Below-ground processes in meadow soil under elevated ozone and carbon dioxide : - Greenhouse gas fluxes, N cycling and microbial communities

This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.

Aerobic carbon-cycle related microbial communities in boreal peatlands: responses to water-level drawdown

Boreal peatlands represent a considerable portion of the global carbon (C) pool. Water-level drawdown (WLD) causes peatland drying and induces a vegetation change, which affects the decomposition of soil organic matter and the release of greenhouse gases (CO2 and CH4). The objective of this thesis was to study the microbial communities related to the C cycle and their response to WLD in two boreal peatlands. Both sampling depth and site type had a strong impact on all microbial communities. In general, bacteria dominated the deeper layers of the nutrient-rich fen and the wettest surfaces of the nutrient-poor bog sites, whereas fungi seemed more abundant in the drier surfaces of the bog. WLD clearly affected the microbial communities but the effect was dependent on site type. The fungal and methane-oxidizing bacteria (MOB) community composition changed at all sites but the actinobacterial community response was apparent only in the fen after WLD. Microbial communities became more similar among sites after long-term WLD. Litter quality had a large impact on community composition, whereas the effects of site type and WLD were relatively minor. The decomposition rate of fresh organic matter was influenced slightly by actinobacteria, but not at all by fungi. Field respiration measurements in the northern fen indicated that WLD accelerates the decomposition of soil organic matter. In addition, a correlation between activity and certain fungal sequences indicated that community composition affects the decomposition of older organic matter in deeper peat layers. WLD had a negative impact on CH4 oxidation, especially in the oligotrophic fen. Fungal sequences were matched to taxa capable of utilizing a broad range of substrates. Most of the actinobacterial sequences could not be matched to characterized taxa in reference databases. This thesis represents the first investigation of microbial communities and their response to WLD among a variety of boreal peatland habitats. The results indicate that microbial community responses to WLD are complex but dependent on peatland type, litter quality, depth, and variable among microbes.

Carbon dynamics in peatlands under changing hydrology : Effects of water level drawdown on litter quality, microbial enzyme activities and litter decomposition rates

Pristine peatlands are carbon (C) accumulating wetland ecosystems sustained by a high water level (WL) and consequent anoxia that slows down decomposition. Persistent WL drawdown as a response to climate and/or land-use change directly affects decomposition: increased oxygenation stimulates decomposition of the old C (peat) sequestered under prior anoxic conditions. Responses of the new C (plant litter) in terms of quality, production and decomposability, and the consequences for the whole C cycle of peatlands are not fully understood. WL drawdown induces changes in plant community resulting in shift in dominance from Sphagnum and graminoids to shrubs and trees. There is increasing evidence that the indirect effects of WL drawdown via the changes in plant communities will have more impact on the ecosystem C cycling than any direct effects. The aim of this study is to disentangle the direct and indirect effects of WL drawdown on the new C by measuring the relative importance of 1) environmental parameters (WL depth, temperature, soil chemistry) and 2) plant community composition on litter production, microbial activity, litter decomposition rates and, consequently, on the C accumulation. This information is crucial for modelling C cycle under changing climate and/or land-use. The effects of WL drawdown were tested in a large-scale experiment with manipulated WL at two time scales and three nutrient regimes. Furthermore, the effect of climate on litter decomposability was tested along a north-south gradient. Additionally, a novel method for estimating litter chemical quality and decomposability was explored by combining Near infrared spectroscopy with multivariate modelling. WL drawdown had direct effects on litter quality, microbial community composition and activity and litter decomposition rates. However, the direct effects of WL drawdown were overruled by the indirect effects via changes in litter type composition and production. Short-term (years) responses to WL drawdown were small. In long-term (decades), dramatically increased litter inputs resulted in large accumulation of organic matter in spite of increased decomposition rates. Further, the quality of the accumulated matter greatly changed from that accumulated in pristine conditions. The response of a peatland ecosystem to persistent WL drawdown was more pronounced at sites with more nutrients. The study demonstrates that the shift in vegetation composition as a response to climate and/or land-use change is the main factor affecting peatland ecosystem C cycle and thus dynamic vegetation is a necessity in any models applied for estimating responses of C fluxes to changes in the environment. The time scale for vegetation changes caused by hydrological changes needs to extend to decades. This study provides grouping of litter types (plant species and part) into functional types based on their chemical quality and/or decomposability that the models could utilize. Further, the results clearly show a drop in soil temperature as a response to WL drawdown when an initially open peatland converts into a forest ecosystem, which has not yet been considered in the existing models.

