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.

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Reports

Biodiversity in hot springs in high-temperature areas in Iceland. Overall summary prepared for the Framework Program. Final report

Published:

01/12/2010

Authors:

Sólveig K. Pétursdóttir, Snædís H. Björnsdóttir, Guðmundur Óli Hreggviðsson, Sólveig Ólafsdóttir

Contact

Guðmundur Óli Hreggviðsson

Strategic Scientist

gudmundo@matis.is

Biodiversity in hot springs in high-temperature areas in Iceland. Overall summary prepared for the Framework Program. Final report

Research was carried out on the ecosystem in hot springs in five high-temperature areas in Iceland under the framework of the Framework Program in the years 2004-2009. The aim was to answer questions about the extent and nature of the variability in microbial flora between high-temperature areas in Iceland, which were studied mainly in terms of diversity and rare groups. This report summarizes the results obtained from the above studies. The areas that were examined were the Hengill area, Torfajökull area, Krafla / Námafjall, Krísuvík and Vonarskarð. Furthermore, the results of a similar study carried out for the environmental assessment of the geothermal areas at Þeistareykir and Gjástykki were included in this summary. A total of 115 samples were taken in the six areas and a species composition in 80 of them was analyzed. Species-identifying bacterial and archaeal bacteria in the DNA from the samples were amplified and sequenced. The sequences were classified by affinity and classified by species or genus by comparison with comparable sequences in Genbank. A total of 4424 bacterial sequences and 1006 ancient bacterial sequences were detected from the samples. The distribution of bacteria and ancient bacteria in the samples was examined and it was found that 16 bacterial assemblages were found in most areas and species of the Aquificae array were most common, as they are often primary producers in hot springs. Species of the β- and γ-proteobacterial and Deinococcus-Thermus factions were also found to a significant extent in all areas except Krísuvík. In addition, several other factions were found in individual areas. Within the ancient bacteria, Crenarcheota species were found in all areas, Euryarchaeota species were found in Vonarskarð and Þeistareykir, Thaumarchaeota was found in Vonarskarð and in Krafla / Námafjall and Nanoarchaeota in the Torfajökull area. Calculations of biodiversity (H) microorganisms in the six areas showed that the Krafla area was the most diverse, then Torfajökull, then Vonarskarð, Þeistareykir, Hengill and finally Krísuvík. The assessment of biodiversity by calculating the collection curves largely supported this conclusion. Biological specificity was assessed on the basis of rare species by ≤96% corresponding to close relatives in Genbank. A total of 74 rare species or genera were found in the samples and they seemed for the most part regional and it is not unlikely that some of them are endemic. Most new species or genera were found in the Torfajökull area. Vonarskarð, Krafla / Námafjall and Þeistareykir had slightly fewer rare species or genera. New tribes were also found in the Hengill area, but not to the same extent as in the former.

Culture independent methods were used to study the microbial composition of hot springs in five geothermal areas in Iceland in 2004-2009. The aim was to answer questions on the degree of biodiversity and to what extend the species found were unique to the sites investigated. In this report the site specific research results were combined and compared. The geothermal sites investigated were the Hengill area, the Torfajökull area, the Krafla / Námafjall area, Krísuvík and Vonarskarð. Results from a similar research from an environmental assessment of the geothermal areas of Þeistareykir and Gjástykki were also used. A total of 115 samples were collected from the six geothermal areas and the microbial species composition was estimated in 80 of them. The 16S rRNA genes were amplified from DNA from the samples and partially sequenced. The obtained sequences were classified and identified to the species or genus level by comparison to similar sequences in Genbank. The total of 4424 bacterial sequences and 1006 archaeal sequences were analyzed. The distribution of bacterial and archaeal phyla of the samples was investigated and revealed that 16 bacterial phyla were represented in all areas. Also, that the phylum of the primary producers of hot springs - Aquificae - was dominating. Species belonging to β‐ and γ ‐ proteobacteraa and Deinococcus - Thermus were also found in considerable amounts in all areas except Krísuvík. Several bacterial phyla were only found at one or two geothermal areas. Species belonging to Crenarchaeota were found in all six areas, Euryarchaeota were found in Vonarskarð and Þeistareykir, Thaumarchaeota was found in Vonarskarð as well as in the Krafla / Námafjall area and Nanoarchaeota in the Torfajökull area. Calculation of the biodiversity index (H) of microbial species of the six geothermal areas revealed that the index for the Krafla / Námafjall area was highest, then Torfajökull, Vonarskarð, Þeistareykir, Hengill and finally Krísuvík. The estimate of biodiversity based on Rarefaction curves confirmed the results. The estimation of uniqueness of the areas was based on the number of novel species found using ≤96% similarity to closest relative in Genbank as the cutoff value. The total of 74 novel species or genera were found in the samples most of which were only found in one or at most two areas. Most of these were from the Torfajökull area. A considerable number of novel species were also found in Vonarskarð, Námafjall and Þeistareykir. Novel species or genera were also found in the Hengill geothermal area.

