Molecular phylogeny of Archaea in boreal forest soil, freshwater and temperate estuarine sediment

Suurin osa luonnossa havaitsemistamme mikrobeista on sellaisia, joita emme edelleenkään osaa kasvattaa laboratorio-oloissa, vaikka tietomme mikrobien monimuotoisuudesta paranevat koko ajan. Luonnontilaisen mikrobieliöstön kokoonpano eri ympäristöissä on paljolti epäselvä ja ymmärrämme vielä hyvin puutteellisesti mikrobien ekologiaa ja niiden rooleja eliöyhteisöissä. Nykyaikaiset molekulaariset tutkimusmenetelmät auttavat selvittämään mikrobien monimuotoisuutta kokonaisvaltaisesti ja nopeasti. Ympäristöstä kemiallisesti puhdistetut ribosomaalista RNA:ta koodaavat geenit edustavat periaatteessa kaikkia eliöyhteisön geneettisesti toisistaan poikkeavia eliöitä. Niistä voidaan valikoida halutut genomit jatkotutkimuksia varten. Uusien menetelmien käyttö on tuonut esiin sen merkittävän seikan, että "tavanomaisten" elinympäristöjen eliöyhteisöihin kuuluu suuri joukko entuudestaan tuntemattomia arkkieliöitä. Aiemmin kuviteltiin, että arkkieliöt asuttavat vain sellaisia "epätavallisia" tai "äärimmäisiä" elinympäristöjä, joita luonnehtii joku seuraavista ominaisuuksista: hyvin korkea lämpötila, korkea suolapitoisuus, korkea happamuus tai emäksisyys, hapettomuus. Tutkijat ovat viimeisen noin kymmenen vuoden aikana osoittaneet, että arkkieliöt asuttavat hyvin monenlaisia kylmän ja lauhkean vyöhykkeen ympäristöjä, yhtä hyvin maaperää kuin suolaisen ja makean veden pohjaa tai pintakerroksia. Nämä löydöt ovat avanneet uuden alun arkkieliöiden tutkimukselle, erityisesti sen selvittämiselle, mitkä ovat niiden fysiologiset ja ekologiset roolit monimuotoisissa mikrobiyhteisöissä. Tämä väitöskirja kuvaa entuudestaan tuntemattomien arkkieliöiden löytymistä havumetsävyöhykkeen metsämaasta. Arkkieliöitä löytyi myös lauhkean vyöhykkeen vuorovesialueelta, murtoveden huuhtelemasta pohjasta. Nämä löydöt ovat perustavalaatuisia vuorovesialueen eliöyhteisöjen ymmärtämiseksi. Suomalaisen metsäjärven vedestä määritettiin molempien arkkieliöiden pääryhmien - tieteellisiltä nimiltään Crenarchaeota ja Euryarchaeota - edustajia. Euryarchaeota-ryhmän edustajia voitiin havainnoida myös fluoresenssi-mikroskopoinnilla. Löydöt viittaavat siihen, että arkkieliöillä on oma biogeokemiallinen roolinsa makeanveden ravintoketjujen hiilen käytössä. Tässä työssä määritetyt uudet arkkieliöiden genomien nukleotidisekvenssit on toimitettu ARB-tietokantaan, jonka kasvava vertailuaineisto edelleen parantaa uusien arkkieliösekvenssien analyysiä ja auttaa hybridisaatiokoetinten ja polymeraasiketjureaktioalukkeiden suunnittelussa ja arvioinnissa. Tässä väitöskirjassa esitellyt tulokset yhdessä lukuisien vesi-, maaperä- ja muiden ympäristöjen arkkieliöitä käsittelevien julkaisujen kanssa osoittavat, että arkkieliöt asuttavat monia erilaisia elinympäristöjä ja että ne ovat ekologisesti paljon menestyneempiä, kuin tieteenalalla on kuviteltu. Voimme olettaa, että heti kun joitain näistä eliöistä onnistutaan kasvattamaan ja ylläpitämään laboratorio-oloissa, niiden joukosta löydetään aivan uusia, entuudestaan tuntemattomia fysiologisia fenotyyppejä, jotka avaavat mielenkiintoisia näkymiä aineenvaihdunnan ja perinnöllisten ominaisuuksien tutkimukselle.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Analysis of forest soil chemistry and hydrology with a dynamic model ACIDIC.