Flux and turnover of fixed carbon in soil microbial biomass of limed and unlimed plots of an upland grassland ecosystem

The influence of liming on rhizosphere microbial biomass C and incorporation of root exudates was studied in the field by in situ pulse labelling of temperate grassland vegetation with (13)CO(2) for a 3-day period. In plots that had been limed (CaCO(3) amended) annually for 3 years, incorporation into shoots and roots was, respectively, greater and lower than in unlimed plots. Analysis of chloroform-labile C demonstrated lower levels of (13)C incorporation into microbial biomass in limed soils compared to unlimed soils. The turnover of the recently assimilated (13)C compounds was faster in microbial biomass from limed than that from unlimed soils, suggesting that liming increases incorporation by microbial communities of root exudates. An exponential decay model of (13)C in total microbial biomass in limed soils indicated that the half-life of the tracer within this carbon pool was 4.7 days. Results are presented and discussed in relation to the absolute values of (13)C fixed and allocated within the plant-soil system.

Organo-mineral complexation alters carbon and nitrogen cycling in stream microbial assemblages

Inland waters are of global biogeochemical importance receiving carbon inputs of ~ 4.8 Pg C y^-1. Of this 12 % is buried, 18 % transported to the oceans, and 70 % supports aquatic secondary production. However, the mechanisms that determine the fate of organic matter (OM) in these systems are poorly defined. One important aspect is the formation of organo-mineral complexes in aquatic systems and their potential as a route for OM transport and burial vs. their use potential as organic carbon (C) and nitrogen (N) sources. Organo-mineral particles form by sorption of dissolved OM to freshly eroded mineral surfaces and may contribute to ecosystem-scale particulate OM fluxes. We tested the availability of mineral-sorbed OM as a C & N source for streamwater microbial assemblages and streambed biofilms. Organo-mineral particles were constructed in vitro by sorption of ¹³C:¹⁵N-labelled amino acids to hydrated kaolin particles, and microbial degradation of these particles compared with equivalent doses of ¹³C:¹⁵N-labelled free amino acids. Experiments were conducted in 120 ml mesocosms over 7 days using biofilms and streamwater sampled from the Oberer Seebach stream (Austria), tracing assimilation and mineralization of ¹³C and ¹⁵N labels from mineral-sorbed and dissolved amino acids.Here we present data on the effects of organo-mineral sorption upon amino acid mineralization and its C:N stoichiometry. Organo-mineral sorption had a significant effect upon microbial activity, restricting C and N mineralization by both the biofilm and streamwater treatments. Distinct differences in community response were observed, with both dissolved and mineral-stabilized amino acids playing an enhanced role in the metabolism of the streamwater microbial community. Mineral-sorption of amino acids differentially affected C & N mineralization and reduced the C:N ratio of the dissolved amino acid pool. The present study demonstrates that organo-mineral complexes restrict microbial degradation of OM and may, consequently, alter the carbon and nitrogen cycling dynamics within aquatic ecosystems.

Impact of carbon/nitrogen feeding strategy on polyhydroxyalkanoates production using mixed microbial cultures

Polyhydroxyalkanoates (PHA) production using mixed microbial cultures (MMC) requires a multi-stage process involving the microbial selection of PHA-storing microorganisms, typically operated in sequencing batch reactors (SBR), and an accumulation reactor. Since low-cost renewable feedstocks used as process feedstock are often nitrogen-deficient, nutrient supply in the selection stage is required to allow for microbial growth. In this context, the possibility to uncouple nitrogen supply from carbon feeding within the SBR cycle has been investigated in this study. Moreover, three different COD:N ratios (100:3.79, 100:3.03 and 100:2.43) were tested in three different runs which also allowed the study of COD:N ratio on the SBR performance. For each run, a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5 gCOD L-1 d-1 was used as carbon feedstock, whereas ammonium sulfate was the nitrogen source in a lab-scale sequence batch reactor (SBR) with 1 L of working volume. Besides, a sludge retention time (SRT) of 1 d was used as well as a 6 h cycle length. The uncoupled feeding strategy significantly enhanced the selective pressure towards PHA-storing microorganisms, resulting in a two-fold increase in the PHA production (up to about 1.3 gCOD L-1). A high storage response was observed for the two runs with the COD:N ratios (gCOD:gN) of 100:3.79 and 100:3.03, whereas the lowest investigated nitrogen load resulted in very poor performance in terms of polymer production. In fact, strong nitrogen limitation caused fungi to grow and a very poor storage ability by microorganisms that thrived in those conditions. The COD:N ratio also affected the polymer composition, indeed the produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) showed a variable HV content (1-20 %, w/w) among the three runs, lessening as the COD:N increased. This clearly suggests the possibility to use the COD:N ratio as a tool for tuning polymer properties regardless the composition of the feedstock.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

The role of soil microbes in the global carbon cycle: tracking the below-ground microbial processing of plant-derived carbon for manipulating carbon dynamics in agricultural systems.

