Kvantitatiivse polümeraasi ahelreaktsiooni modifikatsioonide mõju märklaudgeeni kvantifitseerimisele keskkonnaproovidest bioremediatsiooni hindamisel

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Keskkonna reostumine erinevate saasteainetega (nt. naftasaadused, kloororgaanilised ühendid) on muutunud kriitiliseks probleemiks üle maailma kahjustades inimtervist, kahandades puhta joogivee varusid ning mõjutades terveid ökosüsteeme. Et klassikalised „pumpa-ja-töötle“ saastuse eemaldamise meetodid on töömahukad ja kallid, on viimastel kümnenditel hakatud aina enam otse reostuspaigas rakendama erinevaid bioloogilise tervenduse ehk bioremediatsiooni tehnoloogiaid, mis tuginevad suuresti saasteainete mikroobsele lagundamisele. Toimuvate protsesside käiku ja jätkusuutlikkust vastusena muutuvatele keskkonnatingimustele hinnatakse seejuures enamasti keemiliste ja mikrobioloogiliste parameetrite pikaajalise seire abil. Kvantitatiivne polümeraasi ahelreaktsioon (qPCR) on aina sagedamini kasutust leidev metoodika saasteaineid lagundava mikroobikoosluse esinemise ja arvukuse hindamiseks bioremediatsiooni seirel. QPCR on kiire ja tundlik meetod, mis võimaldab nii taksonoomiliste kui funktsionaalsete märklaudgeenide arvukuse määramist hõimkonna tasemest liigi tasandini. Määratud arvukusi muude seireparameetritega (nt. saasteaine kontsentratsioon) kõrvutades on võimalik hinnata bioremediatsiooniprotsesside efektiivsust ja kulgemist uuritavas kohas. Et märklaudgeenide arvukuse määramise edukus keskkonnaproovidest sõltub mitmetest faktoritest, näiteks mikroobikoosluse DNA eraldamise meetodist ja kvaliteedist, inhibiitorite esinemisest eraldatud DNA-s, qPCR reaktsioonikeemia tüübist, märklaudjärjestuse amplifikatsiooni efektiivsusest ja tulemuste analüüsi kvaliteedist, hindasimegi erinevate qPCR reaktsiooni- ja analüüsiprotsessi modifikatsioonide mõju keskkonnaproovidest märklaudgeeni absoluutse ja suhtelise arvukuse määramisele. Leidsime, et varieeruvused qPCRi töövoos ja analüüsiprotsessis mõjutavad oluliselt märklaudgeeni absoluutse ja suhtelise arvukuse määramist keskkonnaproovidest ning seeläbi ka bioremediatsiooniga rakendamisega seotud otsuseid.The increasingly industrialized global economy that has emerged over the last century has led to dramatically elevated releases of anthropogenic chemicals into the environment impacting whole ecosystems (i.e. the Gulf of Mexico oil spill), drinking water supplies or directly human health. Concurrently with increasing pollution levels, avid interest in developing strategies for remediation of environmental contaminants has emerged. As classic “suck and truck” strategies followed by off-site treatments are expensive, the in situ bioremediation processes like monitored natural attenuation (MNA), biostimulation, bioaugmentation and rhizoremediation have become an attractive way to rehabilitate contaminated sites. These bioremediation techniques rely extensively on the presence of an active microbial degrader population able to transform the bioavailable contaminants into harmless or less dangerous compounds. Bioremediation processes need to be continuously monitored to ensure their efficiency and sustainability. One of the increasingly used methods in bioremediation monitoring is quantitative polymerase chain reaction (qPCR) which enables quantification of the abundance of gene markers within the environment. The quantitative data generated can be used to relate variation in gene abundances with variation in abiotic and biotic factors and process rates. However, target gene quantification results from environmental samples depend on a number of factors such as the method and quality of DNA extraction, the subsequent presence of inhibitory substances in the extracted microbial community DNA, the qPCR chemistry used, the amplification efficiency achieved and the overall quality of the resultant datasets. We evaluated the scope of these aspects affecting gene enumerations by qPCR and found that modifications in qPCR workflow and analysis procedure steps can significantly influence the target gene quantification and normalization results from environmental samples and consequently also bioremediation related decision-making. For environmental monitoring purposes the most suitable method workflow relating to the characteristics of individual experiment conducted should be chosen to ensure the quality and truthfulness of obtained results

Differences in microbial community structure and nitrogen cycling in natural and drained tropical peatland soils