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Reports

Microbial diversity in hot springs in Vonarskarð / Microbial diversity in hot springs in Vonarskarð

Published:

01/03/2009

Authors:

Sólveig K. Pétursdóttir, Snædís H. Björnsdóttir, Sólveig Ólafsdóttir, Guðmundur Óli Hreggviðsson

Supported by:

Orkustofnun due to the Master Plan

Contact

Guðmundur Óli Hreggviðsson

Strategic Scientist

gudmundo@matis.is

Microbial diversity in hot springs in Vonarskarð / Microbial diversity in hot springs in Vonarskarð

Samples were taken in the geothermal area east of Eggja in Vonarskarð. A total of 32 samples were taken from different hot springs, streams and soils at different temperatures and acidity levels. Elements were measured in several water samples. The species composition of bacteria and ancient bacteria was determined by molecular biological methods. A total of 1052 16S rRNA gene sequences of bacteria were detected, spreading across 23 factions. More than 50 new bacterial species were found in the samples, of which 11 are probably representatives of new genera. The genome of ancient bacteria was 155 in number. Ten new species of ancient bacteria were found, including five representatives of new genera. The ecosystem of the geothermal area as a whole must therefore be considered very special. The most common were the primitive species of the array Aquificae. Primitive and non-primitive Proteobacteria species were found in significant quantities and were both known and new species. Typical species of bluish-green bacteria and Chloroflexi were found in the samples. Firmicutes, Bacteroidetes and Chlorobi species were found mainly in samples taken at lower temperatures. Ancient bacteria found in the samples were spread over two largest groups of ancient bacteria, i.e. Crenarcheota and Euryarchaeota. The biological diversity of bacteria and archaeal bacteria in the samples was most often in the range Nt / Nmax = 1.0 -3.0, which is typical of peripheral ecosystems. In some cases it was higher, especially in samples where the temperature was relatively low and therefore more viable for more species. Seven bacterial species were cultured from the samples, including one new species of the genus Sediminibacter from the Bacteroidetes class. Several Thermus species were bred, including T.islandicus which is native to Iceland. The proteobacteria Thermomonas hydrothermalis and Tepidimonas ignava were also grown from several samples and the thermophilic Firmicutes species Anoxybacillus kualawohkensis.

Samples were taken from the geothermal area east of Eggja in Vonarskarð. A total of 32 samples were collected from different sites at various temperature and pH values. The concentration of 72 elements were estimated in water samples. Species composition of Bacteria and Archaea was estimated using molecular methods. A total of 1052 16S rRNA gene sequences belonging to 23 bacterial phyla were detected. Roughly 50 novel bacterial species were found of which 11 represent new genera. Ten novel archaeal species were found, five of which represent new genera. Species belonging to the autotrophic phylum of Aquificae dominated many samples. Species of different subphyla of Proteobacteria were also represented in high ratios in the samples, both described and novel species. Common species of Cyanobacteria and Chloroflexi were also detected. Species of the Firmicutes, Bacteroidetes and Chlorobi phyla were common in samples taken at lower temperatures. Archaeal species in the samples belonged to both Crenarchaeota and Euryarchaeota. The calculated biodiversity index for bacteria and archaea in the samples was 1,0-3,0 which is in concordance with values obtained for extreme ecosystems. It was higher in a few samples which were taken at lower temperatures and thus represent habitats acceptable for more diverse organisms. Seven bacterial species were isolated from the samples. One of these represents a novel species of the genus Sediminibacter within the phylum of Bacteroidetes. Several Thermus species were cultivated, ie T.islandicus which has so far only been found in Iceland. The Proteobacteria species Themomonas hydrothermalis and Tepidimonas ignava were also isolated as well as a thermophilic Firmicutes species, Anoxybacillus kualawohkensis.

