Reports

Research on thermophilic microorganisms in high-temperature areas in Reykjanes, Hengill area and Fremrinámur. Prepared for Expert Group 1 in Framework Plan 3 / Thermophilic microorganisms from geothermal areas at Reykjanes, Hengill and Fremrinámar.

Published:

30/05/2016

Authors:

Edda Olgudóttir, Sólveig K. Pétursdóttir

Supported by:

Framework Program 3 (RÁ3)

Research on thermophilic microorganisms in high-temperature areas in Reykjanes, Hengill area and Fremrinámur. Prepared for Expert Group 1 in Framework Plan 3 / Thermophilic microorganisms from geothermal areas at Reykjanes, Hengill and Fremrinámar.

The current study was carried out under the auspices of Framework Plan 3 and included high-temperature areas that declined in the waiting category in RÁ2. The areas were Trölladyngja and Austurengjahver in Reykjanes, Fremrinámar and Þverárdalur and Innstidalur in the Hengill area. The aim of the study was to assess the diversity and rarity of thermophilic microorganisms in the above areas. The methodology was based on DNA analysis independent of culture. DNA was isolated from samples and species-identifying genes amplified and sequenced and the sequences compared to sequences in gene banks and from previous studies. A total of 118 samples were collected in 2015 and were able to be sequenced approx. Their 59%. A total of 10 million 16S gene sequences were obtained from sequencing, which dropped to almost six million after all quality and length conditions had been met. Most rows were obtained from Innstadalur, or 2,176,174, but fewest 286,039 from Trölladyngja. The diversity of microorganisms in each region was assessed by comparing the number of arrays, number of species, collection curves, and Shannon's diversity coefficient. The total number of samples and sequences of each area was very different and reflect its diversity. When the series were classified as species with 97% matching, it was found that most species came from Þverárdalur, or about 42 thousand, but the fewest from Trölladyngja, or about 9 thousand. More than 12 thousand species were found in samples from Fremrinámur, which came as a surprise as the area and the samples seemed homogeneous and such diversity was not expected. All major thermal bacteria were found in the samples, both Ancient Bacteria and Bacteria. Special groups were found especially within the Thaumarchaeota faction. The assessment of diversity in individual areas with collection curves and diversity coefficient was based on the smallest number of rows or 286,039 from Trölladyngja. The greatest diversity of species was in Þverárdalur and Innstadalur and the least at Trölladyngja and Austurengjahver, but Fremrinámar was in between. Collection curves gave the same result. Biodiversity (H) gave a different ranking. There were Þverárdalur and Fremrinámar with the most diversity (H = 8 and 7.7), then Innstidalur but Trölladyngja and Austurengjahver (H = 6) ran the train. Rarity was assessed on the basis of the number and proportion of unknown species in comparison with the Silva database. The number of unknown species was greatest in Þverárdalur and Innstadalur, both with over 1000, Fremrinámar with 756 and Trölladyngja and Austurengjahver with over four hundred unknown species. National rarity was assessed by comparing data from individual regions in previous studies and examining whether a match occurred. It turned out that the geothermal area in Fremrinámur contains a large number of species that have no equivalent in other thermal areas.

