples. LULU identifies ASVs that consistently satisfy some user selected criteria as errors of more abundant ASVs and merges these. Four different combinations of criteria were tested, minimum_match of 80 or 90 and minimum_relative_co-occurrence of 0.1 or 0.9 (figure1). After looking at the number of genera, 4 unique genera were removed in 16S and 3 genera in 18S with criteria1 with a minimum_match of 80 and minimum_relative_co-occurrence of 0.1.


From the different pipelines we can conclude that it is possible to extract ASVs from fastq files, filter the data and assign a taxonomy to the ASVs. We also managed to extract the contaminants using 2 different methods, very little contamination was detected. It was also shown that total DNA can be used for these experiments in the future as results were similar to iDNA because it requires less work in the laboratory. With LULU, we have also successfully clustered similar ASVs which lead to reduction of 1.3% for 16S and 1.4% for 18S of genera. The next step the experiment would be to perform tag switching on the data.



Human activities like sand extraction at sea are increasing and this can be a problem for the marine ecosystem and its biodiversity. Sediment composition can change, resulting in a different biodiversity. It is very important that human activities are regulated so that the marine system remains healthy. In order to manage human activities, there must be a thorough scientific basis to support any actions that could be taken for the benefit of the marine ecosystem.

The aim of this study is to investigate whether there is an impact of sand extraction on the benthic bacterial community and if the benthic bacterial community can recover when sand extraction has stopped. Samples from two sandbanks, the Thortonbank and the Buitenratel, were analysed in this work. For the Thortonbank, we investigated whether there is an impact on the bacterial community in impacted zones compared to reference zones without impact and whether the impact differs between different depth layers of the sediment. In addition, we compared bacterial communities based on intracellular DNA (iDNA) and extracellular DNA from the sediment. When detritus sinks to the bottom of the sea, DNA is released that is no longer intracellular. Extracellular DNA needs to be removed in order to avoid misrepresentation of DNA and so of impact. For the Buitenratel, a previous study showed differences in bacterial community between the highly impacted zones and the reference zones. Since then, sand extraction has stopped five years ago, so we investigated whether the benthic bacterial community recovered during this time.

The chosen method is DNA-metabarcoding due to its cost-efficiency. First, the DNA is extracted from the sediment samples. From the Thortonbank, the extracellular DNA and iDNA is extracted separated using a different method and kit. From the Buitenratel total DNA is extracted using a kit. Amplicon PCR is performed on all the samples using 16S primers with an adapter overhang to bind with the flow cells of the Illumina sequencer. After the amplicon PCR, the contaminants (for example the primers) are removed with AMPure magnetic beads. Index PCR is performed on the purified amplicon PCR to identify each sample with an index (barcode) and purified again with AMPure magnetic beads. The index PCR samples are equimolarly pooled, using the Quantus to measure the concentration, and quality control is performed with capillary electrophoresis. An external company sequences the samples with Illumina Miseq bridge amplification. Data-analysis is performed in R-studio with the Dada2 pipeline and the Phyloseq package.

The capillary electrophoresis shows that the DNA-extraction, the adapter PCR and the index PCR were successful. The sequences from the Thortonbank contain on average approximately 50000 reads with a few outliers. The sequences from the Buitenratel contain on average approximately 85000 reads with some small variation. The number of reads should be the same because the index PCR-products are pooled equimolar, but due to Quantus-, PCR- and pipetting mistakes there can be a variation. Biodiversity is measured with the number of amplicon sequence variants (ASV’s). The more biodiversity, the more species, the more ASV’s. From the samples of the Thortonbank, the reference impact group contains the highest ASV’s, less ASV’s are found in the moderate impact group and the high impact group contains the least ASV’s. This means that the more sand extraction has happened, the less species are found. No correlation has been found between the number of reads and extracellular DNA and iDNA, nor between the depths. This is the same for the number of ASV’s. Heatmaps were made to look at taxonomy clusters. For the Thortonbank three clusters were seen, one for each impact group illustrating that the bacterial communities are impacted by sand extraction activity. The non-metric multidimensional scaling (nMDS) plot, which shows the genetic distances between locations, shows three clusters corresponding to the impact groups in the Thortonbank. A few samples from the high impact group belonged to the moderate impact group cluster, as seen in the heatmap. No clustering was found between the extracellular DNA and the iDNA samples. There is a clustering based on the different depths, meaning that comparisons can only be made between samples with the same depth. For the Buitenratel, the number of ASV’s do not differ between the reference groups and the impact groups. In addition, no clusters were seen between the impact groups in the heatmap. The nMDS plot also did not contain separated clusters between the impact groups. All results strongly indicate that the bacterial community has recovered from the sand extraction activities.

This study shows that sand extraction has impact on the benthic bacterial communities and that the benthic bacterial communities can recover from it after 5 years. In the future, separating extracellular DNA and iDNA is not needed. Vigilance is still needed to protect the biodiversity. 

Abstract Bachelor Project 2 FBT 2019-2020: Chemical taste characterization of algae by analyzing free amino acids with HPLC-MS/MS

Approximately 90% of algae is used for food purposes, while in Europe this is only 9%. One of the bottlenecks in Europe is the lack of consumer acceptance. Little is known about the flavor of algae and how it can be used as food or added to food products. When algae are to be integrated in foodstuffs, it is important to consider the taste and aroma of the algal biomass. In the framework of the Interreg project ValgOrize, taste and aroma profiles of several seaweed and microalgae species will be investigated.

The aim of this study is to determine the free amino acids composition in microalgae which can be related to their taste. A hydrophilic interaction liquid chromatography (HILIC) method was validated for analyzing the free amino acids in two different microalgae species, Nannochloropsis and Tetraselmis, by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS).

Precision, accuracy and limits of detection and quantification were evaluated for all individual amino acids. The results shows an acceptable accuracy and precision for most amino acids of Tetraselmis. All amino acids can be detected from Tetraselmis. However, it’s not possible to quantify phenylalanine, methionine, valine and threonine due to the low concentration in this species. The remaining amino acids are quantifiable.

For Nannochloropsis is was not possible to detect following amino acids: taurine, tyrosine, leucine, methionine, isoleucine, threonine, alanine, glycine, aspartic acid, histidine, glutamine, asparagine and tryptophan. Phenylalanine, valine and proline can be detected but not quantified. Only glutamic acid and lysine can be quantified.

The highest concentrations of free amino acids of Tetraselmis are alanine and glutamic acid which are both known to give an umami taste. It can therefore be concluded that Tetraselmis will have an umami taste. Nannochloropsis also has an umami taste because of the high concentration of glutamic acid.

Abstract Bachelor Project 1 FBT 2018-2019: Detection of fraud in sole and cod through DNA barcoding and qPCR in the Belgian fish industry

Sole (Solea solea) and cod (Gadus morhua) are both very known and important fish in the Belgian fish industry. The combination of sole being expensive and cod being a public’s favorite comes with the opportunity for fraudsters to emerge. Both fishes are sensitive to fraud, previous research showed that mislabeling occurred in 13,1 % of the cod samples and in 11,1 % of the sole samples in Belgium. The main aim of this study is to detect fraud in the Belgian fish industry using DNA barcoding and qPCR. In this study 97 cod samples and 20 sole samples were collected.