Funding Information: This was supported by the Estonian Research Council (grant IUT2-16); and the EU through the European Regional Development Fund through Centre of Excellence EcolChange and the European Social Fund (Doctoral School of Earth Sciences and Ecology). We would like to thank the PhD students participating in the field works.Peer reviewedPublisher PD

Soil Bacterial and Archaeal Communities and Their Potential to Perform N-Cycling Processes in Soils of Boreal Forests Growing on Well-Drained Peat

Funding Information: This study was supported by the Estonian Forest Management Centre, the Estonian Research Council grants PRG548, PRG916, and PRG352, WaterJPI-JC-2018_13 project, and Centres of Excellence Environ and EcolChange.Peer reviewedPublisher PD

Microbial community changes in TNT spiked soil bioremediation trial using biostimulation, phytoremediation and bioaugmentation

Trinitrotoluene (TNT), a commonly used explosive for military and industrial applications, can cause serious environmental pollution. 28-day laboratory pot experiment was carried out applying bioaugmentation using laboratory selected bacterial strains as inoculum, biostimulation with molasses and cabbage leaf extract, and phytoremediation using rye and blue fenugreek to study the effect of these treatments on TNT removal and changes in soil microbial community responsible for contaminant degradation. Chemical analyses revealed significant decreases in TNT concentrations, including reduction of some of the TNT to its amino derivates during the 28-day tests. The combination of bioaugmentation-biostimulation approach coupled with rye cultivation had the most profound effect on TNT degradation. Although plants enhanced the total microbial community abundance, blue fenugreek cultivation did not significantly affect the TNT degradation rate. The results from molecular analyses suggested the survival and elevation of the introduced bacterial strains throughout the experiment. First published online: 15 Feb 201

Microbial Community Dynamics during Biodegradation of Crude Oil and Its Response to Biostimulation in Svalbard Seawater at Low Temperature

The development of oil exploration activities and an increase in shipping in Arctic areas have increased the risk of oil spills in this cold marine environment. The objective of this experimental study was to assess the effect of biostimulation on microbial community abundance, structure,dynamics, and metabolic potential for oil hydrocarbon degradation in oil-contaminated Arctic seawater.The combination of amplicon-based and shotgun sequencing, together with the integration of genome-resolved metagenomics and omics data, was applied to assess microbial community structure and metabolic properties in naphthenic crude oil-amended microcosms. The comparison of estimates for oil-degrading microbial taxa obtained with different sequencing and taxonomic assignment methods showed substantial discrepancies between applied methods. Consequently,the data acquired with different methods was integrated for the analysis of microbial community structure, and amended with quantitative PCR, producing a more objective description of microbial community dynamics and evaluation of the effect of biostimulation on particular microbial taxa. Implementing biostimulation of the seawater microbial community with the addition of nutrients resulted in substantially elevated prokaryotic community abundance (103-fold), a distinctly different bacterial community structure from that in the initial seawater, 1.3-fold elevation in the normalized abundance of hydrocarbon degradation genes, and 12% enhancement of crude oil biodegradation.The bacterial communities in biostimulated microcosms after four months of incubation were dominated by Gammaproteobacterial genera Pseudomonas, Marinomonas, and Oleispira, which were succeeded by Cycloclasticus and Paraperlucidibaca after eight months of incubation. The majority of 195 compiled good-quality metagenome-assembled genomes (MAGs) exhibited diverse hydrocarbon degradation gene profiles. The results reveal that biostimulation with nutrients promotes naphthenic oil degradation in Arctic seawater, but this strategy alone might not be sufficient to effectively achieve bioremediation goals within a reasonable timeframe.publishedVersio

Assessment of Hydrocarbon Degradation Potential in Microbial Communities in Arctic Sea Ice

