Effect of CO2 enrichment on bacterial metabolism in an Arctic fjord (update)

The anthropogenic increase of carbon dioxide (CO₂) alters the seawater carbonate chemistry, with a decline of pH and an increase in the partial pressure of CO₂ (pCO₂). Although bacteria play a major role in carbon cycling, little is known about the impact of rising pCO₂ on bacterial carbon metabolism, especially for natural bacterial communities. In this study, we investigated the effect of rising pCO₂ on bacterial production (BP), bacterial respiration (BR) and bacterial carbon metabolism during a mesocosm experiment performed in Kongsfjorden (Svalbard) in 2010. Nine mesocosms with pCO₂ levels ranging from ca. 180 to 1400 μatm were deployed in the fjord and monitored for 30 days. Generally BP gradually decreased in all mesocosms in an initial phase, showed a large (3.6-fold average) but temporary increase on day 10, and increased slightly after inorganic nutrient addition. Over the wide range of pCO₂ investigated, the patterns in BP and growth rate of bulk and free-living communities were generally similar over time. However, BP of the bulk community significantly decreased with increasing pCO₂ after nutrient addition (day 14). In addition, increasing pCO₂ enhanced the leucine to thymidine (Leu : TdR) ratio at the end of experiment, suggesting that pCO₂ may alter the growth balance of bacteria. Stepwise multiple regression analysis suggests that multiple factors, including pCO₂, explained the changes of BP, growth rate and Leu : TdR ratio at the end of the experiment. In contrast to BP, no clear trend and effect of changes of pCO₂ was observed for BR, bacterial carbon demand and bacterial growth efficiency. Overall, the results suggest that changes in pCO₂ potentially influence bacterial production, growth rate and growth balance rather than the conversion of dissolved organic matter into CO₂.

Ocean acidification effects on marine microbial communities

Anthropogenic CO2 emissions are causing an acidification of the world’s oceans. The consequences for marine organisms and especially heterotrophic bacteria remain under debate, and almost nothing is known concerning marine fungi. Both microbial groups are important players in organic matter decomposition and nutrient cycling, and their pH tolerance is known to be broad in relation to the predicted acidification. So far, ocean acidification effects on marine bacterial communities have mainly been investigated in large-scale mesocosm studies. In these systems, indirect effects mediated through complex food web interactions come into play. Until now, these experiments were not carried out in sufficient replication. In this thesis, we chose an alternative approach and investigated bacterial and fungal communities in highly replicated microcosm experiments (1-1.6 L). The duration of the experiments was four weeks. We incubated the natural microbial community from Helgoland Roads (North Sea) at in situ seawater pH, pH 7.82 and pH 7.67. These pH levels represent the present-day situation and acidification at atmospheric CO2 of 700 or 1000 ppm, projected for the southern North Sea for the year 2100. For the bacterial community, different dilution approaches were used to select for different ecological groups. Seasonality was accounted for by repeating the experiment four times (spring, summer, autumn, winter). In a second experiment repeated in two consecutive years, we investigated direct pH effects on marine fungal communities. We additionally isolated marine yeasts and identified them by Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and partial sequencing of the large subunit (LSU) rRNA gene. To reveal changes in community structure, we applied the culture-independent fingerprint method automated ribosomal intergenic spacer analysis (ARISA) for both bacteria and fungi. Bacterial communities were furthermore analyzed by 16S ribosomal amplicon pyrosequencing. Abundances were determined by flow cytometry (bacteria) and colony forming unit counts (fungi). To be able to interpret results comprehensively, we determined the natural variability of the carbonate system at Helgoland Roads over a yearly cycle. We found that from September 2010 to September 2011, pH at Helgoland Roads ranged from 8.06 to 8.43, corresponding to partial pressures of carbon dioxide (pCO2) of 215-526 µatm. The acidification predicted for the year 2100 consequently represents a strong perturbation of the system. Bacterial communities developing in the microcosms were primarily influenced by season and dilution, demonstrating that diverse communities had been generated. We predominantly found pH-dependent shifts in bacterial community structure already at pH 7.82. Groups involved in these shifts were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. While Rhodobacteraceae were consistently less characteristic for reduced pH, Campylobacteraceae profited from pH reduction. For most other bacterial groups however, pH effects were context-dependent, i.e. dependent on season, dilution or an interaction of effects. Regarding bacterial abundance, no pH effect was found. Fungal community structure was significantly different between both years of the experiment, hinting at inter-annual variability. Shifts in response to pH occurred predominantly only at pH 7.67. In contrast, a strong pH effect was observed on fungal abundance. In comparison to in situ pH, fungal numbers were on average 9 times higher at pH 7.82 and 34 times higher at pH 7.67. Concerning marine yeasts, Leucosporidium scottii, Rhodotorula mucilaginosa and related species, as well as Cryptococcus sp. and Debaromyces hansenii reacted positively to low pH. Our findings demonstrate that already small reductions in pH have direct effects on both bacterial and fungal communities. A tipping point for community shifts appears to be reached earlier for bacteria than for fungi. Regarding bacteria and yeasts, both naturally abundant groups and rare species were affected by pH reductions. The strong increase in fungal numbers at reduced pH suggests that with ocean acidification, marine fungi may reach higher importance in marine biogeochemical cycles and as infectious agents. Using a microcosm approach, a robust analysis of direct ocean acidification effects on marine bacterial and fungal communities was accomplished. Results yield valuable hypotheses to test in future large-scale and long-term studies.