The samples were collected by various staff members at ILVO. The samples were all bought in Belgium and cover the different branches of the Belgian fish industry such as retail, catering and specialist stores. The kit used for DNA extractions was the NucleoSpin® food kit, this kit gave the best results for processed samples. The DNeasy kit was tested as well, but the results were not quite as good for processed samples. To amplify the DNA, either the qPCR or the regular PCR was used. Three primer sets were tested using the regular PCR, one for the CO1 gene, one for the cytb gene and one for the cytb gene with small fragments (mini-barcoding). The samples were sent to an external company for Sanger Sequencing. 80,3 % of the samples sent for Sanger sequencing resulted in a high quality CO1 sequence. For the cytb gene the standard primers gave in 75 % of the cases a valid result. The best result came from the mini-barcoding cytb primers, with a success ratio of 84,7 %. The cytb mini-barcoding primers were able to deliver a result for samples that failed using the CO1 primers or the standard cytb primers. The preferred technique for analyzing cod was qPCR, because this technique is faster and cheaper. The fraudulent qPCR results always need to be double checked with DNA barcoding to prevent a false result. At the time of the investigation there was no qPCR kit available for sole, so every sole sample was analyzed using DNA barcoding. Two sole samples failed the sequencing and did not have a valid barcoding result. Five out of the eighteen successfully sequenced sole samples were mislabeled or 27,78 %. For cod there was only one sample out of 97 that was mislabeled or 1,03 %, this low percentage might be due to cod being mostly imported and undergoing several controls before the fish reaches the Belgian fish market. The fraud rate for sole was a lot higher than what previous research showed, this may be explained by the high delivery price of sole in Belgium. The substitutes for both cod and sole are cheaper fish. This translates to financial gain for the seller, thus fraud is present in the Belgian fish industry.

Abstract Bachelor Project 2 FBT 2018-2019: Booster biocide determination at the Belgian part of the North Sea

Antifouling paints with booster biocides are used to avoid fouling on the hull of ships. Booster biocides can be toxic to the marine environment and some can also be persistent in marine sediment. Since there is no information available on booster biocide concentrations at the Belgian part of the North Sea, the goal of this thesis is to validate a method for analysis of 6 booster biocides and to apply it to measure these compounds in marine sediment of the Belgian part of the North Sea. Booster biocides selected were irgarol, diuron, sea-nine, tolylfluanide, dichlofluanide and medetomidine.

The total organic carbon content (TOC) and grain size distribution was determined on 29 samples by respectively a redox titration by Mebius and a sieving procedure followed by laserdiffraction by a Malvern Mastersizer. For the analysis of the booster biocides, pressurized liquid extraction (PLE) was used to extract the booster biocides, after which they were analysed with an HPLC-device coupled with a tandem MS-detector. The method passed the validation for the booster biocides irgarol, diuron, sea-nine 211, dichlofluanide and tolyfluanide as limits for trueness and reproducibility were met. The limit of detection ranged from 0,04-1,14 ng/g. For medetomidine, the method will not be applied as validation failed. For this compound, too high variability was noted between samples, probably due to matrix effects.

The TOC and grain size analysis showed that samples from the dredging spoil disposal sites LZO and LOO along with the shipping track near port Zeebrugge had a high amount of silt and organic components. Three boosterbiocides, irgarol, diuron and sea-nine 211 were detected at these locations with a respective normalized maximum concentration of 15 ng/g, 4 ng/g and <LOQ. The presence of these boosterbiocides confirms that risk assessment is needed to have a view on the impact of these compounds on the marine environment.

Abstract 2018-2019 advanced bachelor of bioinformaticsMining MinION long read metatranscriptome and ribosomal RNA data as a primer-free method to characterize microbial and eukaryotic diversity from marine sediments
Nematodes are often used as bioindicators for the health of a marine area because nematode species show different responses to environmental stressors and because their small sizes prevent escape from the impacted area. However, their small body size and lack of easily observable morphological characters complicates their identification. It is also important to look at the Bacteria present in the marine area, because they are responsible for multiple metabolic processes. To characterize the microbial and eukaryotic diversity in marine sediments, RNA is extracted from the samples and sequenced with MinIon. Two different marine sediment samples are used: North Sea (18S RNA) and Vietnam (RNA) samples. The quality characteristics of the sequencing runs are evaluated with the Nanopore summary statistics and Basic QC tutorial. The goal of this project is to construct a pipeline to filter the ribosomal RNA and determine the taxonomy for the sequenced reads. Also the functional genes are studied by executing the Nanopore Transcriptome Tutorial. Two data collections are processed: 18S/16S rRNA data from North Sea samples and total RNA data from Vietnam samples. Two different marine sediment samples are used: North Sea (18S RNA) and Vietnam (RNA) samples. The quality characteristics of the sequencing runs are evaluated with the Nanopore summary statistics and Basic QC tutorial. 16S and both ribosomal and messenger types The Nanopore summary statistics and QC tutorial is used to assess the quality characteristics of the MinIon sequencing run for both the 16S/18S/16S RNA data and the total RNA data. The Rmarkdown script that is provided in the tutorial, is executed in Rstudio with the sequence and barcode summary files of the data as input. An HTML document is created that contains the analysis and exploration of the data. For the total RNA data 51,8% of the reads passed the QC filter. For the 16S/18S/16S data 41.4% of the reads passed, but only 32.8% of them are assigned to a barcode (classified). 16S/18S/16S ribosomal RNA data (North Sea) To detect if there is 18S rRNA in the 18S/16S RNA data and determine the taxonomy for these reads, the data are aligned against different databases: SSU Ref NR99 database (comprising Bacteria, Archaea and Eukarya 18S/16S rRNA) from the SILVA project and the own 18S RNA long Nematoda database. The Silva database is downloaded and filtered using PhyloFlash. The classified reads are blasted against the databases using blastn on the command line. The results are processed by creating an R script that assigns the taxonomy to the hits and designscreates barplots for the different domains, genera and Bilateria. There are 603 hits for Nematoda against the own 18S RNA long Nematoda database and only 190 against the Silva database. As expected, the Blast results contain multiple marine Nematoda species. Total RNA data (Vietnam) To detect if there is rRNA in the total RNA data and determine the taxonomy for these reads, the data is mapped against the filtered Silva database per barcode (BC01-BC06) with SortmeRna. The Fasta file of a random sample (BC02) is also blasted against the Silva database to compare the results of Blastn and SortmeRna. An R script is created to process the results. The taxonomy of the hits is assigned, the alignment percentages are calculated and barplots for the different domains per barcode are designed. For the different barcodes, most hits belong to Bacteria and only a few belong to Archaea or Eukaryota. For BC02 SortmeRna gets 1553 hits and Blastn only gives 1126 hits. There are no nematode species found. To examine the functional genes and in the RNA data the Nanopore Transcriptome Tutorial is followed. The first step in the tutorial is to run snakemake. This will uses Minimap2, Samtools and Salmon. The next step is knitting an Rmarkdown script to process the results. Originally the tutorial is constructed for mapping against the human transcriptome from the FTP Ensembl website. However the data that is used for this project comes from marine sediment samples. It contains RNA from different Bacteria and other organisms. Therefore NCBI Assembly is used to construct and download a reference database that contains all the prokaryotic genomes that are available. The snakemake file is adjusted, because the reference database and the complementary GFF3 file are saved locally. The output contains BAM and count files from the mapped reads per barcode. The first part of the Rmarkdown script is executed to examine the raw cDNA sequences and the cDNA read mapping against the different genomes. The results show that 100% of the reads are mapped against the reference genomes. This is probably because the reads map to multiple genomes and also multiple times against the same genome. Conclusion Blasting against the own 18S RNA long Nematoda database is the best way to characterize the nematode diversity. Using SortmeRna to map against the filtered Silva database, gives the best results for examining the microbial diversity. To examine the functional genes it is probably better to map to only one transcriptome, instead of multiple genomes, to get reliable results. 
Marine litter that ends up in oceans breaks down into smaller fragments namely microplastics due to UV light, wind and waves. These fragments stay on the sea bottom and are regarded as food for many marine species. For the analysis of microplastics in food items, laboratory method was further optimized and validated. Detection of microplastics is done in four steps: sampling, digestion with potassium hydroxide for biodegradation of the samples, active filtration and detection is done with a stereomicroscope. Method optimization and validation was exclusively done with spiked samples and procedures blanks, as no certified samples exist. During the optimization, various test designs were tested, in which the original test design was the most efficient. Positive results were established with colorless and red granules of 400-600 µm. Colorless granules of 105-125 µm were difficult to visualize under the stereomicroscope. The validation indicated that precision and accuracy were not adequate for small, colorless granules. More tests need to be performed on a broad variety of matrices, ensuring the feasibility of the analytical protocol for multiple food items.
Abstract traineeship advanced bachelor of bioinformatics 2017-2018: The studying of the effects of sand extraction on bacterial communities in the sea floor along a disturbance gradient

The Belgian Part of the North Sea (BPNS) is used extensively by humans. The activities focused in this research are sand and gravel extraction. To assist with spatial planning, granting exploitation licences and implementation of European regulations, it’s imperative to understand the individual and cumulative impacts of these various human activities on the marine ecosystem. Our goal was to determine if sand extraction activities have influenced the “Buitenratel” (a part of the BPNS) on a microbiological scale. Sediment samples of the “Buitenratel” have been collected on the 25th of September 2015. Samples were procured from thirteen areas of high extraction and six high extraction reference locations. Other samples were taken from two areas with moderate extraction and seven reference locations for moderate extraction. After DNA extraction, 16S RNA libraries were prepared to sequence on the Illumina Miseq sequencer (PE 300bp).

