The microbial control of phosphorus fluxes in marine sediments

This thesis explores how microorganisms affect the release of the key nutrient phosphorus from marine sediments. A detailed understanding of the controls on regeneration of phosphorus from sediments is important because phosphorus availability in surface waters can regulate primary productivity. Oxygen depletion can enhance sediment phosphorus release and intensify phosphorus cycling between the sediment and overlying water, thereby sustaining eutrophication in marine systems. In addition, increased phosphorus release can exacerbate anoxic conditions, as the degradation of the subsequent higher flux of organic matter to the sediment places an extra demand on the oxygen supply. At present, many coastal ecosystems are facing the joint effects of hypoxia and eutrophication. As yet, our knowledge of benthic phosphorus cycling is insufficient to quantitatively predict future trends in the coupled cycles of carbon, phosphorus and oxygen in coastal systems and this strongly hampers the development of strategies for remediation and restoration. In this thesis we show that, although redox conditions have a profound impact on the phosphorus release from sediments, bacterial communities in Baltic Sea sediments from contrasting redox conditions show a large overlap. In addition, the bacteria in these carbon-limited sediments express phosphatase enzymes under both oxic and anoxic conditions. The role of phosphatases very likely is to relieve carbon limitation by removing phosphate groups from organic compounds prior to uptake of the remainder of the molecule. By labeling phosphatase-expressing cells, we determined that the phosphatase-expressing bacterial community coarsely reflects the total bacterial community. The expression of phosphatases by carbon-limited microorganisms provides a mechanism that explains the ongoing release of phosphate from anoxic sediments. The release of more phosphate from organic matter than is required by microorganisms can increase pore water phosphate concentrations, which can lead to a higher release of phosphate from the sediment to the overlying water. Because the phosphatase activity was lower in anoxic than in oxic sediments, phosphatase activity by itself cannot explain the higher release of P from anoxic sediments. However, phosphate is more efficiently retained in oxic sediments, for example by adsorption to, or co-precipitation with, iron and by microbial polyphosphate accumulation. We measured – to our knowledge for the first time – C:P ratios of individual sediment microorganisms (by X-Ray MicroAnalysis). Surprisingly, the microorganisms were phosphorus poor in comparison to the Redfield ratio (C:P ratio of approximately 400:1). The relative phosphorus content was the same for microorganisms from oxic and anoxic sediment incubations. The formation of new microbial biomass in sediments thus requires less phosphorus relative to carbon than would be needed for the growth of phosphorus-rich organisms, and less than supplied in incoming organic matter. The measured C:P ratios can explain up to approximately 70% of the enhanced phosphorus release observed in the study area, depending on microbial biomass accumulation. The results from this thesis need to be scaled up from the microbial realm to global cycles with the help of (mathematical) models. Further cooperation between microbiologists and geochemists is necessary to resolve the remaining questions regarding the microbial control of benthic phosphorus cyclin

Effect of redox conditions on bacterial community structure in Baltic Sea sediments with contrasting Phosphorus fluxes

Phosphorus release from sediments can exacerbate the effect of eutrophication in coastal marine ecosystems. The flux of phosphorus from marine sediments to the overlying water is highly dependent on the redox conditions at the sediment-water interface. Bacteria are key players in the biological processes that release or retain phosphorus in marine sediments. To gain more insight in the role of bacteria in phosphorus release from sediments, we assessed the effect of redox conditions on the structure of bacterial communities. To do so, we incubated surface sediments from four sampling sites in the Baltic Sea under oxic and anoxic conditions and analyzed the fingerprints of the bacterial community structures in these incubations and the original sediments. This paper describes the effects of redox conditions, sampling station, and sample type (DNA, RNA, or whole-cell sample) on bacterial community structure in sediments. Redox conditions explained only 5% of the variance in community structure, and bacterial communities from contrasting redox conditions showed considerable overlap. We conclude that benthic bacterial communities cannot be classified as being typical for oxic or anoxic conditions based on community structure fingerprints. Our results suggest that the overall structure of the benthic bacterial community has only a limited impact on benthic phosphate fluxes in the Baltic Sea

Effect of redox conditions on bacterial community structure in Baltic Sea sediments with contrasting redox conditions