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Reports

Biodiversity in hot springs at Þeistareykir and Gjástykki / Biodiversity in hot springs at Þeistareykir and Gjástykki

Published:

01/12/2008

Authors:

Sólveig K. Pétursdóttir, Snædís Huld Björnsdóttir, Sólveig Ólafsdóttir, Guðmundur Óli Hreggviðsson

Supported by:

Þeistareykir ehf

Contact

Guðmundur Óli Hreggviðsson

Strategic Scientist

gudmundo@matis.is

Biodiversity in hot springs at Þeistareykir and Gjástykki / Biodiversity in hot springs at Þeistareykir and Gjástykki

The project was part of an environmental assessment for the planned geothermal utilization and involved a study of the ecosystem in the hot springs at Þeistareykir and Gjástykki. A total of 27 samples were taken. Temperatures and acidity at the sampling points ranged from 33-96 ° C to pH 1.9-8.6. The species composition of the micro-organisms and their proportions were determined by nucleic acid amplification and sequencing of the 16S rRNA species-determining gene using bacterial and antimicrobial markers. A total of 21 bacteria and / or ancient bacteria were detected in 21 samples. DNA sequences were classified into species according to 98% affinities and compared with Genbank sequences for species analysis. In acidic hot springs at Þeistareykir, species within the bacterial assemblages β-, δ-, and γ-Proteobacteria and Aquificae were most common, especially acidic and / or primitive species that use sulfur and iron compounds and bind CO2. In vapors in lava caves at Þeistareykir at higher acidity levels (pH 6.7-8.6), the species Acidobacteria, Actinobacteria, Chloroflexi and Deinococcus-Thermus were prominent. Deinococcus-Thermus and Verrucomicrobium were the most common in samples from the hot springs in Gjástykki (pH 4.4-6.9). Many of these species are non-primitive. Ancient bacteria were found mainly in acidic areas at Þeistareykir, and in all samples from Gjástykki, but not in steam eyes in lava at Þeistareykir, as the acidity level is higher there. Species within the Crenarchaeota group were found in all of these samples, but species within the Euryarchaeota were bound to samples from the surface formations and acid soils. Most ancient bacteria can live primitive life. Biodiversity (Nt / Nmax) bacteria was most often between 1-3 and 1-2 among ancient bacteria. These low values are typical of peripheral ecosystems, where one species is in a very high proportion. Numerous new species were found in the samples, especially bacteria in steam eyes in lava at Þeistareykir and in hot springs in Gjástykki. Also, species of Euryarchaeota within ancient bacteria often showed a low kinship percentage and are therefore considered new species.

Due to future plans for utilizing the geothermal power at Þeistareykir and Gjástykki, an environmental assessment of the biodiversity in hot springs from these sites was carried out. A total of 27 samples were taken from various sites at temperatures of 33-96 ° C and pH 1.9-8.6. The species composition and ratios of thermophiles were estimated by PCR and sequencing of the 16S rRNA genes using bacterial and archaeal primers. Microbial species were detected in 21 samples. DNA sequences were grouped at the 98% similarity species level and compared with available sequences in Genbank for species determination. Species belonging to the bacterial phyla of β-, δ-, and γ-Proteobacteria and Aquificae were dominating in samples from the solfatara fields of Þeistareykir. These were mainly acidophiles and autotrophs capable of utilizing sulfur- and iron compounds and fixing CO2. A totally different pattern of species composition was observed in samples from fumaroles at the lava fields of Þeistareykir at higher pH (6,7-8,6) than in the solfataras. These were mainly Acidobacteria, Actinobacteria, Chloroflexi and DeinococcusThermus. In Gjástykki, (pH 4.4-6.9) Deinococcus-Thermus and Verrucomicrobium sp. were dominating. These are mainly heterotrophs. Archeal species were found as well in the solfatara fields at Þeistareykir and also in hot springs at Gjástykki, but not in the high pH fumaroles at Þeistareykir lava fields. Species from the Crenarchaeota group were found in the samples, but species belonging to the Euryarchaeota group were only detected in solfatara soil samples and sulfur / iron precipitates. These were mainly autotrophs. Biodiversity (Nt / Nmax) was calculated for all samples and estimated at 1-3 among the Bacteria and 1-2 among the Archaea. These low values are typical for extreme environments where one species is highly dominating. Many novel species were found in the samples, especially in soil from fumaroles at the lava field at Þeistareykir and in hot springs at Gjástykki. Euryarchaeal species within the Archaea domain often showed low similarity to known species and most likely represent new species.