Until recently operational taxonomic unit (OTU) clustering was standard in the pipeline for the determination of bacteriological communities. The now recently developed DADA2 pipeline presents many improvements. It records the number of times each exact amplicon sequence variant was observed in each sample.  It makes models and corrects amplicon errors. This is to improve the overview and quality of the results. As the last step of the DADA2 pipeline the sequences are assigned a taxonomy and are put into an abundance table. The DADA2 pipeline was optimised for the “Buitenratel” dataset.  

The outputted table is used to build a taxonomic plot and a multidimensional scaling (MDS) plot to see differences between conditions. To determine the credibility of the produced plots the following statistical test were used: permanova, permdisp and posthoc pairwise comparison. The pipeline and the tests were conducted on normalised data and on rarefied data.

The taxonomic assignments were preformed using three different microbiological databases namely Silva 132, Greengenes 16_8 and the Ribosomal database project training set 16 (RDP). This was to see if the taxonomy of a sequence was equal in each database, which wasn’t the case. Some taxonomic assignments were different when other databases were used. Other sequences could only be predicted with only one of the three databases. On the genus rank, the differences between each dataset increased. The eucarya were removed from the Silva database to decrease interference of non-important sequences. Silva is still the best database as it is large, manually curated and recently updated.

On the MDS there is a clear difference between areas of high sand extraction and their references sites without sand extraction. On the other hand, there is no difference between the areas of moderate sand extraction and their references sites without sand extraction. There is also a clear contrast between high and moderate areas. The results of the statistical tests confirm this. It can be clearly seen that excavating sandbanks changes the bacteriological communities, both the rarefied data and the normalised data produced the same result. In the future an indicator analysis could be preformed to identify which taxa are most affected.

The taxonomic assignment could be changed. It’s still unclear what the true taxonomy is of some sequences. Adjustments could be done by limiting each dataset to only the bacteria present in the sea or sediment. This lowers the interference of non-relevant sequences.

Abstract bachelor project 2 FBT 2017-2018: The determination of booster biocides in the marine environment: method development

In the marine environment, there is vegetation on the hull of ships and boats of algae and aquatic animals. To counteract this, various antifouling products are applied to the hull of ships and boats. These products contain booster biocides that can be toxic to the marine environment. Within this bachelor’s report, a method was developed to determine seven booster biocides in the sediment matrix: medetomidine, zinc pyrithione, Irgarol 1051, diuron, Sea-Nine 211, tolylfluanid and dichlofluanid.

The sediment is extracted with pressurized liquid extraction (PLE). Booster biocides are then separated using the ultra-high-performance liquid chromatography (UHPLC) which is linked to a tandem mass spectrometry (MS/MS) as detector. Two ionization methods are used, namely electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

The LC-MS settings, including the multiple reaction monitoring (MRM) settings with determination of mass/charge (m/z) ratios and fragmentation patterns has already been optimized. The derivatisation method of zinc pyrithione is also created, since it does not give a response without derivatisation. In this bachelor’s report, the PLE solvent was optimized to hexane:acetone 2:1. Purification was tested with gel permeation chromatography (GPC), aluminium oxide (Al2O3) and silicon dioxide (SiO2). The latter purification technique gave the best results, but was not appropriate for medetomidine. When the analysis was done without purification, good linear responses were obtained for medetomidine as well as other booster biocides, while the extract was pure enough for injection onto the UHPLC.

The method was finalized for following booster biocides with ESI: medetomidine, Irgarol 1051, diuron and Sea-Nine 211, and with APCI: tolylfluanid and dichlofluanid. Precise results were obtained by quantification by standard addition, except for medetomidine, for which the spiked amount still must be increased. The derivatisation for zinc pyrithione with ESI is successful, but should be further improved to apply on real samples.

Abstract bachelor project 1 FBT 2017-2018: Optimization of a qPCR TaqMan assay for the identification of tuna species in processed food products

On the Belgian market many fish products are sold whose origin are not always known. This allows food processors to add cheaper fish varieties to their products without the consumer knowing. To prevent this, products must be checked. This is often done by DNA control. Each product, in this case fish, has unique DNA sequences that can be checked. But this is not always easy in stored products. These products are often sold after they have been processed. This means that products are steamed, smoked or they are stored in oils and sauces, among other things. Because products are processed, the DNA of the fish in the product is processed or broken down. Because the DNA is often broken in very short sequences, is it difficult to find specific sequences for certain species. This makes fraud much more difficult to detect. This study looks for a way in which this fraud can be detected. This research is mainly done for the interest of the consumer. A consumer pays for quality and expects to receive this quality. As mentioned earlier, food processors can use cheaper variants instead of their more expensive variety that is offered so to speak.

In this study, several tuna samples are bought in a supermarket where a DNA extraction will take place with two different DNA extraction kits. The DNA extraction will also determine which DNA extraction kit is the most efficient to extract the most DNA from these different products. There will also be an extra step included in de DNA extraction protocol with a chloroform/methanol/water solution. This will be checked by comparing DNA concentrations and the purity of the DNA. This DNA extraction will also run on a gel electrophoresis. Next, primers and probes are developed tested to investigate the fraud in tuna products on the Belgian market based on Real-Time Polymerase Chain Reaction (qPCR). To make sure fraud is excluded the result should match with the label of the product.

For processed samples we can decide that de DNeasy® Food kit with a chloroform step is the best method. The Food kit is also the best option for extracting DNA from pure samples. In case of pure samples, no chloroform step is needed. We developed our own probes and primers for identification of specific tuna species. There are three species who can already be identified. This at an amplification temperature of 62°C and a 100/400 probe/primer concentration. With those conditions T. alalunga, T. albacares and K. pelamis can be identified out of an unidentified sample. Also in mixed samples with different tuna species, each species can be successfully identified while the concentration of sample is only 50% or 33%. As a conclusion we can say that different tuna species can be identified by using qPCR and a TaqMan assay.

Abstract bachelorproef 1 FBT 2016-2017Determination of the quality and stability of two types of Belgian fish silage 

On January 1, 2014, a new law was introduced, namely the landing obligation. The landing obligation requires all catches of regulated commercial species of fish on-board to be landed and counted against quota. Because of this new law there is a big amount of fish and fish waste that cannot be used for human consumption. A solution for this problem is to use the fish materials as food for animals. One of the processes that has been done is with chemical preservation by acid addition. The product of this process is called fish silage. Several studies on fish silage have been done in Northern Europe but it’s unique in Belgium.

The aim of this study is to analyze the stability and quality of fish silage made in Belgium.

The parameters were analyzed for different kinds of fish silage. These go from raw material to pasteurized (wet sample) and dried silages (concentrated sample). The dry matter (DM) was determined with a freeze drier by using a temperature of -84°C and vacuum. The overall degradation was determined based on the total volatile basic nitrogen (TVBN), the method is based on an extraction with perchloric acid, a steam distillation and titration with hydrochloric acid. The bacterial degradation is based on the measurement of trimethylamine (TMA). The method is the same as for TVBN except that formaldehyde needs to be added to block the primary and secondary amines. The degree of hydrolysis is defined as the percentage of peptide bonds in a protein which have been cleaved during hydrolysis. It’s based on the reaction of TNBS and N-terminal amino groups that is measured spectrophotometric. The quality of lipids is based on the lipid oxidation. The method is based on a distillation and reaction of malonaldehyde (MA) with TBA that is measured spectrophotometric.