The current project was requested by the Master Plan for Nature Protection and Energy Utilization and aimed at geothermal areas which had not been classified for preservation or utilization during Masterplan 2. The geothermal areas investigated were Trölladyngja and Austurengjahver at Reykjanes, Fremrinámar and Þverárdalur and Innstidalur at Hengill. The goal of the project was to estimate biodiversity and rarity of thermophiles inhabiting the areas mentioned. The methods used were DNA based and were performed on DNA extracted from primary samples (culture independent). Microbial species identification was performed by amplification and sequencing of 16S rRNA genes and comparison with sequence databases and previous research. A total of 118 samples were collected in 2015 of which 59% were sequenced. The total sequencing yield was 10 million reads, of which 6 million passed quality assessment and were used for downstream analyzes. The largest proportion of the reads were obtained from Innstidalur samples, 2,176,174 reads, and the lowest proportion from Trölladyngja, 286,036 reads. The biodiversity of microorganisms within each area was estimated using the number of phyla and species, rarefaction curves and Shannons' biodiversity index. The total number of species identified varied between sites and reflected the diversity of the geothermal area and the total amount of sequences obtained. Using a cut-off value of 97% similarity, the sequences were classified to the species level. The highest number of species, approximately 42.000, were identified in samples from Þverárdalur and the lowest number, approximately 9.000, in samples from Trölladyngja. Roughly 12,000 species were found in samples from Fremrinámar, which was surprising as the area and the samples appeared rather homogenous and such diversity was therefore not expected. All the main thermophilic taxa of the Bacteria and Archaea domains were identified in the samples. Unknown groups were found especially within the phylum of Thaumarchaeota. For the rarefaction and biodiversity index estimates the lowest number of sequence reads, Trölladyngja, was used as reference. The species diversity was found to be highest in Þverárdalur and Innstidalur, the lowest in Trölladyngja and Austurengjahver, and intermediate in Fremrinámar. Rarefaction curves showed the same results. The calculated biodiversity index (H) gave different results, indicating highest diversity in Þverárdalur and Fremrinámar (H = 8 and 7,7 respectively), intermediate in Innstidalur (H = 7.0) and lowest in Trölladyngja and Austurengjahver (H = 6). Rarity was estimated as the number of species which could not be identified by comparison to the Silva database. The highest number of unidentified species was roughly 1000 in Þverárdalur and Innstidalur, 756 in Fremrinámar and between 300- 400 in Trölladyngja and Austurengjahver. The rarity was also estimated by comparing data obtained in the current project with data from previous projects. The analysis revealed a particularly high number of unique species in Fremrinámar that have not been identified in other geothermal areas in Iceland.

View report

Reports

Microbial diversity in the Icelandic fishing grounds

Published:

01/12/2011

Authors:

Eyjólfur Reynisson, Sveinn Haukur Magnússon, Árni Rafn Rúnarsson, Kristinn Guðmundsson, Erla Björk Örnólfsdóttir, Viggó Þór Marteinsson

Supported by:

Fisheries Project Fund

Contact

Viggó Marteinsson

Research Group Leader

viggo.th.marteinsson@matis.is

Microbial diversity in the Icelandic fishing grounds

The diversity of microorganisms in the ocean around Iceland is largely unknown, but little or no research has been done to date. In this study, samples of the sea around the country were collected for analysis of microbial concentrations by flow cytometry and their diversity using molecular biological methods. A total of 504 sea samples were collected; 483 samples from the MRI's spring rally, 16 samples from Breiðfjörður and 5 samples were collected north of the Westfjords with specially equipped bottom trawling equipment. From the spring rally, selected samples were further investigated, they came from Selvogsbanki, Siglunes and Langanes as well as each sample west of Látrabjarg and Hornbanki. The microbial concentration was highest in Selvogsbanki, where the total count was about 1.6 million cells / mL of seawater. However, the calculated mean of all samples was 0.68 million cells / mL. The species composition of the microflora was determined by amplification and sequencing of the 16S bacterial gene. A total of 528 sequences were sequenced showing 174 different 16S bacterial sequences in the samples and 52% were found to belong to previously unknown bacterial species. The diversity of the microbial flora was generally high, with the exception of trawl tails. Eight batches of bacteria were detected in the samples to varying degrees. Cyanobacteria and Cyanobacteria-like sequences were predominant in all samples except for the 353-0m sample at Selvogsbanki, where their proportion was only 4%. Alpha and Gamma ‐ protebacteria predominated there. Of the other groups, Flavobacteria was regularly diagnosed together with other groups in a smaller proportion. Differences in the microbial composition of marine samples were assessed using a key factor analysis of the fingerprints of microbial communities obtained using t-RFLP technology. Overall, the main difference was in samples from each sample cycle, ie. from vorralli, Breiðafjörður and the tail of a trawler. The variability within the samples from the spring rally went hand in hand with the sampling point where samples from Selvogsbanki showed greater mutual similarity compared to the samples north of Siglufjörður and west of Iceland which were more different from each other. Data from the fingerprints of microbial communities in different samples showed a correlation between certain variables in the fingerprints of the communities with environmental factors that were measured during the sampling. Temperature had the most to say but also fluorescence and salinity. Vibrio was screened. Paramaemolyticus in warm seawater samples from the south of the country where the sea surface temperature was between 8-10 ° C. No V. parahaemolyticus was detected in these samples. The results of this project can be regarded as an important basis for further research on microorganisms in Icelandic waters. Ongoing research in this field can be of great significance in the long term to assess the impact of foreseeable environmental changes due to global warming on marine life and fish stocks in Icelandic waters. 