It is well known that atmospheric concentrations of carbon dioxide (CO2) (and other greenhouse gases) have increased markedly as a result of human activity since the industrial revolution. It is perhaps less appreciated that natural and managed soils are an important source and sink for atmospheric CO2 and that, primarily as a result of the activities of soil microorganisms, there is a soil-derived respiratory flux of CO2 to the atmosphere that overshadows by tenfold the annual CO2 flux from fossil fuel emissions. Therefore small changes in the soil carbon cycle could have large impacts on atmospheric CO2 concentrations. Here we discuss the role of soil microbes in the global carbon cycle and review the main methods that have been used to identify the microorganisms responsible for the processing of plant photosynthetic carbon inputs to soil. We discuss whether application of these techniques can provide the information required to underpin the management of agro-ecosystems for carbon sequestration and increased agricultural sustainability. We conclude that, although crucial in enabling the identification of plant-derived carbon-utilising microbes, current technologies lack the high-throughput ability to quantitatively apportion carbon use by phylogentic groups and its use efficiency and destination within the microbial metabolome. It is this information that is required to inform rational manipulation of the plant–soil system to favour organisms or physiologies most important for promoting soil carbon storage in agricultural soil.

Land use change to perennial energy crops in Northern Italy: Effects on soil organic carbon sequestration and distribution, soil enzyme activities and microbial communities.

Studies on soil organic carbon (SOC) sequestration in perennial energy crops are available for North-Central Europe, while there is insufficient information for Southern Europe. This research was conducted in the Po Valley, a Mediterranean-temperate zone characterised by low SOC levels, due to intensive management. The aim was to assess the factors influencing SOC sequestration and its distribution through depth and within soil fractions, after a 9-year old conversion from two annual systems to Miscanthus (Miscanthus × giganteus) and giant reed (Arundo donax). The 13C natural abundance was used to evaluate the amount of SOC in annual and perennial species, and determine the percentage of carbon derived from perennial crops. SOC was significantly higher under perennial species, especially in the topsoil (0-0.15 m). After 9 years, the amount of C derived from Miscanthus was 18.7 Mg ha-1, mostly stored at 0-0.15 m, whereas the amount of C derived from giant reed was 34.7 Mg ha-1, evenly distributed through layers. Physical soil fractionation was combined with 13C abundance analysis. C derived from perennial crops was mainly found in macroaggregates. Under giant reed, more newly derived-carbon was stored in microaggregates and mineral fraction than under Miscanthus. A molecular approach based on denaturing gradient gel electrophoresis (DGGE) allowed to evaluate changes on microbial community, after the introduction of perennial crops. Functional aspects were investigated by determining relevant soil enzymes (β-glucosidase, urease, alkaline phosphatase). Perennial crops positively stimulated these enzymes, especially in the topsoil. DGGE profiles revealed that community richness was higher in perennial crops; Shannon index of diversity was influenced only by depth. In conclusion, Miscanthus and giant reed represent a sustainable choice for the recovery of soils exhausted by intensive management, also in Mediterranean conditions and this is relevant mainly because this geographical area is notoriously characterised by a rapid turnover of SOC.

Plant diversity drives soil microbial biomass carbon in grasslands irrespective of global environmental change factors

Soil microbial biomass is a key determinant of carbon dynamics in the soil. Several studies have shown that soil microbial biomass significantly increases with plant species diversity, but it remains unclear whether plant species diversity can also stabilize soil microbial biomass in a changing environment. This question is particularly relevant as many global environmental change (GEC) factors, such as drought and nutrient enrichment, have been shown to reduce soil microbial biomass. Experiments with orthogonal manipulations of plant diversity and GEC factors can provide insights whether plant diversity can attenuate such detrimental effects on soil microbial biomass. Here, we present the analysis of 12 different studies with 14 unique orthogonal plant diversity × GEC manipulations in grasslands, where plant diversity and at least one GEC factor (elevated CO2, nutrient enrichment, drought, earthworm presence, or warming) were manipulated. Our results show that higher plant diversity significantly enhances soil microbial biomass with the strongest effects in long-term field experiments. In contrast, GEC factors had inconsistent effects with only drought having a significant negative effect. Importantly, we report consistent non-significant effects for all 14 interactions between plant diversity and GEC factors, which indicates a limited potential of plant diversity to attenuate the effects of GEC factors on soil microbial biomass. We highlight that plant diversity is a major determinant of soil microbial biomass in experimental grasslands that can influence soil carbon dynamics irrespective of GEC.