There was a big difference in DM content between the different silages going from 22,55% for raw material to 26,44% for pasteurized silages. The dried sample varies from 90,17% to 94,68% DM. The protein content decreases slightly over time. The TVBN value for raw material was under the limit of 50 mg N/100g but increased over time. The dried silages contained more than 195 mg N/100g, this because the TVBN values are more concentrated. TMA values were above the limit of 10 mg N/100g but were relatively stable. This means that the increase of TVBN is mainly due to NH3 production, which corresponds with the protein decrease. The degree of hydrolysis reached a maximum of 68,04%. The lipid oxidation reached a value of 10,62 mg MA/kg for the hydrolyzed sample, the other silages were below the limit.

More research needs to be done to improve the quality and stability of fish silage. Fresher raw material should be used to minimize the TMA values. An anti-oxidant can be added to prevent lipid oxidation. To produce a more stable product, the oil fraction can be removed from the fish silage.

Abstract bachelorproef 2 FBT 2016-2017Digital image analysis of flatfish injury – development of a protocol and device
Vitality status of beam-trawled flatfish is typically determined by external visual examination. This involves observers (or so called raters) who may use semi-quantitative indices to describe for example reflex responsiveness and the type and extent of external injury. One of the drawbacks of such scoring methods is that they are prone to bias given a potentially subjective interpretation of scoring criteria by individual and possibly independent raters. The purpose of this study was to develop a method and device which can eliminate this subjectivity by taking standardized, high resolution images to allow for automated calculation of the injury surface area relative to the whole fish. Injuries of interest are visible multifocal cutaneous petechiae (hereafter termed ‘point bleeding’), and suffusion or haemorrhaging (termed ‘bruising’). To develop a protocol and device for taking digital colour images of commercially caught fish, Common plaice (Pleuronectes platessa; n=23 fish), Common dab (Limanda limanda; n=24), Common sole (Solea solea, n=8), Mediterranean scaldfish (Arnoglossus laterna; n=6), European Flounder (Platichthys flesus; n=3) and turbot (Scophthalmus maximus, n=1) were sourced from the research vessel R/V Simon Stevin while beam-trawling in the Belgian coastal zone of the Southern North Sea. Out of these 66 fish, 53 fish were photographed a day or two after their capture (kept on ice, fresh), and 13 fish were defrosted (after being stored in a freezer). Using an already established laboratory-based prototype set-up, key light exposure parameters were established per species, length range and freshness status. Under tested lightening conditions, Canon EOS D6 shutter speed and aperture values were species specific (e.g. scaldfish were more translucent than plaice, regardless of their length), or length-specific, in particular for plaice. The freshness level did not seem to significantly affect the intensity values of the red-green-blue (RGB) colour channels. Exposure and set-up parameters from this pilot trial facilitated the design of a portable device consisting of a light table, dome and monopod, which may be used on-board both research and commercial vessels; to maximize on-board sampling capacity without having to bring each fish ashore for further examination. By accurately recording the coverage of externally visible bleeding injury, the device may find its applications in measuring whole fish or fillet quality, the effect of different capture techniques, and in improving vitality assessments as part of the implementation of the European landing obligation. 
Abstract bachelorproef 3 FBT 2016-2017DNA barcoding as a tool for detecting mislabelling of seafood

Fishery and aquaculture products are an important food source for humans. One of the major concerns in the seafood trading is renaming and mislabelling of species. Mislabelling involves providing inaccurate information about the identification of the product, most often because the product is a cheaper or a more easily available species. The results of which include degradation of fisheries resources, consumer losses, undermining of the ecological market, and the adverse effects on human health.

This paper examines the application of the correct commercial and scientific names and the authenticity of 70 Belgian seafood samples using DNA barcoding.  Furthermore, the focus of this research is the optimization of the DNA barcoding method to identify a greater range of seafood species on the Belgian market.

The fresh, frozen and processed seafood samples were purchased from different Belgian supermarkets and retailers. The extracted DNA, using the Spin Column method and the Chelex method, was used for amplification of the mitochondrial COI, cytb and 16S rRNA and the nuclear rhod genes. The obtained PCR products were then loaded on a 1,5 % agarosegel. After electrophoresis the PCR products that result in one band were purified. The purified fragments were then send to Macrogen Europe Laboratories for Sanger sequencing. The resulting sequences are further processed with BioNumerics 7.6 software and analyzed with the BLAST tool in the NCBI database. After identification of the seafood products the usage of the commercial designation and the scientific name on the package was checked using the World Register of Marine Species and the Policy Informative Note: Scientific and trade names for fishery and aquaculture products on the Belgian market.

The DNA barcoding method revealed that the use of 16S rRNA primers yielded the most identifications of fishes, bivalves, crustaceans and cephalopods. The 16S rRNA primers do not always provide identification to species level, which makes the use of other primers necessary. Of the examined seafood samples in this study 52 % contained an invalid or an unacceptable commercial designation or scientific name and 6 % of these species were genetically mislabelled. The retail chains should be supported more so that the last accepted commercial designation or scientific names can be applied. The use of one name per species could provide a solution for better traceability and transparency in the fisheries sector.

Abstract bachelorproef 2015-2016Een vergelijkende studie van variant calling tools voor de identificatie van genomische variatie in Lolium perenne

Perennial ryegrass (Lolium perenne) is an important grass species, utilized as hay, silage and pasture. It’s a plant that has a rapid establishment, high yields, tolerance of grazing and a long growing season. ILVO is strongly involved in ryegrass breeding, but this is still a challenge. L. perenne is an outbreeding species, which makes the genome highly polymorphic. Therefore genetic variation has been identified by sequencing a large collection of individual plants. Because the genome of L. perenne is too large, targeted resequencing was used. For targeted resequencing by probe capture in 746 genotypes, 500 genes known to regulate plant growth and quality were selected. The raw sequencing data was first prepared, by trimming the adapter, mapping the reads on a reference genome, sorting the reads, marking duplicates and ultimately with an InDel realignment.

Variant calling is a process used to identify genetic variation. This process is very hard. Firstly, because different tools give different results, and secondly some forms of variation are very hard to detect correctly. That’s why in this project a comparing study is done for variant calling tools to identify genetic variation in L. perenne.

For the variant calling BCFtools, VarScan, Platypus and GATK were used. Variant calling was ultimately done for 67 genes. For BCFtools, VarScan and Platypus different methods were tested in order to select parameter settings that are best suited for our dataset. Results from best suited methods were then compared. The average variant density over all gene regions was calculated per tool.

Platypus performed the worst, by not even being able to identify variants for 21 genes, where all other tools did. Platypus also found much less SNPs compared to the other tools. BCFtools has the biggest overlap from SNPs with the other tools, but also found ten times more unique SNPs than GATK. This big number can be an indication that BCFtools identified a lot of false positive variants. VarScan has the biggest overlap of InDels with the other tools and identified the lowest number of unique InDels, which may be an indication that this tool is precise when calling InDels. This can be checked in a genome browser, by manually looking for traces of called InDels in the reads. GATK however has the biggest overall variant-density, and the biggest InDel-density.

More research is needed to be able to make a good and reliable variant dataset. Future steps are required, and may involve taking into account variant call quality and genotype call quality and verifying through a genome browser if variants are true- or false-positive.