Phosphorus release from sediments can exacerbate the effect of eutrophication in coastal marine ecosystems. The flux of phosphorus from marine sediments to the overlying water is highly dependent on the redox conditions at the sediment-water interface. Bacteria are key players in the biological processes that release or retain phosphorus in marine sediments. To gain more insight in the role of bacteria in phosphorus release from sediments, we assessed the effect of redox conditions on the structure of bacterial communities. To do so, we incubated surface sediments from four sampling sites in the Baltic Sea under oxic and anoxic conditions and analyzed the fingerprints of the bacterial community structures in these incubations and the original sediments. This paper describes the effects of redox conditions, sampling station, and sample type (DNA, RNA, or whole-cell sample) on bacterial community structure in sediments. Redox conditions explained only 5% of the variance in community structure, and bacterial communities from contrasting redox conditions showed considerable overlap. We conclude that benthic bacterial communities cannot be classified as being typical for oxic or anoxic conditions based on community structure fingerprints. Our results suggest that the overall structure of the benthic bacterial community has only a limited impact on benthic phosphate fluxes in the Baltic Sea.

Biphasic kinetics of a methanotrophic community is a combination of growth and increased activity per cell

Since methane-oxidizing bacteria (MOB) are the only biological sink for the greenhouse gas methane, knowledge about the functioning of these bacteria in various ecosystems is needed to understand the dynamics observed in global methane emission. The activity of MOB is commonly assessed by methane oxidation assays. The resulting methane depletion curves often follow a biphasic pattern of initial and induced methane oxidation activity, often interpreted as representing the in situ active and total MOB community, respectively. The application of QPCR on soil incubations, that were stopped before, at, and after the transition point in the methane-depletion curve demonstrated that both pmoA-mRNA was produced as well as substantial cell growth took place already in the initial phase. In addition, type Ia and II MOB displayed markedly different behaviour which can be interpreted as ecologically different strategies. For the correct interpretation of methane oxidation assays, it is recommend using small time windows to calculate methane oxidation activities as to avoid substantial cell growth

MICROBIAL DIVERSITY AS A CONTROLLING FACTOR OF AEROBIC METHANE CONSUMPTION

Background. Aerobic methane oxidizing bacteria (MOB) play a vital role in the global climate by degrading the greenhouse gas CH4. The process of CH4 consumption is sensitive to disturbance leading to strong variability in CH4 emission from ecosystems. Mechanistic explanations for variability in CH4 emission from soil and sediment ecosystems may be found in the diversity and ecology of these microbes. The objective of the presented work is to link the community composition and ecology of these microbes to the environmental variability observed in CH4 consumption. Methods. Methane consumption was investigated in a river floodplain along the river Rhine. Methane oxidation kinetics were determined in vitro in slurry incubations as well as on intact cores. Methanotrophic community composition was assessed using pmoA-based micro array and QPCR on mRNA as well as DNA. Stable isotope probing (SIP) of lipids and mRNA was applied to detect the active methanotrophic species. Results. The flooding regime in the Rhine floodplain established a distinct CH4 consumption pattern with a maximum exactly in the part of the floodplain between permanently and irregularly flooded sites. This pattern was mirrored by the MOB community composition. Diversity index as assessed by micro array and activity components (initial consumption, Vmax, Vmax/Km) were positively correlated. These analyses as well as SIP showed that -proteobacterial MOB were responsible for the observed kinetics with a distinct optimum in the gradient whereas α-proteobacterial MOB increased with decreasing flooding intensity. Conclusion. In general it can be concluded that the environmental disturbances shaped the CH4-consuming microbial community leading to differential eco-distribution of MOB. The relative abundance of specific subgroups controlled CH4 consuming activity which makes it evident that knowledge on the microbial community composition is necessary to predict effects of environmental change on methane cycling

Phylogenetic characterization of phosphatase-expressing bacterial communities in Baltic Sea sediments

Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is largely unknown. We herein presented a culture-independent method to phylogenetically characterize phosphatase-expressing bacteria in sediments. We labeled whole-cell extracts of Baltic Sea sediments with an artificial phosphatase substrate and sorted phosphatase-expressing cells with a flow cytometer. Their phylogenetic affiliation was determined by Denaturing Gradient Gel Electrophoresis. The phosphatase-expressing bacterial community coarsely reflected the whole-cell bacterial community, with a similar dominance of Alphaproteobacteria

Does microbial stoichiometry modulate eutrophication of aquatic ecosystems?