In this study we analyze how the ion concentrations in forest soil solution are determined by hydrological and biogeochemical processes. A dynamic model ACIDIC was developed, including processes common to dynamic soil acidification models. The model treats up to eight interacting layers and simulates soil hydrology, transpiration, root water and nutrient uptake, cation exchange, dissolution and reactions of Al hydroxides in solution, and the formation of carbonic acid and its dissociation products. It includes also a possibility to a simultaneous use of preferential and matrix flow paths, enabling the throughfall water to enter the deeper soil layers in macropores without first reacting with the upper layers. Three different combinations of routing the throughfall water via macro- and micropores through the soil profile is presented. The large vertical gradient in the observed total charge was simulated succesfully. According to the simulations, gradient is mostly caused by differences in the intensity of water uptake, sulfate adsorption and organic anion retention at the various depths. The temporal variations in Ca and Mg concentrations were simulated fairly well in all soil layers. For H+, Al and K there were much more variation in the observed than in the simulated concentrations. Flow in macropores is a possible explanation for the apparent disequilibrium of the cation exchange for H+ and K, as the solution H+ and K concentrations have great vertical gradients in soil. The amount of exchangeable H+ increased in the O and E horizons and decreased in the Bs1 and Bs2 horizons, the net change in whole soil profile being a decrease. A large part of the decrease of the exchangeable H+ in the illuvial B horizon was caused by sulfate adsorption. The model produces soil water amounts and solution ion concentrations which are comparable to the measured values, and it can be used in both hydrological and chemical studies of soils.

Bacillus pallidus sp nov., isolated from forest soil

A Gram-positive bacterium, designated strain CW 7(T), was isolated from forest soil in Anhui Province, south-east China. Cells were strictly aerobic, motile with peritrichous flagella and rod-shaped. The strain grew optimally at 30-37 degrees C and pH 7.0-8.0. The major fatty acids of strain CW 7(T) were anteiso-C-15:0, iso-C-15:0 and anteiso-C-17:0. The predominant menaquinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The G + C content of the genomic DNA was 42.3 mol%. Phylogenetic analysis indicated that strain CW 7(T) belonged to a monophyletic cluster within the genus Bacillus and showed 16S rRNA gene sequence similarities of less than 96.5% to recognized species of the genus Bacillus. The results of the polyphasic taxonomic study, including phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain CW 7(T) represents a novel species of the genus Bacillus, for which the name Bacillus pallidus sp. nov. is proposed. The type strain is CW 7(T) (=KCTC 13200(T)=CCTCC AB 207188(T)=LMG 24451(T)).

Enchytraeid worms retard polycyclic aromatic hydrocarbon degradation in a coniferous forest soil

In this study the fate of naphthalene, fluorene and pyrene were investigated in the presence and absence of enchytraeid worms. Microcosms were used, which enabled the full fate of ¹⁴C-labelled PAHs to be followed. Between 60 and 70% of naphthalene was either mineralised or volatilised, whereas over 90% of the fluorene and pyrene was retained within the soil. Mineralisation and volatilisation of naphthalene was lower in the presence of enchytraeid worms. The hypothesis that microbial mineralisation of naphthalene was limited by enchytraeids because they reduce nutrient availability, and hence limit microbial carbon turnover in these nutrient poor soils, was tested. Ammonia concentrations increased and phosphorus concentrations decreased in all microcosms over the 56 d experimental period. The soil nutrient chemistry was only altered slightly by enchytraeid worms, and did not appear to be the cause of retardation of naphthalene mineralisation. The results suggest that microbial availability and volatilisation of naphthalene is altered as it passes through enchytraeid worms due to organic material encapsulation. © 2004 Elsevier Ltd. All rights reserved.