The world fish consumption per capita has been increasing steadily, from an average of 9.9 kg in the 1960s to 18,5 kg in 2014. The fisheries industry are treating the fish for functional and sensory properties with a number of cheap additives. One of these treatments is to soak the fish or inject them with a saline solution. The uptake of sodium has an influence on the health of the consumer. Scientists found a connection between the uptake of sodium (NaCl) and cardiovascular diseases (CVD). The central question is whether it’s healthy to eat fish that is treated with additives and how many salt the consumer ingests by consuming the meal. Therefore it’s important to take these features into account in order to properly evaluate the daily salt intake to assess how fit the diet is.
The principal company has selected seven species of fishery products that were analyzed. The selected fish are sole (Solea solea), plaice (Pleuronectes platessa), great mediterranean scallop (Pecten jacobaeus), black tiger shrimp (Penaeus monodon), cod (Gadus morhua), Atlantic salmon (Salmo salar) and saithe (Pollachius virens).
To determine which samples are modified by treatment, they were tested on seven factors which are: dry matter (moisture content), pH, conductivity, protein content, sodium percentage , chloride percentage and Aw (water activity).
The three main parameters are dry matter, conductivity and sodium percentage. When fish fillets are injected with a saline solution the dry matter declines and the sodium percentage rises along with the conductivity. When cod filet is treated with a saline solution (1,8%) for 4 hours he achieves a constant value in sodium content by reaching its saturation point. When the consumer consumes 100 grams of the processed fish, he will absorb 1/6 to 1/5 of the daily salt intake. The plaice requires further study to take a conclusion.
Samenvatting eindwerk 2 2014-2015: Determination of microbial diversity on marine sediments through DGGE profiles for investigation of the potential impact of anthropogenic activities
Sand extraction and the disposal of dredged material could have an effect on the microbial communities in the Belgian part of the North Sea (BPNS). The bacterial profiles are evaluated in sediments of sand extraction zones and dredged zones in BPNS. This thesis examines the method based on the clustering of denaturing gradient gel electrophoresis (DGGE) bacterial fingerprints, and is optimized for the use in monitoring.
The Van Veen grab collected 55 sediment samples at different locations on the BPNS. Extracted DNA from the sediments was used for the amplification of the V3 region of the 16S rRNA. The obtained PCR product was loaded on a DGGE gel, which resulted in a DGGE fingerprint with a lot of bands. The program Bionumerics, makes the clustering between different locations.
An important observation is that the bacterial biodiversity is remarkably higher in the autumn compared with the spring. The genetic fingerprints from muddy sediments as well as from sandy sediments showed a high bacterial diversity. The results indicate that sediment characteristics, like grain size and presence of sludge, will influence the  bacterial communities. The proposed method is certainly useful, but it should be further optimized in the future, for example to identify environment specific bacterial groups and indicator bacteria.. Bacterial species or families could also be identified by using NGS (next generation sequencing). The results of this thesis, recommend a PCR-DGGE method combined with metagenomics (using NGS, for the sequence of many bacteria) on all the sediments. In this way, it’s possible to collect all the necessary information for monitoring based on bacterial communities. Although a lot is known about the used methods, still there are unanswered questions. So it stays a challenge to gather more knowledge about the techniques by scientific investigation and practice-oriented experience.
Samenvatting eindwerk 3 2014-2015: Equation of microbiological, chemic and sensory quality of plaice (Pleuronectes platessa) after storage on flake ice and slurry ice
Worldwide, different kind of methods are used for conservation of fresh fish. The purpose of this experiment was to determine the quality of fresh plaice (Pleuronectes platessa), stored in two different icing techniques, the classic flake ice method and slurry ice method. The goal of this project was to examine if these cooling techniques have an influence of the shelf life of the fish. These techniques were evaluated and analyzed by sensory, microbiological and chemical methods.
Slurry ice is reported for being a promising technique in the storage of aquatic food products. The main characteristic of slurry ice is store the material at a temperature slightly below 0°C, this method is a faster cool technique than classic flake ice. This because of the microscopic particles that have a spherical shape which are typical for the slurry ice which covers the entire surface of the material. This protects the fish from the action of oxygen. (Losada, Rodríguez, Miranda, Barros-Velázquez, & Aubourg, 2006)
The Quality Index Method was used for the sensory evaluation, this method is used to determine the quality of plaice. Evaluation is done by using a demerit score- system (0-3) of different parts of plaice (gill, skin, eyes, …). The scores for all attributes are added to give an overall sensory score, the so-called quality index. The quality index increases linearly with the storage ice. Therefore the total demerit score can also be used to predict the remaining shelf life. (QIM Eurofish, 2008)
In the microbial analyse , the concentration of Pseudomonas, Shewanella and other psychrotrophic marine count systems was evaluated by using Pseudomonas Agar Base and Lyngby Iron Agar. The biochemical breakdown, what cause these bacteria in fish, is evaluated by a chemical analysis. For the chemical analyse, the steam destillation was used to determine the concentration of different kinds of breakdown components (total volatile basic nitrogen (TVB-N) and trimethylamine). An Ultra High Pressure (Performance) Liquid Chromatography (UHPLC) was used for the determination of biogenic amine .
The obtained results during this experiment are different from the results found in literature. The obtained values exhibit no or hardly any difference between the two conservation methods. During storage, an increase is noticed in the score that was obtained in the sensory analysis. At the end of the experiment, the score reached the maximum of 24. There was a marked increase in the microbiological count of Pseudomonas spp., Shewanella colonies and total bacterial counts. At the start of the experiment, there was only a concentration measured of Pseudomonas. There was a clear zone of growth of these bacteria. There was no concentration measured of Shewanella.
The concentration of the total volatile bases increased remarkably at the end of the experiment. This also applies for the concentration of TMA, which increased after day eleven of the storage process. After the procedures with the UHPLC, it became clear that very low levels of biogenic amines are represented in plaice during the storage process. These concentrations are usually measured under the detection limit.
Finally, there can be inferred that trimethylamine is formed in fish by Shewanella colonies. The concentration of Shewanella rises at day eleven, while there is a clear increasing observed at the level of trimethylamine in plaice. Pseudomonas spp. forms no trimethylamine but ketones, aldehydes and esters. For both the sensory, microbiological and chemical analysis, there are no differences detected between the various conservation methods. It is recommended to repeat the experiment during summer, since the body temperature of the fish is higher. Therefore the fast cooling effect of slurry ice might play a more important role in prolonging shelf life.
Losada, V., Rodríguez, Ó., Miranda, J. M., Barros-Velázquez, J., & Aubourg, S. P. (2006).  Development of different damage pathways in Norway lobster (Nephrops norvegicus) stored under different chilling systems. Journal of the Science of Food and Agriculture, 86(10), 1552–1558.
QIM Eurofish (2008). The principle of QIM. Geraadpleegd 18 mei 2015, van
Samenvatting eindwerk 1 2013-2014: Development of a microsatellite panel for paternity analysis in soy-breeding
