Microbial communities in Pb contaminated boreal forest soil

Abstract

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.

Paikallinen lyijykontaminaatio esim. ampumarata-alueilla on saanut suhteellisen vähän huomiota verrattuna laaja-alaiseen teollisuusperäiseen kontaminaatioon. Ampumarata-alueille päätynyt lyijy muodostaa riskin eliöille, pohjavesille ja myös ihmisten terveydelle. Metsittyneet ampumaradat eivät kuitenkaan usein erotu ulkonäöltään läheisistä terveistä ympäristöistä.

Tässä väitöskirjassa tutkittiin Hälvälän ampumarata-aluetta Etelä-Suomessa. Saastuneen maaperän oli aiemmin osoitettu vaikuttavan mm karikkeenhajotukseen. Maan akuutista toksisuudesta saatiin kuitenkin ristiriitaista tietoa, esim. änkyrimatoihin vaikutus oli selvä mutta bakteeritestit osoittivat, että lyijyn biosaatavuus on vähäistä. Lyijyn vesiliukoisuus oli myös pientä. Lyijyresistenttien bakteerien osuus yhteisössä oli kasvanut.

Väitöskirjatyössä käytettiin useita erilaisia DNA-tekniikoita tutkittaessa maaperän bakteereiden ja sienten, erityisesti ektomykorritsasienten yhteisöijä. Bakteerien ja sienten diversiteetti kontaminoituneessa maaperässä oli suuri eikä bakteerien osalta poikennut puhtaasta maasta. Sienten diversiteetti sen sijaan oli pienentynyt kontaminoituneessa maaperässä. Tämä näkyi erityisesti kantasienissä (Basidiomycota). Oordinaatioanalyysillä osoitettiin, että ektomykorritsasienten (EcM) yhteisörakenne oli muuttunut. Thelephora- suku selkeästi yleistyi kontaminoituneessa maassa, kun Pseudotomentella, Suillus ja Tylospora -sukujen frekvenssit vähenivät.

Väitöskirjan yhtenä osatyönä kehitettiin tietokoneohjelma suurten DNA-aineistojen käsittelyyn ja analysoimiseen.