On sampling collection procedure effectiveness for forest soil characterization

One of the most important measures to prevent wild forest fires is the use of prescribed and controlled burning actions as it reduce the fuel mass availability. The impact of these management activities on soil physical and chemical properties varies according to the type of both soil and vegetation. Decisions in forest management plans are often based on the results obtained from soil-monitoring campaigns. Those campaigns are often man-labor intensive and expensive. In this paper we have successfully used the multivariate statistical technique Robust Principal Analysis Compounds (ROBPCA) to investigate on the sampling procedure effectiveness for two different methodologies, in order to reflect on the possibility of simplifying and reduce the sampling collection process and its auxiliary laboratory analysis work towards a cost-effective and competent forest soil characterization.

Prescribed burning impact on forest soil properties – a fuzzy boolean nets approach

The Portuguese northern forests are often and severely affected by wildfires during the Summer season. These occurrences significantly affect and negatively impact all ecosystems, namely soil, fauna and flora. In order to reduce the occurrences of natural wildfires, some measures to control the availability of fuel mass are regularly implemented. Those preventive actions concern mainly prescribed burnings and vegetation pruning. This work reports on the impact of a prescribed burning on several forest soil properties, namely pH, soil moisture, organic matter content and iron content, by monitoring the soil self-recovery capabilities during a one year span. The experiments were carried out in soil cover over a natural site of Andaluzitic schist, in Gramelas, Caminha, Portugal, which was kept intact from prescribed burnings during a period of four years. Soil samples were collected from five plots at three different layers (0–3, 3–6 and 6–18) 1 day before prescribed fire and at regular intervals after the prescribed fire. This paper presents an approach where Fuzzy Boolean Nets (FBN) and Fuzzy reasoning are used to extract qualitative knowledge regarding the effect of prescribed fire burning on soil properties. FBN were chosen due to the scarcity on available quantitative data. The results showed that soil properties were affected by prescribed burning practice and were unable to recover their initial values after one year.

Fuzzy Boolean nets investigation of the alteration of some physical forest soil properties due to a prescribed fire action

Portuguese northern forests are often and severely affected by wildfires during the summer season. Some preventive actions, such as prescribed (or controlled) burnings and clear-cut logging, are often used as a measure to reduce the occurrences of wildfires. In the particular case of Serra da Cabreira forest, due to extremely difficulties in operational field work, the prescribed (or controlled) burning technique is the the most common preventive action used to reduce the existing fuel load amount. This paper focuses on a Fuzzy Boolean Nets analysis of the changes in some forest soil properties, namely pH, moisture and organic matter content, after a controlled fire, and on the difficulties found during the sampling process and how they were overcome. The monitoring process was conducted during a three-month period in Anjos, Vieira do Minho, Portugal, an area located in a contact zone between a two-mica coarse-grained porphyritic granite and a biotite with plagioclase granite. The sampling sites were located in a spot dominated by quartzphyllite with quartz veins whose bedrock is partially altered and covered by slightly thick humus, which maintains low undergrowth vegetation.

Forest soil short term response to prescribed fire: an approach by Principal Components Analysis