The diversity of microorganisms in the ocean around Iceland is largely unknown and little or no research has been conducted to date. In this study, seawater samples around the country were gathered for analysis concentration and diversity of microorganisms using flow ‐ cytometry and molecular methods. A total of 504 samples were collected. All samples were analyzed with regards to microbial counts while samples from selected areas were investigated further, from Selvogsbanki, Siglunes and Langanes. Microbial concentrations were highest at Selvogsbanki, where the total counts were around 1.6 million cells / ml. Arithmetic mean of all samples was o.68 million cells / ml. Species composition of microbial flora was determined by amplification and sequencing of the 16S bacterial gene. A total of 528 16S sequences were sequenced, and showed 174 different bacterial sequences. 52% of the sequences belonged to previously unknown bacterial species. Eight divisions of bacteria were detected in the samples. Cyanobacteria and cyanobacteria ‐ like sequences were predominant in all samples except sample 353‐0m in Selvogsbanki where the ratio was only 4% and alpha and gamma‐ proteobacteria were predominant. Of other ranks identified, Flavobacterium were regularly detected along with other less frequent groups. The difference in microbial composition in the sea samples was assessed by principal component analysis of the microbial community fingerprint obtained by t ‐ RFLP technique. Variability within the samples was dependent upon sampling point, samples from Selvogsabanki showed more correlation with other samples from that area - than with samples from Siglunes or the west coast of Iceland that showed more intrinsic diversity. The community fingerprint and changes in the fingerprint shows correlation to changes in environmental factors measured at sampling. Temperature was the most important environmental factor, along with fluorescence and salinity. Samples from the warmer waters off the south coast were screened for Vibrio parahaemolyticus, but none was detected. The results of this project can be seen as an important basis for further studies of microorganisms in Icelandic waters. Continued research in this area can be of great importance for the evaluation environmental change and the effects of global warming on the marine environment and exploitable marine species in Icelandic waters.  

View report

Reports

Optimization of sample preparation - filtration and DNA extraction - for the analysis of sea water samples

Published:

01/11/2010

Authors:

Eyjólfur Reynisson, Árni Rafn Rúnarsson, Sveinn Haukur Magnússon, Desiree Seehafer, Viggó Þór Marteinsson

Supported by:

Fisheries Project Fund, Ministry of Fisheries and Agriculture

Contact

Viggó Marteinsson

Research Group Leader

viggo.th.marteinsson@matis.is

Optimization of sample preparation - filtration and DNA extraction - for the analysis of sea water samples

Little is known about microorganisms or the diversity of microbial communities in Icelandic waters, but they play an important role in the marine ecosystem. It is necessary to study the microbiology of the ocean around Iceland with new and powerful methods based on molecular biology. In such work, the quality of the samples and sample preparation are very important. In this study, a preliminary survey of sea samples, sampling and sample handling was performed before large quantities of samples are taken. First, samples were taken from the marina in Reykjavík for preliminary study and then we continued with samples from the open sea. Yields were examined for DNA levels and how well the microorganisms' genes were amplified by PCR. The results showed that the best method was a purchased DNA isolation kit that isolated most of the DNA and was quantifiable by PCR. A cheaper and faster method with an automatic isolator and home-made substrates also proved to be very successful, as comparable results were obtained from PCR amplification, although lower DNA recovery was obtained. Based on these results, it is possible to set up procedures based on automatic DNA isolation of samples but the use of purchased isolation kits on more difficult samples. It is planned to use these results for sea samples from the Marine Research Institute's spring survey.

