Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO2

Elevated atmospheric CO₂ can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritus-based ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO₂ would result in changes in the size and composition of microbial communities colonizing leaves in a woodland stream. Three tree species, Populus tremuloides, Salix alba, and Acer saccharum, were grown under ambient (360 ppm) or elevated (720 ppm) CO₂, and their leaves were incubated in a woodland stream. Elevated-CO₂ treatment resulted in significant increases in the phenolic and tannin contents and C/N ratios of leaves. Microbial effects, which occurred only for P. tremuloides leaves, included decreased fungal biomass and decreased bacterial counts. Analysis of fungal and bacterial communities on P. tremuloides leaves via terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing revealed that fungal community composition was mostly unchanged by the elevated-CO₂ treatment, whereas bacterial communities showed a significant shift in composition and a significant increase in diversity. Specific changes in bacterial communities included increased numbers of alphaproteobacterial and cytophaga-flavobacter-bacteroides (CFB) group sequences and decreased numbers of betaproteobacterial and firmicutes sequences, as well as a pronounced decrease in overall Gram-positive bacterial sequences.The concentration of atmospheric CO₂ has been increasing for the last 150 years, from 270 ppm prior to the industrial revolution (49) to the current level of approximately 388 ppm (http://www.mlo.noaa.gov), and is projected to exceed 700 ppm by the end of the century (57). This ongoing increase in atmospheric CO₂ is believed to be due to the extensive use of fossil fuels and changes in land use patterns (5). Elevated atmospheric CO₂ has global climate implications due to its role in the greenhouse effect (39), and it has also been shown to have direct biological effects. Specifically, elevated CO₂ can increase the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco) (13), resulting in increased carbon fixation by C3 plants (49). This increased carbon fixation can result in increased above- and below-ground plant biomass (21, 47, 63, 72), as well as altered foliar chemical composition (31, 46, 58, 70).Elevated atmospheric CO₂ is unlikely to have direct impacts on forested headwater streams, as they are primarily heterotrophic systems (2) in which CO₂ is typically supersaturated (41). However, changes in leaf chemistry may have an impact, as forested headwater streams are detritus-based ecosystems that derive up to 99% of their carbon inputs from terrestrial organic matter (71), which is mainly leaf litter (29). Microbes play a key role in the entry of this allochthonous organic material into stream food webs. Fungi and bacteria colonize leaf litter after its deposition in a stream and begin decomposition of the leaf material (34). The resulting growth of microbial assemblages associated with leaf litter provides a critical food resource for detritus-feeding invertebrate consumers (6, 18, 23, 44), which through their feeding activities help facilitate the further transformation and breakdown of plant litter and the flow of carbon and nutrients to higher-trophic-level organisms, including fish. Prior research has demonstrated that aquatic invertebrates show a clear preference to eat leaves that have been extensively colonized, or “conditioned,” by microbes (4, 18, 65). This is likely due to the fact that microbial colonization significantly increases the nutrient content of detritus, as microbes can incorporate soluble nutrients from stream water (e.g., nitrogen) into the microbial biomass (64, 66). In addition, microbes convert indigestible leaf components (e.g., lignin and cellulose) into microbial biomass, which invertebrates can digest more efficiently (6). Therefore, fungi and bacteria are significant contributors to the transfer of carbon and nutrients from terrestrial to aquatic ecosystems.Microbial decomposition of leaves in streams is influenced by the chemical composition of the leaf material. This has been illustrated by a number of studies comparing decomposition of leaves from different tree species (for a review, see reference 62). These studies have demonstrated that leaves from species, such as oaks and conifers, that are relatively high in polyphenolic compounds, including lignin and tannins, tend to decompose more slowly than leaves from species with lower concentrations of these compounds, such as alder (62). The leaf carbon-to-nitrogen (C/N) ratio also impacts decomposition rates; leaf litter with a high C/N ratio tends to decompose more slowly than litter with a low C/N ratio (62). These trends are relevant to atmospheric CO₂ concentrations because elevated atmospheric CO₂ has been shown to increase the concentrations of phenolic compounds (lignin and tannins), as well as the C/N ratio of leaves of C3 plants (31, 46, 58, 70). Therefore, it is reasonable to hypothesize that growth of trees under elevated CO₂ could have negative impacts on microbial colonization and decomposition of leaves. Rier et al. (58) tested this hypothesis with one tree species, Populus tremuloides (quaking aspen), and found that leaves produced under elevated CO₂ decomposed more slowly in streams and supported less fungal and bacterial biomass than leaves produced under ambient conditions (58).In addition to impacting microbial community size, it is reasonable to hypothesize that changes in leaf chemistry caused by growth of trees under elevated CO₂ could impact microbial community composition. Several studies have demonstrated that the compositions of microbial communities colonizing leaves in streams can differ based on tree species (36, 45). No study we are aware of has examined the effects of tree growth under elevated atmospheric CO₂ on the compositions of microbial communities colonizing leaf litter in streams; however, such changes in microbial community composition could be highly relevant to stream food webs. For example, different groups of fungi and bacteria differ in their abilities to degrade various components of leaf litter (1, 67), so the species compositions of microbial communities could potentially impact rates of decomposition and production of microbial biomass (26). This in turn could impact the transfer of carbon and energy to higher-trophic-level organisms. In addition, different groups of fungi and bacteria differ in chemical composition (9, 32), and thus, they may differ in their nutritional values to aquatic invertebrates.In the current study, we tested the hypothesis that changes in leaf chemistry caused by elevated CO₂ would result in changes in the biomass and composition of detrital microbial communities by growing three tree species under ambient or elevated CO₂, collecting leaves after abscission, incubating the leaves in a woodland stream, and determining the biomass and composition of the microbial communities colonizing the leaves.