In the current context of serious climate changes, where the increase of the frequency of some extreme events occurrence can enhance the rate of periods prone to high intensity forest fires, the National Forest Authority often implements, in several Portuguese forest areas, a regular set of measures in order to control the amount of fuel mass availability (PNDFCI, 2008). In the present work we’ll present a preliminary analysis concerning the assessment of the consequences given by the implementation of prescribed fire measures to control the amount of fuel mass in soil recovery, in particular in terms of its water retention capacity, its organic matter content, pH and content of iron. This work is included in a larger study (Meira-Castro, 2009(a); Meira-Castro, 2009(b)). According to the established praxis on the data collection, embodied in multidimensional matrices of n columns (variables in analysis) by p lines (sampled areas at different depths), and also considering the quantitative data nature present in this study, we’ve chosen a methodological approach that considers the multivariate statistical analysis, in particular, the Principal Component Analysis (PCA ) (Góis, 2004). The experiments were carried out in a soil cover over a natural site of Andaluzitic schist, in Gramelas, Caminha, NW Portugal, who was able to maintain itself intact from prescribed burnings from four years and was submit to prescribed fire in March 2008. The soils samples were collected from five different plots at six different time periods. The methodological option that was adopted have allowed us to identify the most relevant relational structures inside the n variables, the p samples and in two sets at the same time (Garcia-Pereira, 1990). Consequently, and in addition to the traditional outputs produced from the PCA, we have analyzed the influence of both sampling depths and geomorphological environments in the behavior of all variables involved.

Forest soil carbon cycle under elevated CO2 – a case of increased throughput?

Forest soils account for a large part of the stable carbon pool held in terrestrial ecosystems. Future levels of atmospheric CO2 are likely to increase C input into the soils through increased above- and below-ground production of forests. This increased input will result in greater sequestration of C only if the additional C enters stable pools. In this review, we compare current observations from four large-scale Free Air FACE Enrichment (FACE) experiments on forest ecosystems (EuroFACE, Aspen-FACE, Duke FACE and ORNL-FACE) and consider their predictive power for long-term C sequestration. At all sites, FACE increased fine root biomass, and in most cases higher fine root turnover resulted in higher C input into soil via root necromass. However, at all sites, soil CO2 efflux also increased in excess of the increased root necromass inputs. A mass balance calculation suggests that a large part of the stimulation of soil CO2 efflux may be due to increased root respiration. Given the duration of these experiments compared with the life cycle of a forest and the complexity of processes involved, it is not yet possible to predict whether elevated CO2 will result in increased C storage in forest soil.

Effect of air-drying pre-treatment on the characterization of forest soil carbon pools

Archived soils could represent a valuable resource for the spatio-temporal inventory of soil carbon stability. However, archived soils are usually air-dried before storage and the impact of a drying pretreatment on physically and chemically-defined C fractions has not yet been fully assessed. Through the comparison of field-moist and corresponding air-dried (at 25oC for 2 weeks) forest soil samples, we examined the effect of air-drying on: a) the quantity and the quality of cold- (CWEC) and hot-water (HWEC) extractable C and b) the concentration of C in physically isolated fractions (free- and intra-aggregate light and organo-mineral). Soil samples were collected from the organic (O) and mineral (A and B) horizons of three different forest soils from southeastern England: (i) Cambisol under Pine (Pinus nigra); (ii) Cambisol under Beech (Fagus sylvatica) and (iii) Gleysol under oak (Quercus robur). CWEC concentrations for dry samples were up to 2 times greater than for corresponding field moist samples and had significantly (p < 0.001) higher phenolic content. However, the effect of drying pretreatment on HWEC, its phenolic content was not significant (p > 0.05) for most samples. Dried soils had significantly (p < 0.001) higher concentrations of free light-C while having lower concentrations of intra-aggregate-C when compared to moist samples (p < 0.001). However, fine silt and clay fractions were not significantly affected by the drying pretreatment (p=0.789). Therefore, based on the results obtained from gleysol and cambisol forest soils studied here, C contents in hot-water extractions and fine particle size physical fractions (< 25µm) seem to be robust measurements for evaluating C fractions in dried stored forest soils. Further soil types should be tested to evaluate the wider generality of these findings.

Bacterial diversity of terra preta and pristine forest soil from the Western Amazon

The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic, terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two soils were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG) and by DNA sequencing. The results showed that both soils had similar bacterial community compositions over a range of phylogenetic distances, among which Acidobacteria were predominant, but that terra preta supported approximately 25% greater species richness. The survey provides the first detailed analysis of the composition and structure of bacterial communities from terra preta anthrosols using noncultured-based molecular methods. (c) 2006 Elsevier Ltd. All rights reserved.