The stoichiometry of prokaryotes (Bacteria and Archaea) can control benthic phosphorus (P) fluxes relative to carbon (C) and nitrogen (N) during organic matter remineralization. This paper presents the first experimental data on benthic microbial stoichiometry. We used X-ray microanalysis to determine C:N:P ratios of individual prokaryotes from C-limited Baltic Sea sediments incubated under oxic or anoxic conditions. At approximately 400:1, C:P ratios of prokaryotes from both oxic and anoxic incubations were higher than the Redfield ratio for marine organic matter (106:1), whereas prokaryotic C:N ratios (6.4:1) were close to the Redfield ratio. We conclude that high microbial C:P ratios contribute to the enhanced remineralization of P from organic matter relative to C and N observed in many low oxygen marine settings.

Phosphatases relieve carbon limitation of microbial activity in Baltic Sea sediments along a redox-gradient

The relationship between phosphatase activity and the element limiting microbial activity (carbon [C], nitrogen [N], or phosphorus [P]) was studied experimentally in sediment from four stations in the Baltic Sea located along a depth transect from oxic to anoxic bottom waters. The role of extracellular phosphatases was assessed by determining the percentages of intact cells that could be labeled with an artificial substrate for phosphatases (i.e., enzyme-labeled fluorescence 97 phosphatase substrate [ELF]) using a flow cytometer. Phosphatase activity was detected in sediment slurries from all sites either with or without prior incubation under oxic or anoxic conditions. In addition, ELF-labeled cells were detected in all incubated sediments, indicating that intact cells bearing phosphatases contribute to the phosphatase activity. Phosphatase activities and percentages of ELF-labeled cells were lower for the anoxic than for the oxic slurry incubations. Phosphatases are likely used to relieve the limitation of microbial activity by utilizable C in these recently deposited, organic C–rich sediments in the Baltic Sea. In marine sediments overlain by anoxic bottom waters, the biological and chemical mechanisms of P retention are often less efficient than in oxic settings and the P released to relieve C limitation escapes to the overlying water. This explains the ongoing higher P fluxes from sediments overlain by anoxic bottom waters.

Beyond nitrogen: the importance of phosphorus for CH4 oxidation in soils and sediments

Wetlands, lakes and agricultural soils are important sources and sinks of the greenhouse gas methane. The only known methane sink of biological nature is the oxidation by methanotrophic microorganisms, these organisms therefore provide an important ecosystem service. To protect this ecosystem service, it is important to maintain methanotrophic microorganism diversity, especially under increased anthropogenically-induced environmental pressures, such as imbalanced input of nutrients to ecosystems. There is therefore an urgent need to understand how N and P affect the structure and activity of methane oxidizing communities. Numerous research studies have already shown variable effects of N-addition on methane oxidation: small additions tend to stimulate methane oxidation, whereas large additions are inhibitory. There is however still a large knowledge gap concerning effects of P on methane oxidation. Here, we present data on the relation between methane oxidation and various measures of P in 50 drainage ditches, and summarize literature reporting relations between P and methane oxidation in wetlands and soils. Additionally, we review experiments on effects of P, N and N + P addition on both low affinity and high affinity methane oxidation. In our set of drainage ditches, as well as studies on wetland and permafrost soils, P content is positively correlated to methane oxidation, though it also co-correlates with many other variables. However, results from P-additions in rice paddies, agricultural soils, landfills, peat bogs, permafrost soils and forests were more variable: sometimes inhibiting (2 studies), other times stimulating methane oxidation (4 studies), and sometimes showing no effect (5 studies). Two studies report increased methanotroph (pmoA) abundance following P-fertilization, but little is known about effects of P on methanotroph community structure and its consequences for methane consumption. By mining methanotrophic genomes for genes involved in N and P-related processes, we demonstrate that variability in N/P related traits (influencing acquisition, uptake and metabolism) does not reflect DNA-based phylogeny. This review points to a need for better mechanistic understanding of the effects of P on methane oxidation, and the role of traits of methanotrophic community members in regulating this proces

Phylogenetic characterization of phosphatase-expressing bacterial communities in Baltic Sea sediments

Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is largely unknown. We herein presented a culture-independent method to phylogenetically characterize phosphatase-expressing bacteria in sediments. We labeled whole-cell extracts of Baltic Sea sediments with an artificial phosphatase substrate and sorted phosphatase-expressing cells with a flow cytometer. Their phylogenetic affiliation was determined by Denaturing Gradient Gel Electrophoresis. The phosphatase-expressing bacterial community coarsely reflected the whole-cell bacterial community, with a similar dominance of Alphaproteobacteria