Interactive effects of global climate change and pollution on marine microbes: the way ahead

Global climate change has the potential to seriously and adversely affect marine ecosystem functioning. Numerous experimental and modeling studies have demonstrated how predicted ocean acidification and increased ultraviolet radiation (UVR) can affect marine microbes. However, researchers have largely ignored interactions between ocean acidification, increased UVR and anthropogenic pollutants in marine environments. Such interactions can alter chemical speciation and the bioavailability of several organic and inorganic pollutants with potentially deleterious effects, such as modifying microbial-mediated detoxification processes. Microbes mediate major biogeochemical cycles, providing fundamental ecosystems services such as environmental detoxification and recovery. It is, therefore, important that we understand how predicted changes to oceanic pH, UVR, and temperature will affect microbial pollutant detoxification processes in marine ecosystems. The intrinsic characteristics of microbes, such as their short generation time, small size, and functional role in biogeochemical cycles combined with recent advances in molecular techniques (e.g., metagenomics and metatranscriptomics) make microbes excellent models to evaluate the consequences of various climate change scenarios on detoxification processes in marine ecosystems. In this review, we highlight the importance of microbial microcosm experiments, coupled with high-resolution molecular biology techniques, to provide a critical experimental framework to start understanding how climate change, anthropogenic pollution, and microbiological interactions may affect marine ecosystems in the future.

The response of abyssal organisms to low pH conditions during a series of CO2-release experiments simulating deep-sea carbon sequestration

The effects of low-pH, high-pCO₂ conditions on deep-sea organisms were examined during four deep-sea CO₂ release experiments simulating deep-ocean C sequestration by the direct injection of CO₂ into the deep sea. We examined the survival of common deep-sea, benthic organisms (microbes; macrofauna, dominated by Polychaeta, Nematoda, Crustacea, Mollusca; megafauna, Echinodermata, Mollusca, Pisces) exposed to low-pH waters emanating as a dissolution plume from pools of liquid carbon dioxide released on the seabed during four abyssal CO₂-release experiments. Microbial abundance in deep-sea sediments was unchanged in one experiment, but increased under environmental hypercapnia during another, where the microbial assemblage may have benefited indirectly from the negative impact of low-pH conditions on other taxa. Lower abyssal metazoans exhibited low survival rates near CO₂ pools. No urchins or holothurians survived during 30–42 days of exposure to episodic, but severe environmental hypercapnia during one experiment (E1; pH reduced by as much as ca. 1.4 units). These large pH reductions also caused 75% mortality for the deep-sea amphipod, Haploops lodo, near CO₂ pools. Survival under smaller pH reductions (ΔpH<0.4 units) in other experiments (E2, E3, E5) was higher for all taxa, including echinoderms. Cephalopods, gastropods, and fish were more tolerant than most other taxa. The gastropod Mohnia vernalis and octopus Benthoctopus sp. survived exposure to pH reductions that episodically reached −0.3 pH units. Ninety percent of abyssal zoarcids (Pachycara bulbiceps) survived exposure to pH changes reaching ca. −0.3 pH units during 30–42 day-long experiments.

Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean

We examined the combined effects of light and pCO₂ on growth, CO₂-fixation and N₂-fixation rates by strains of the unicellular marine N₂-fixing cyanobacterium Crocosphaera watsonii with small (WH0401) and large (WH0402) cells that were isolated from the western tropical Atlantic Ocean. In low-pCO₂-acclimated cultures (190 ppm) of WH0401, growth, CO₂-fixation and N₂-fixation rates were significantly lower than those in cultures acclimated to higher (present-day 385 ppm, or future 750 ppm) pCO₂ treatments. Growth rates were not significantly different, however, in low-pCO₂-acclimated cultures of WH0402 in comparison with higher pCO₂ treatments. Unlike previous reports for C. watsonii (strain WH8501), N₂-fixation rates did not increase further in cultures of WH0401 or WH0402 when acclimated to 750 ppm relative to those maintained at present-day pCO₂. Both light and pCO₂ had a significant negative effect on gross : net N₂-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated light and pCO₂. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing light and pCO₂, emphasizing the need for examining the effects of global change on a range of isolates within this biogeochemically important genus. In general, however, our data suggest that cellular N retention and CO₂-fixation rates of C. watsonii may be positively affected by elevated light and pCO₂ within the next 100 years, potentially increasing trophic transfer efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the marine biota.

Response of Nodularia spumigena to pCO2 – Part 3: Turnover of phosphorus compounds (update)

Diazotrophic cyanobacteria form extensive summer blooms in the Baltic Sea driving the surrounding surface waters into phosphate limitation. One of the main bloom-forming species is the heterocystous cyanobacterium Nodularia spumigena. N. spumigena exhibits accelerated uptake of phosphate through the release of the extracellular enzyme alkaline phosphatase whose activity also serves as an indicator of the hydrolysis of dissolved organic phosphorus (DOP). The present study investigated the utilisation of DOP and its compounds (e.g., ATP) by N. spumigena during growth under different CO₂ concentrations, in order to estimate potential consequences of ocean acidification on the cell’s supply with phosphorus (P). Cell growth, the phosphorus pool, and four DOP compounds (ATP, DNA, RNA, and phospholipids) were determined in three setups with different CO₂ concentrations (average 341 μatm, 399 μatm, and 508 μatm) during a 15-day batch experiment. The results showed stimulated growth of N. spumigena and a rapid depletion of dissolved inorganic phosphorus (DIP) in all pCO₂ treatments. DOP uptake was enhanced by a factor of 1.32 at 399 μatm and of 2.25 at 508 μatm compared to the lowest CO₂ concentration. Among the measured DOP compounds, none was found to accumulate preferentially during the incubation or in response to a specific pCO₂ treatment. However, at the beginning 61.9 ± 4.3% of total DOP were not characterised but comprised the most utilised fraction. This is demonstrated by the decrement of this fraction to 27.4 ± 9.9% of total DOP during the growth phase with a preference at high pCO₂. Our results indicate a stimulated growth of diazotrophic cyanobacteria at increasing CO₂ concentrations which is accompanied by increasing utilisation of DOP as an alternative P source.

Impact of temperature and species interaction on filamentous cyanobacteria may be more important than salinity and increased pCO2 levels

A future business-as-usual scenario (A1FI) was tested on two bloom-forming cyanobacteria of the Baltic Proper, Nodularia spumigena and Aphanizomenon sp., growing separately and together. The projected scenario was tested in two laboratory experiments where (a) interactive effects of increased temperature and decreased salinity and (b) interactive effects of increased temperature and elevated levels of pCO₂ were tested. Increased temperature, from 12 to 16 °C, had a positive effect on the biovolume and photosynthetic activity (F _v/F _m) of both species. Compared when growing separately, the biovolume of each species was lower when grown together. Decreased salinity, from 7 to 4, and elevated levels of pCO₂, from 380 to 960 ppm, had no effect on the biovolume, but on F _v/F _m of N. spumigena with higher F _v/F _m in salinity 7. Our results suggest that the projected A1FI scenario might be beneficial for the two species dominating the extensive summer blooms in the Baltic Proper. However, our results further stress the importance of studying interactions between species.

Ecological aspects of marine Vibrio bacteria – exploring relationships to other organisms and a changing environment