There are a lot of reasons to introduce soy in Flanders. The biggest problem here is the climate. Soy needs a short photoperiod and simply can’t grow in every part of the world. That’s why the people of the Institute for Agricultural and Fisheries Research started a breeding program to develop races which are suitable for growing in Belgium. Because the growing season is shorter here, this country needs a race growing fast and is ready to harvest earlier. Last year, a first selection was made. However, it’s only this year that the development of a new race really starts.
This thesis focusses now on the molecular aspect to see if the crosses are successful. Because without a cross, no new race can be made. This research occurs by doing a paternity analysis using microsatellites, little pieces of genetic material that can be compared between the parents and the offspring.
First, a DNA-extraction is necessary. Unfortunately, there is no easy protocol available so different methods were tested. After this step, the microsatellites can be easily detected using Polymerase Chain Reaction and capillary electrophoresis. The primers must be chosen correctly to create a good microsatellite-panel that can be used in the paternity analysis. To save some money, a new PCR-technique, in which all of the microsatellites can be detected with a single labeled primer, was evaluated.
9 microsatellites were selected to test on the parents and the offspring. Only 19 of the 128 crosses (15%) were certainly cross-fertilizations.
Samenvatting eindwerk 2 2013-2014: Tube worm reefs as feeding grounds: genetic analysis of wader faeces
Intertidal areas are known to attract massive amounts of waders and other birds by providing resting area, space for breeding and feeding grounds. The latter is increasingly important in mega-tidal beaches such as in the Bay of Mont Saint-Michel. The research line proposed here is about waders of this particular area. In the bay there are Lanice conchilega reefs that are biologically very important: many species tend to associate in high densities with Lanice reefs. The research focus on determing the prey DNA in the wader faeces. By means of genetic analysis of faeces available in ethanol and prey DNA available in formol/ethanol samples. This study will investigate three methods of DNA extraction: Invisorb tissue mini kit from Stratec, Wizard Genomic DNA Purification Protocol and CTAB method. After the DNA extraction there will be a PCR using universal Cytochrome oxidase subunit 1 Primers and the samples of prey and faeces will be analyzed by an agarose gel electrophoresis. After the electrophoresis the COI fragment (~700bp) will be cut out and purified using a gel extraction kit. The samples will undergo a new PCR which contains a DGGE primer so the GC clamp will be added to the DNA. The PCR extract will be purified and loaded on a Denaturing gradient gel electrophoresis. Using DNA fingerprinting of the faeces samples with the prey samples we can identify which prey the wader bird had eaten. This information is important because both birds and reef building organism receive international attention in the framework of marine conservation and their interaction is in great interest of the management of beaches. After testing the three methods we came to the conclusion that for the DNA extraction of faeces the Invisorb tissue mini kit from Stratec didn’t work to extract faecal DNA. The Wizard Genomic DNA Purification Protocol  wasn’t optimal too extract DNA but it is possible with a pretreatment of the faeces. The CTAB method wasn’t optimal either but had the best results of the three tested methods. Using DNA fingerprinting to investigate which prey the wader had eaten is possible but the DNA extractions were to impure to be sequenced and therefore we can’t form a conclusion of which prey the wader had eaten.
Samenvatting eindwerk 3 2013-2014: Determination of microbial diversity on marine sediments using DGGE fingerprinting
The increase of anthropogenic activities such as aggregate extraction and the discharge of dredging material at the Belgian Part of the North Sea (BPNS) have an effect on the health status of the marine ecosystem. Based on molecular biotechnology techniques sediment samples are investigated to assess possible impact of anthropogenic activities. The microbial diversity on this sediment is examined using DGGE fingerprinting. These profiles are compiled for reference sites and will be used in the future for routine monitoring.
Sediment samples were collected with the ‘Van Veen’ grab on board of the research vessel (RV) ‘Simon Stevin’. After the storage of the samples at -80°C, a DNA extraction will be performed. The microbial DNA of the several sediment samples at different points is amplified with various PCR-techniques. In general, nested PCR provides a higher DNA yield and purity than the conventional and Touchdown PCR. These PCR products are loaded on DGGE using an optimized protocol, which results in a clear DGGE fingerprint. The punched bands are sequenced and are leading to the ability to identify the microbial communities. Furthermore, the influence of the different types of sediment (mud and sand) and the locations on the Belgian Part of the North Sea are also being evaluated.
The DGGE fingerprinting method revealed a higher bacterial diversity on mud compared to sand and the differences between the two sandbanks are remarkably lower than the differences between sand and mud. The results confirm that the used PCR-technique has a clear influence on the finale fingerprint, so it must be stressed that a standard protocol for monitoring is important. A method for evaluating the bacterial diversity in sediment based on DGGE profiles is presented and draft methods for Archaea and protists are suggested. This method is useful for the preparation of microbial DGGE profiles and the future assessment of anthropogenic impact on the BPNS.
Samenvatting eindwerk 1 2012-2013: Microbiologische grondstof –en omgevingsanalyses in droge en natte procesomgeving in levensmiddelenbedrijven
Deze bachelorproef omvat een oriënterende studie in verschillende levensmiddelenbedrijven. Enerzijds wordt de droge procesomgeving van zoetwarenbedrijven onderzocht, anderzijds de natte procesomgeving van garnaal –en visverwerkende bedrijven. Op zich hebben deze twee projecten niets met elkaar te maken maar de bedrijven hebben wel het zelfde doel: de houdbaarheid van hun producten verlengen voor de consument.
In de zoetwarenbedrijven heerst er een problematiek rond xerofiele schimmels. Deze kunnen namelijk voor bederf zorgen bij producten met een lage aw-waarde, waar de groei van bacteriën geen probleem is. Om de export naar overzeese gebieden te doen stijgen wil men dat de producten bij aankomst nog lang genoeg houdbaar blijven. In vele oosterse landen is dit een probleem omdat men alcohol in onder andere pralines grotendeels verbied. Dit zorgt ervoor dat een antimicrobieel product verdwijnt en de houdbaarheid ingeperkt wordt. Ook heeft de consument liever geen additieven in hun pralines. Om die redenen wil men de initiële microbiota in de producten beperken en onderzoekt men de mogelijke contaminatiebronnen.
Grondstoffen (fruitpasta, noten, ganache en suiker) werden uit zes zoetwarenbedrijven verzameld om te onderzoeken welke het meest bijdragen tot contaminatie. De grondstoffen werden uitgeplaat op DG-18 en MYA50G gedurende drie weken op 21°C om de groei van xerofiele schimmels mogelijk te maken. Vooral de noten bleken hier het grootste probleem te zijn. Met dezelfde voedingsmedia werden in één van de bedrijven luchtstalen genomen in drie verschillende ruimtes en oppervlaktes geanalyseerd op de aanwezigheid van schimmels via rodacplaatjes.
Garnaal –en visverwerkende bedrijven hebben geen schimmels, maar wel bacteriën als boosdoener. Consumenten willen de garnalen of vis zo lang mogelijk kunnen bewaren met zo weinig mogelijk additieven toegevoegd. Reiniging en desinfectie spelen in deze bedrijven een veel grotere rol dan in de zoetwarenbedrijven. Het effect hiervan wordt onderzocht aan de hand van swabs die men voor en na reiniging en desinfectie neemt van verschillende oppervlakken uit de procesomgeving. Vervolgens worden de swabs uitgeplaat op een aantal selectieve en niet-selectieve voedingsmedia: PCA, MA, VRBGA en MRS. Dit deel van het onderzoek leed tot heel uiteenlopende resultaten die voor verscheidene hypothesen zorgen. Concrete besluiten kunnen pas na enkele herhalingen getrokken worden. Naast de omgevingsanalyses werden er ook ongepelde en machinaal gepelde garnalen vergeleken en met de hand op steriele wijze, ontvelde vis en machinaal ontvelde vis. Dit gebeurde aan de hand van dezelfde voedingsmedia gebruikt bij de swabs plus IA. In beide gevallen bleek dat de machines een bijdrage hebben tot de contaminatie van deze levensmiddelen.