The anthropogenic release of oil hydrocarbons into the cold marine environment is an increasing concern due to the elevated usage of sea routes and the exploration of new oil drilling sites in Arctic areas. The aim of this study was to evaluate prokaryotic community structures and the genetic potential of hydrocarbon degradation in the metagenomes of seawater, sea ice, and crude oil encapsulating the sea ice of the Norwegian fjord, Ofotfjorden. Although the results indicated substantial differences between the structure of prokaryotic communities in seawater and sea ice, the crude oil encapsulating sea ice (SIO) showed increased abundances of many genera-containing hydrocarbon-degrading organisms, including Bermanella, Colwellia, and Glaciecola. Although the metagenome of seawater was rich in a variety of hydrocarbon degradation-related functional genes (HDGs) associated with the metabolism of n-alkanes, and mono- and polyaromatic hydrocarbons, most of the normalized gene counts were highest in the clean sea ice metagenome, whereas in SIO, these counts were the lowest. The long-chain alkane degradation gene almA was detected from all the studied metagenomes and its counts exceeded ladA and alkB counts in both sea ice metagenomes. In addition, almA was related to the most diverse group of prokaryotic genera. Almost all 18 goodand high-quality metagenome-assembled genomes (MAGs) had diverse HDGs profiles. The MAGs recovered from the SIO metagenome belonged to the abundant taxa, such as Glaciecola, Bermanella, and Rhodobacteracea, in this environment. The genera associated with HDGs were often previously known as hydrocarbon-degrading genera. However, a substantial number of new associations, either between already known hydrocarbon-degrading genera and new HDGs or between genera not known to contain hydrocarbon degraders and multiple HDGs, were found. The superimposition of the results of comparing HDG associations with taxonomy, the HDG profiles of MAGs, and the full genomes of organisms in the KEGG database suggest that the found relationships need further investigation and verification

Impact of Reed Canary Grass Cultivation and Mineral Fertilisation on the Microbial Abundance and Genetic Potential for Methane Production in Residual Peat of an Abandoned Peat Extraction Area.

This study examined physiochemical conditions and prokaryotic community structure (the bacterial and archaeal 16S rRNA genes and mcrA gene abundances and proportions), and evaluated the effect of reed canary grass cultivation and mineral fertilisation on these factors, in the 60 cm thick residual peat layer of experimental plots located on an abandoned peat extraction area. The archaeal proportion was 0.67-39.56% in the prokaryotic community and the methanogens proportion was 0.01-1.77% in the archaeal community. When bacterial abundance was higher in the top 20 cm of peat, the archaea were more abundant in the 20-60 cm layer and methanogens in the 40-60 cm layer of the residual peat. The bacterial abundance was significantly increased, but archaeal abundance was not affected by cultivation. The fertiliser application had a slight effect on peat properties and on archaeal and methanogen abundances in the deeper layer of cultivated peat. The CH4 emission was positively related to mcrA abundance in the 20-60 cm of the bare peat, while in case of reed canary grass cultivation these two parameters were not correlated. Reed canary grass cultivation mitigated CH4 emission, although methanogen abundance remained approximately the same or even increased in different layers of residual peat under cultivated sites over time. This study supports the outlook of using abandoned peat extraction areas to produce reed canary grass for energy purposes as an advisable land-use practice from the perspective of atmospheric impact in peatland-rich Northern Europe

Assessment of Hydrocarbon Degradation Potential in Microbial Communities in Arctic Sea Ice

The anthropogenic release of oil hydrocarbons into the cold marine environment is an increasing concern due to the elevated usage of sea routes and the exploration of new oil drilling sites in Arctic areas. The aim of this study was to evaluate prokaryotic community structures and the genetic potential of hydrocarbon degradation in the metagenomes of seawater, sea ice, and crude oil encapsulating the sea ice of the Norwegian fjord, Ofotfjorden. Although the results indicated substantial differences between the structure of prokaryotic communities in seawater and sea ice, the crude oil encapsulating sea ice (SIO) showed increased abundances of many genera-containing hydrocarbon-degrading organisms, including Bermanella, Colwellia, and Glaciecola. Although the metagenome of seawater was rich in a variety of hydrocarbon degradation-related functional genes (HDGs) associated with the metabolism of n-alkanes, and mono- and polyaromatic hydrocarbons, most of the normalized gene counts were highest in the clean sea ice metagenome, whereas in SIO, these counts were the lowest. The long-chain alkane degradation gene almA was detected from all the studied metagenomes and its counts exceeded ladA and alkB counts in both sea ice metagenomes. In addition, almA was related to the most diverse group of prokaryotic genera. Almost all 18 goodand high-quality metagenome-assembled genomes (MAGs) had diverse HDGs profiles. The MAGs recovered from the SIO metagenome belonged to the abundant taxa, such as Glaciecola, Bermanella, and Rhodobacteracea, in this environment. The genera associated with HDGs were often previously known as hydrocarbon-degrading genera. However, a substantial number of new associations, either between already known hydrocarbon-degrading genera and new HDGs or between genera not known to contain hydrocarbon degraders and multiple HDGs, were found. The superimposition of the results of comparing HDG associations with taxonomy, the HDG profiles of MAGs, and the full genomes of organisms in the KEGG database suggest that the found relationships need further investigation and verification.Assessment of Hydrocarbon Degradation Potential in Microbial Communities in Arctic Sea IcepublishedVersio