Heterotrophic bacteria of the genus Vibrio are indigenous in the marine environment although environmental cues regulate their growth and distribution. The attention brought to this genus is due to its many species/strains that are pathogenic to humans and other organisms. Vibrio abundances are strongly coupled to water temperature and salinity but abundance dynamics occur even where these hydrographical parameters are stable. In this thesis, I have studied Vibrio dynamics in relation to other organisms such as phytoplankton (papers I, II and III) and a bivalve host-organism (paper IV) in a changing environment where increasing temperature (paper III) and ocean acidification (paper IV) may influence survival and proliferation of these bacteria. In particular paper I showed that in a tropical coastal area, where the water temperature and salinity were stable across seasons, abundances of Vibrio were tightly coupled to phytoplankton biomass and community composition. A diatom bloom during December seemed to support high numbers of vibrios in waters with otherwise low levels of dissolved organic carbon. Paper II further supports that some phytoplankton can favor Vibrio growth while others seem to have a negative influence on Vibrio abundances. For instance, Skeletonema tropicum, a common diatom in Indian coastal waters, easily eradicated Vibrio parahaemolyticus from sea water in our experiments. In temperate marine areas culturable Vibrio predominantly occurs in the water column during the warmer months. Sediments are suggested to be potential reservoirs when conditions in the water-column are harsh. Accordingly, in paper III we showed that cold-water sediments from geographically separate areas in a boreal region of Scandinavia all contained relative high abundances of total Vibrio spp. and that all sediments also included culturable Vibrio. In agreement with paper I, the fresh input of organic material from phytoplankton blooms, for which chlorophyll a was used as a proxy, seemed to positively influence Vibrio abundances also in the sediments (paper III). Therefore, the pelagic-benthic coupling which can supply the sediments with biomass from the primary production could influence the abundance of Vibrio spp. Increasing temperature had variable influence on sediment-associated Vibrio abundance, with a significant increase in abundances in sediments originating from one area when the temperature reached over 21°C and a generally negative influence of increasing temperature on abundances in sediments originating from another area (paper III). This suggests that the sediments contained different Vibrio communities with varying temperature tolerance traits. Rising levels of carbon dioxide in the atmosphere does not only lead to higher water temperature through the green house effect, but also to acidification of the oceans. Paper IV illustrated how a common bivalve pathogen, Vibrio tubiashii, can be favored in the interaction with a calcifying bivalve host, Mytilus edulis, when this host-pathogen combination was exposed to levels of ocean acidification projected to occur by the end of the 21st century. Thus, global environmental changes may enhance the probability of Vibrio infections in higher organisms.

Arctic microbial community dynamics influenced by elevated CO2 levels (update)

The Arctic Ocean ecosystem is particularly vulnerable to ocean acidification (OA) related alterations due to the relatively high CO₂ solubility and low carbonate saturation states of its cold surface waters. Thus far, however, there is only little known about the consequences of OA on the base of the food web. In a mesocosm CO₂-enrichment experiment (overall CO₂ levels ranged from ~ 180 to 1100 μatm) in Kongsfjorden off Svalbard, we studied the consequences of OA on a natural pelagic microbial community. OA distinctly affected the composition and growth of the Arctic phytoplankton community, i.e. the picoeukaryotic photoautotrophs and to a lesser extent the nanophytoplankton thrived. A shift towards the smallest phytoplankton as a result of OA will have direct consequences for the structure and functioning of the pelagic food web and thus for the biogeochemical cycles. Besides being grazed, the dominant pico-and nanophytoplankton groups were found prone to viral lysis, thereby shunting the carbon accumulation in living organisms into the dissolved pools of organic carbon and subsequently affecting the efficiency of the biological pump in these Arctic waters.

Ocean acidification shows negligible impacts on high-latitude bacterial community structure in coastal pelagic mesocosms (update)

The impact of ocean acidification and carbonation on microbial community structure was assessed during a large-scale in situ costal pelagic mesocosm study, included as part of the EPOCA 2010 Arctic campaign. The mesocosm experiment included ambient conditions (fjord) and nine mesocosms with pCO₂ levels ranging from ~145 to ~1420 μatm. Samples for the present study were collected at ten time points (t–1, t1, t5, t7, t12, t14, t18, t22, t26 to t28) in seven treatments (ambient fjord (~145), 2 × ~185, ~270, ~685, ~820, ~1050 μatm) and were analysed for “small” and “large” size fraction microbial community composition using 16S RNA (ribosomal ribonucleic acid) amplicon sequencing. This high-throughput sequencing analysis produced ~20 000 000 16S rRNA V4 reads, which comprised 7000 OTUs. The main variables structuring these communities were sample origins (fjord or mesocosms) and the community size fraction (small or large size fraction). The community was significantly different between the unenclosed fjord water and enclosed mesocosms (both control and elevated CO₂ treatments) after nutrients were added to the mesocosms, suggesting that the addition of nutrients is the primary driver of the change in mesocosm community structure. The relative importance of each structuring variable depended greatly on the time at which the community was sampled in relation to the phytoplankton bloom. The sampling strategy of separating the small and large size fraction was the second most important factor for community structure. When the small and large size fraction bacteria were analysed separately at different time points, the only taxon pCO₂ was found to significantly affect were the Gammaproteobacteria after nutrient addition. Finally, pCO₂ treatment was found to be significantly correlated (non-linear) with 15 rare taxa, most of which increased in abundance with higher CO₂.