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Reports

Lífríki í kalkríkum hverum á Ölkelduháls / Microbial ecology of calcium rich hot springs at Ölkelduháls geothermal area

Published:

01/10/2008

Authors:

Sólveig K. Pétursdóttir, Snædís Björnsdóttir, Alexandra Klonowski, Sólveig Ólafsdóttir, Guðmundur Óli Hreggviðsson

Supported by:

The Environmental and Energy Research Fund of Orkuveita Reykjavíkur

Contact

Alexandra María Klonowski

Project Manager

alex@matis.is

Lífríki í kalkríkum hverum á Ölkelduháls / Microbial ecology of calcium rich hot springs at Ölkelduháls geothermal area

The ecology of calcareous hot springs is little studied. This study involved analyzing the ecosystem in calcareous hot springs at Ölkelduháls and assessing whether it had a special position compared to the ecosystem of other hot springs in the same area with the same temperature and acidity. Bacteria were isolated from thermal samples by conventional culture methods. Genetic analysis methods were used to analyze the species composition. Many species found in samples from calcareous hot springs are also found in other hot springs. It is noteworthy, however, that species within the Aquificae party were not found in the samples, but they are very common in hot springs and widely prevalent. Elemental measurements showed differences in the concentration of sulfur, iron, carbon and arsenic in calcareous water and other hot springs, which may be an explanation for this. Cultivated methods identified mainly known bacterial species of the genera Thermus and Bacillus. One new species of the Meiothermus genus was isolated. Using genetic analysis methods, 195 true bacterial clones were obtained from calcareous hot springs, which were classified into 60 species based on 98% affinity. These 60 species are spread over nine camps. The species found in the samples were the same between the hot springs, but also unique to the sample from which they came. No ancient bacteria were found in the samples. Biodiversity in samples from calcareous hot springs at Ölkelduháls was slightly higher than comparable values from hot springs with similar properties in the same area. The high proportion of unknown species and genera in samples taken in calcareous hot springs at Ölkelduháls is noteworthy. Of the 60 species found in the samples, a sufficiently close relative of the same species was found in 25 cases. The other 35 species were so closely related to close relatives that they could not be classified except into genera, tribes, clans, or families. Ecosystems in calcareous hot springs at Ölkelduháls must therefore be considered very special.

The ecology of calcium rich hot springs is not well documented. In this study an attempt was made to estimate if microbial species composition in calcium rich hot springs in Ölkelduháls in Iceland was special compared to species composition in other hot springs with similar temperature and pH in the same geothermal area. Isolation methods as well as culture independent methods were used to analyze species composition in the samples. Many species found in the calcium rich hot springs are also found in other hot springs. It is noteworthy that Aquificae species were totally absent in samples from calcium rich hot springs, but these species were abundant and dominating in other hot spring samples. Elemental analysis of hot spring water revealed a difference in the concentration of sulfur, iron, carbon and arsenate between calcium rich hot springs and other hot springs in the area. Known species of Thermus and Bacillus genera were isolated from the samples. A novel Meiothermus species was isolated. Approximately 60 species belonging to nine phyla were identified in the samples using culture independent methods. The species identified in the calcium rich samples were identical between samples but also unique for the sample investigated. No archaea were detected in the samples. Biodiversity calculated for the samples from calcium rich hot springs was slightly higher than in samples from other hot springs. A high ratio of unknown species and genera in the samples from calcium rich hot springs in Ölkelduháls is remarkable. Of the total of 60 species identified only 25 had a close relative from the same species according to Genbank. The remaining 35 species were only distantly related to their closest relative and could only be classified to genera, families, orders or classes. Thus, the ecology of calcium rich hot springs appears to be quite unique.

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