Samenvatting eindwerk 2 2012-2013: Moleculaire identificatie van de dominante bederfbacteriën bij langoustines(Nephrops norvegicus)
Langoustines zijn kreeftachtigen die vooral gevangen worden in de Noordzee. Tot in de jaren ’80 kenden we in Vlaanderen een bloeiende langoustinevisserij. Deze vloot is nagenoeg helemaal verdwenen omdat de focus bij de modernisering van de vissersvloot op de grote boomkorvisserij lag. De aanvoer van langoustines is sindsdien sterk gedaald. Om de langoustinevisserij nieuw leven in te blazen, werd met de financiële steun van de Vlaamse overheid het OOLAVIS (Oostendse Langoustine Visserij) project opgestart door onder andere ILVO-visserij, de Rederscentrale en VLAM. Dit project heeft als doel de kopers, verwerkers en handelaars opnieuw commercieel te motiveren door de hernieuwde en regelmatige aanvoer van langoustines van topkwaliteit in de Vlaamse visveiling.
Langoustines zijn net zoals andere schaaldieren zeer gevoelig voor bederf. Het is belangrijk om de microflora en specifieke bederforganismen (SBO’s) van langoustines in kaart te brengen. Vooral SBO’s zijn van belang omdat deze bacteriën kunnen doorgroeien tijdens de gekoelde bewaring en verantwoordelijk zijn voor bederf.
Langoustines worden op twee manieren aangevoerd in de Vlaamse visveiling: op hun geheel of als langoustinestaarten. Tijdens deze bachelorproef werd nagegaan of er een verschil is in houdbaarheid tussen volledige langoustines en langoustinestaarten op ijs. Om dit verschil te onderzoeken werd het totaal aëroob kiemgetalgedurende 14 dagen opgevolgd op mariene agar en plate count agar (PCA). Het einde van de houdbaarheid, op basis van de bepaling van het kiemgetal op PCA, werd bereikt na 11 dagen voor langoustinestaarten en na 13 dagen voor volledige langoustines.
Om te weten te komen welke micro-organismen er verantwoordelijk zijn voor het bederf bij langoustines werden bacteriën geïdentificeerd aan de hand van moleculaire technieken. Daartoe werden individuele kolonies opgepikt van de platen waarop het totale kiemgetal bepaald werd, gevolgd door PCR en sequenering van het hypervariabele v3-gebied van het 16S-rRNA gen. Er werd eveneens een plate-swab genomen met een steriel entoog, voor het uitvoeren van PCR-DGGE. Deze DGGE werd ook rechtstreeks uitgevoerd op het staal, waarbij de tussenstap van het opkweken van de micro-organismen vermeden werd.
Uit de resultatenkunnen we afleiden dat de dominante microflora vooral bestaat uit de genera Pseudoalteromonas, Pseudomonas en Psychrobacter.
Dit werk is een eerste aanzet tot het bepalen van de bederfbacteriën op langoustines. In de toekomst kan verder ingezet worden op de identificatie van de bacteriën tot op species-niveau. Ook kunnen andere voedingsbodems getest worden en verschillende (sensorische en chemische) kwaliteitsparameters opgenomen worden in het onderzoek op langoustines.
Samenvatting eindwerk 3 2012-2013: Identificatie van de bacteriële gemeenschappen op marien plastic afval
Het dagelijks gebruik van plastic in onze maatschappij blijft toenemen terwijl deze synthetische polymeren nauwelijks degraderen. Daarom is het belangrijk de problematiek rond plastic zwerfvuil in het marien milieu in kaart te brengen. Zeer actueel zijn de trieste foto’s van door plastic afval verstrikte, verwonde, gestikte of uitgehongerde zeedieren. Dieper onderzoek is echter noodzakelijk om ook de impact van de kleinere plastic partikels zoals microplastics en nanoplastics te kunnen evalueren. Deze partikels kunnen opgenomen worden door mariene organismen zoals de mossel waarna ze kunnen accumuleren in het weefsel. Persistente polluenten kunnen accumuleren op deze microplastics en een bijkomende impact uitoefenen op het mariene ecosysteem. Plastic afval en microplastics blijken eveneens als habitat te fungeren voor tal van mariene micro-organismen. Uit onderzoek blijkt dat sommige mariene bacteriën zelfs in staat zouden zijn de synthetische polymeren te biodegraderen. Hoe dit gebeurt en welke effecten dit zal hebben op het ecosysteem is echter nog een groot vraagteken.
Aangezien mariene bacteriën zeer moeilijk te cultiveren zijn, bestaat er een grote interesse naar het isoleren, cultiveren en identificeren van mariene bacteriën. In het kader van de Europese onderzoeksprojecten MICRO (InterReg 2 Zeeën) en CleanSea (7KP) wordt onderzoek verricht naar de identificatie van bacteriële gemeenschappen en bacteriële biodegradatie van marien plastic afval. Dit eindwerk behandelt verschillende methodes voor de identificatie van bacteriële gemeenschappen op microplastics, plastic strandafval en marien plastic zwerfvuil. Vervolgens wordt er nagegaan met een microkosmos experiment welke opportunistische bacteriën zich preferentieel op het plastic zullen hechten. Er zal gebruik gemaakt worden van zowel microbiologische als moleculair biotechnologische technieken om de isolatie, cultivatie en identificatie van mariene bacteriën te verwezenlijken.
Hiervoor worden verschillende opwerkingsmedia, voedingsbodems, incubatietijden en -temperaturen uitgetest. Na optimalisatie wordt besloten gebruik te maken van een isolatie in Maximum Recovery Diluent (MRD) met behulp van een vortex-methode en een cultivatie op Marine Agar (MA) en Plate Count Agar (PCA). Gegroeide kolonies worden vervolgens op twee manieren opgewerkt. In de eerste methode wordt een kolonie PCR uitgevoerd waarbij gescheiden kolonies aangeprikt worden en overgebracht worden in PCR mix. In de tweede methode worden volledige swabs afgenomen van de begroeide platen. Het geëxtraheerde DNA van de swabs wordt vervolgens geamplificeerd en gescheiden via denaturerende gradiënt gelelektroforese (DGGE). Enkele testen werden uitgevoerd waarbij DNA rechtstreeks zonder cultivatie geëxtraheerd werd uit de stalen met behulp van sonificatie of Retsch methode. Voor de identificatie wordt gebruik gemaakt van een amplificatie van de V3 regio op het 16S fragment. Opgezuiverde PCR fragmenten werden tot slot verzonden naar Macrogen waar ze gesequeneerd werden. Binnen de 24 uur waren de resultaten beschikbaar zijn voor een verdere data analyse. De verkregen sequenties werden vervolgens verwerkt met de gepaste software en geanalyseerd in de databank NCBI BLAST.
Het microkosmos experiment werd uitgevoerd in glazen 500ml bokalen die gevuld worden met zeewater of sediment. Hierin werden vervolgens bacterievrije plastic pellets of synthetische vezels gebracht. Gedurende 1 maand werden deze plastic stukjes blootgesteld aan zeewater of sediment, waarbij 4 bemonsteringen plaatsvinden voor de identificatie van aanwezige bacteriën op het plastic.
Voor de cultivatie van mariene bacteriën blijkt MA het beste medium. Hierop werden 20 verschillende species geïdentificeerd terwijl er op PCA slechts 11 verschillende werden aangetroffen.
De geïdentificeerde bacteriën op het plastic afval behoren voornamelijk tot de klassen van de Gammaproteobacteria, Actinobacteria, Bacilli en Flavobacteria. Vele aangetroffen bacteriën zoals Pseudoalteromonas sp., Acinetobacter sp., Alteromonas sp., Vibrio sp. en Psychrobacter sp. kunnen verwacht worden in een aquatisch milieu. Wat opvalt, is dat een aantal bacteriën, behorend tot de klasse van de Actinobacteria, enkel aangetroffen werden op de preproductie pellets van het strand, terwijl ze niet op het strandafval noch op het afval uit zee gevonden werden. Op marien afval werden enkel bacteriën geïdentificeerd die behoren tot de klasse van de Gammaproteobacteria. Enkele mariene soorten zoals Vibrio sp. en Shewanella sp. werden zelfs enkel teruggevonden op het plastic uit zee en kunnen niet in verband gebracht worden met het strandafval.
Uit het microkosmos experiment blijkt dat bacteriën uit het zeewater in staat zijn om het plastic te koloniseren. Bacteriën zoals Shewanella sp., Pseudoalteromonas sp. en Colwellia sp. werden geïdentificeerd als opportunistische soorten.
Dit eindwerk draagt bij tot de studie van de microbiële biodiversiteit en karakterisatie van het marien milieu, wat relevant is voor ondermeer de evaluatie van de zwemwaterkwaliteit en toepassingen binnen de aquacultuur sector. Dit eindwerk vormt een eerste stap in het onderzoek naar de topic bacteriële biodegradatie van marien plastic zwerfvuil.