The knowledge on microbial diversity and community structure in Icelandic seawater is scarce at present despite their important role in ocean ecology. The agenda is to increase our knowledge in this field by applying recent and powerful analytical tools. In order to do that it is essential to have access to high quality samples and sample preparation procedures. In the present study sea sample preparation was studied with aim of comparing different methods and optimizes the workflow. Samples from a harbor in Reykjavík and open sea samples were used for this purpose. The results showed that an extraction method based on an Epicenter kit gave the best results regarding DNA recovery from the samples and suitability in a PCR amplification. However, a method based on semi ‐ automatic protocol and in house reagents proved to be more cost effective and showed comparable performance with PCR suitability of the samples although a lower DNA recovery was obtained. From these results it is now possible to establish an efficient work flow for microbial diversity analysis of sea samples using an automated method as a first choice with the option of more costly method for more challenging samples.

View report

Reports

Bacterial diversity in the processing environment of fish products

Published:

01/03/2010

Authors:

Eyjólfur Reynisson, Sveinn Haukur Magnússon, Árni Rafn Rúnarsson, Viggó Þór Marteinsson

Supported by:

Tækniþróunarsjóður, AVS

Contact

Viggó Marteinsson

Research Group Leader

viggo.th.marteinsson@matis.is

Bacterial diversity in the processing environment of fish products

The report seeks to address the diversity and species composition of micro-organisms in fish processing environments. The research work began with the installation and development of methods for scanning microbial composition using molecular biological methods, and then at a later stage, work began on examining selected environments from the fishing industry. Two fish processing plants were visited, each twice where an evaluation was made of the processing and approx. 20 samples taken in each trip. A diverse community of bacteria was found, where known harmful bacteria were usually in a high proportion along with various other species. Microbial counts showed high levels of bacteria on the surface of production lines during processing with few bacterial groups in excess but also numerous other species in smaller quantities. The main groups of bacteria found belong to Photobacterium phosphoreum, which was in the highest proportion overall throughout the study, along with Flavobacterium, Psychrobacter, Chryseobacter, Acinetobacter and Pseudoalteromonas. All of these species are known fish bacteria that live in the redness and intestines of live fish. This is the first known project where molecular biological methods are used to scan the bacterial ecosystem of fish processing plants. A knowledge base has therefore been laid here for bacterial ecosystems in different conditions in fish processing, which will be used permanently in research and development of improved processing processes and storage methods for fish.

In this report we seek answers on diversity and species composition of bacteria in fish processing environment. The study initiated method development to screen microbial systems using molecular methods followed by analysis of samples from 2 fish processing plants. This research shows the presence of a diverse microbial community in fish processing environment where known spoilage microorganisms are typically in high relative numbers along with various other bacterial species. Total viable counts showed the presence of bacteria in high numbers on processing surfaces during fish processing where few species typically dominated the community. Photobacterium phosphoreum was the most apparent species followed by genera such as Flavobacterium, Psychrobacter, Chryseobacter, Acinetobacter and Pseudoalteromonas. All these species are known fish associated bacteria that live on the skin and in the digestive tract of a living animal. To our knowledge, this is the first study where molecular methods are used to screen microbial communities in fish processing plants. This research has therefore contributed a database on bacterial diversity in fish processing plants that will be used in the future to improve processing and storage methods in the fish industry.

View report
EN