Samenvatting eindwerk 1 2011-2012: Moleculaire identificatie van bederfbacteriën in grijze Noordzeegarnalen (Crangon crangon) gekookt en bewaard onder verschillende condities
Grijze Noordzeegarnalen (Crangon crangon) zijn vier tot negen centimeter groot en hebben een doorzichtige, grijze zandkleur. Ze komen voor in de Noordzee, maar ook in andere zeeën van het Noordelijk halfrond. De garnalen worden gevangen met behulp van boomkorren en daarna worden ze aan boord gesorteerd en gekookt. De grijze Noordzeegarnalen, gevangen door Belgische vissers, worden meestal zonder bewaarmiddelen verkocht onder het kwaliteitslabel ‘Purus’. Aan de garnalen gevangen door Nederlandse vissers worden wel bewaarmiddelen toegevoegd, omdat deze na de vangst gepeld worden in Marokko. De meest gebruikte bewaarmiddelen zijn benzoëzuur en sorbinezuur. Garnalen worden meestal MAP verpakt om een langere houdbaarheid te creëren.
Het bederfproces kan onderverdeeld worden in drie stadia namelijk autolyse, bacterieel bederf en oxidatieve ranzigheid. Pseudomonas en Brochothrix thermosphacta zijn de belangrijkste bedervers. Deze bacteriën kunnen het enzym TMAO-reductase produceren, waardoor TMAO omgezet kan worden naar TMA. Daarna vindt de vorming van ammoniak en aminen plaats, waardoor een onaangename geur en smaak verkregen wordt.
Om de houdbaarheid van de garnalen na te gaan, wordt er geïncubeerd op een aantal standaardmedia (LH, IA, MA en PCA). Na incuberen bij verschillende temperaturen wordt er besloten om drie dagen incubatie op 21°C als standaardmethode te gebruiken. Het gebruik van selectieve media zorgt ervoor dat er een beeld kan gevormd worden van de bederfveroorzakende bacteriën. Pseudomonas is de belangrijkste bederver bij Purus garnalen, terwijl Brochothrix thermospacta een dominante bacterie is bij MAP-verpakte garnalen. Wanneer garnalen gekookt worden in het labo kan de houdbaarheid verlengd worden. Dit wordt enerzijds veroorzaakt door het mijden van zeewater als koelmiddel en anderzijds wordt er in het labo gebruik gemaakt van steriele zakken in plaats van de licht gecontamineerde bakken in de visveiling. De microbiële populatie is eveneens anders, want na twintig dagen wordt een zuivere cultuur van Bacillus verkregen.
Ten slotte worden nog een aantal bacteriën aanwezig op MAP-verpakte garnalen en levende garnalen moleculair onderzocht, want biochemische testen, zoals Api®, zijn vaak niet gevoelig genoeg voor de identificatie van mariene micro-organismen. Via de software van BioNumerics wordt de fingerprint van elke uitgezuiverde kolonie geanalyseerd en indien mogelijk, geclusterd met andere bandenpatronen. De fingerprint wordt eveneens vergeleken met een aantal typestammen. Er kan echter geen bandenpatroon gegroepeerd worden met dat van de typestammen, waardoor sequeneren noodzakelijk wordt. Er is wel een goede clustervorming tussen een aantal fingerprints afkomstig van levende garnalen. Hierdoor kan het aantal te sequeneren kolonies sterk gereduceerd worden. Het sequeneren van de kolonies werd niet meer uitgevoerd.
Samenvatting eindwerk 2 2011-2012: Impact en effect van microplastics op verschillende epibenthos soorten
Het is algemeen geweten dat het plastic tijdperk zware sporen nalaat in oceanen, zeeën en kustzones. Aangezien het gebruik van plastic blijft toenemen en plastic amper degradeert, is het van belang deze problematiek in het marien milieu in kaart te brengen en om de effecten ervan te bestuderen. Gevolgen zoals verstikking, verstrikking, verwonding en uithongering van zeedieren en vogels door plasticafval is een gekend fenomeen, maar de impact van kleine plastic partikels, zoals microplastics en nanoplastics, op het marien ecosysteem vormt nog een groot vraagteken. Zoals al aangetoond werd kunnen microplastics opgenomen worden door mariene organismen zoals de mossel, de zeekomkommer, de wadpier en de strandvlo. Naast eventuele effecten op de spijsverteringsorganen, bestaat de mogelijkheid dat deze partikels translocatie en accumulatie ondergaan in de weefsels. Daarnaast werd al gedemonstreerd dat microplastics schadelijke stoffen uit het marien milieu kunnen adsorberen, waardoor deze toxische componenten kunnen opstapelen met gevolgen voor de ganse voedselketen. Maar ook de additieven, toegevoegd tijdens de productie van plastics, kunnen een gevaar vormen wanneer deze vanuit het plastic in het water of een het organisme emigreren. Deze studie omvat drie grote delen. Een eerste luik omvat de blootstelling van mariene organismen aan microplastics. In een volgend deel wordt sediment onderzocht op de aanwezigheid van microplastics en tot slot wordt er gezocht naar een methode voor het koppelen van huishoudgenen aan pollutie gerelateerde genen.
Om de problematiek van deze microplastics in het Belgisch deel van de Noordzee in kaart te brengen worden microplastics gemonitord in het sediment, de waterkolom en mariene organismen. Verder moet er ook onderzoek verricht worden naar de opname van microplastics door mariene organismen.
In dit eindwerk worden verschillende locaties in het Belgisch deel van de Noordzee, zoals de havengeul van Zeebrugge en de zandbanken Kwintebank, Buitenratel en Oostdyck, gemonitord op microplastics in het sediment. Daarnaast worden ook enkele stalen op het strand van Oostende onderzocht. Hiervoor wordt gebruik gemaakt van het verschil in dichtheid tussen de sedimentkorrels en de microplastics. Uit de onderzochte sedimentstalen kan besloten worden dat microplastics abundant aanwezig zijn in het Belgisch deel van de Noordzee. Ook het onderzoek van enkele wijtingmagen op microplastics illustreert het overvloedig voorkomen van microplastics in de waterkolom in ons marien milieu.
Aan de hand van blootstellingexperimenten met sferische polyethyleen microplastics van 100 µm, 250.µm en 500 µm diameter werd nagegaan of microplastics opgenomen kunnen worden door de Noordzeekrab en de gewone zeester. De experimenten hebben aangetoond dat de microplastics in eerste instantie opgenomen worden door de Noordzeekrab, maar vervolgens ook weer uitgescheiden worden. De microplastics blijken duidelijk te groot voor translocatie naar de weefsels. Bij de zeesterren kon geen inname van deze microplastics waargenomen worden.
Om de biologische effecten, veroorzaakt door microplastics of hun chemische belasting van microplastics te kunnen kwantificeren, werd geopteerd om te werken met genetische biomerkers. Uit voorgaande studies is immers gebleken dat er heel wat genen zijn waarvan de regulatie gekoppeld is aan de effecten (of de metabolisatie) van chemische contaminanten. Voor Asterias rubens zijn tot op heden weinig genetische gegevens beschikbaar. Binnen dit eindwerk werd van start gegaan met het opstellen van een kwantitatieve PCR (real-time PCR) protocol met de doelstelling dit later te kunnen gebruiken voor experimentele doeleinden. Ovaria, maag en darmen van Asterias rubens werden geselecteerd als doelorganen en hierop werden verschillende commerciële kits getest voor DNA, RNA en cDNA bereiding. De resultaten met DNA toonden aan dat het primerkoppel voor de amplificatie van het mitochondriaal DNA cytochroom b goed werkt en bruikbaar is om populaties van zeester te identificeren. Op cDNA werden primerkoppels getest voor vier huishoudgenen. Klassieke PCR amplificatie op deze referentiegenen werd gebruikt voor het bepalen van de invloed van de annealing temperatuur en voor de sequentie controle van het bekomen fragment. Real-time PCR op de referentiegenen werd gebruikt om de praktische bruikbaarheid van deze genen na te gaan in een qPCR protocol. De resultaten toonden aan dat de 18S rRNA merker goed bruikbaar is voor verder onderzoek en dat voor de andere drie referentiegenen nog optimalisatie nodig is. Wegens tijdsgebrek konden geen experimenten worden uitgevoerd met pollutie-gerelateerde biomerker genen.
Binnenkort gaat het project MICRO van start, waarbij ILVO projectleider is. Enkele van de geplande acties zijn het in kaart brengen van plasticvervuiling door microplastics op basis van monitoring en modellen, identificeren van plastic hot-spots, impact/effect op mariene biota, blootstelling van oesters en mosselen aan microplastics, nagaan of microbiële degradatie mogelijk is, … Er valt in de toekomst zeker nog heel wat te onderzoeken rond de problematiek.


Ankerstraat 1
Ankerstraat 1
8400 Oostende


Traineeship supervisor
Johan Robbens
Sebastian Uhlmann
Mike van 't Land
Sofie Derycke
Bavo Dewitte
Via Map