Development of a colorimetric microfluidic pH sensor for autonomous seawater measurements

High quality carbonate chemistry measurements are required in order to fully understand the dynamics of the oceanic carbonate system. Seawater pH data with good spatial and temporal coverage are particularly critical to apprehend ocean acidification phenomena and their consequences. There is a growing need for autonomous in situ instruments that measure pH on remote platforms. Our aim is to develop an accurate and precise autonomous in situ pH sensor for long term deployment on remote platforms. The widely used spectrophotometric pH technique is capable of the required high-quality measurements. We report a key step toward the miniaturisation of a colorimetric pH sensor with the successful implementation of a simple microfluidic design with low reagent consumption. The system is particularly adapted to shipboard deployment: high quality data was obtained over a period of more than a month during a shipboard deployment in northwest European shelf waters, and less than 30 mL of indicator was consumed. The system featured a short term precision of 0.001 pH (n=20) and an accuracy within the range of a certified Tris buffer (0.004 pH). The quality of the pH system measurements have been checked using various approaches: measurements of certified Tris buffer, measurement of certified seawater for DIC and TA, comparison of measured pH against calculated pH from pCO₂, DIC and TA during the cruise in northwest European shelf waters. All showed that our measurements were of high quality. The measurements were made close to in situ temperature (+0.2 °C) in a sampling chamber which had a continuous flow of the ship’s underway seawater supply. The optical set up was robust and relatively small due to the use of an USB mini-spectrometer, a custom made polymeric flow cell and an LED light source. The use of a three wavelength LED with detection that integrated power across the whole of each LED output spectrum indicated that low wavelength resolution detectors can be used instead of the current USB mini spectrophotometer. Artefacts due to the polychromatic light source and inhomogeneity in the absorption cell are shown to have a negligible impact on the data quality. The next step in the miniaturisation of the sensor will be the incorporation of a photodiode as detector to replace the spectrophotometer.

Ecosystem effects of shell aggregations and cycling in coastal waters: an example of Chesapeake Bay oyster reefs

Disease, overharvesting, and pollution have impaired the role of bivalves on coastal ecosystems, some to the point of functional extinction. An underappreciated function of many bivalves in these systems is shell formation. The ecological significance of bivalve shell has been recognized; geochemical effects are now more clearly being understood. A positive feedback exists between shell aggregations and healthy bivalve populations in temperate estuaries, thus linking population dynamics to shell budgets and alkalinity cycling. On oyster reefs a balanced shell budget requires healthy long-lived bivalves to maximize shell input per mortality event thereby countering shell loss. Active and dense populations of filter-feeding bivalves couple production of organic-rich waste with precipitation of calcium carbonate minerals, creating conditions favorable for alkalinity regeneration. Although the dynamics of these processes are not well described, the balance between shell burial and metabolic acid production seems the key to the extent of alkalinity production vs. carbon burial as shell. We present an estimated alkalinity budget that highlights the significant role oyster reefs once played in the Chesapeake Bay inorganic-carbon cycle. Sustainable coastal and estuarine bivalve populations require a comprehensive understanding of shell budgets and feedbacks among population dynamics, agents of shell destruction, and anthropogenic impacts on coastal carbonate chemistry.

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool-scenario

Rising atmospheric CO₂-concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO₂-concentrations in a rockpool scenario. U. lactuca was cultured under aeration with air containing either preindustrial pCO₂ (280 μatm) or the pCO₂ predicted by the end of the 21st century (700 μatm) for 31 days. We addressed the following question: Will elevated CO₂-concentrations affect photosynthesis (net photosynthesis, maximum relative electron transport rate (rETR(max)), maximum quantum yield (Fv/Fm), pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1–4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO₂ condition by continuous aeration with target pCO₂ values. In the second phase (day 4–31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO₂-concentrations, whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO₂-concentrations. At low CO₂-concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO₂-concentrations most thalli remained integer until day 31. Thallus disintegration at low CO₂-concentrations was mirrored by a drastic decline in seawater dissolved inorganic carbon and HCO₃⁻. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO₂-concentrations. The accelerated thallus disintegration at high CO₂-concentrations under conditions of limited water exchange indicates additional CO₂ effects on the life cycle of U. lactuca when living in rockpools.

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.

Long-term trends in calcifying plankton and pH in the North Sea

Relationships between six calcifying plankton groups and pH are explored in a highly biologically productive and data-rich area of the central North Sea using time-series datasets. The long-term trends show that abundances of foraminiferans, coccolithophores, and echinoderm larvae have risen over the last few decades while the abundances of bivalves and pteropods have declined. Despite good coverage of pH data for the study area there is uncertainty over the quality of this historical dataset; pH appears to have been declining since the mid 1990s but there was no statistical connection between the abundance of the calcifying plankton and the pH trends. If there are any effects of pH on calcifying plankton in the North Sea they appear to be masked by the combined effects of other climatic (e.g. temperature), chemical (nutrient concentrations) and biotic (predation) drivers. Certain calcified plankton have proliferated in the central North Sea, and are tolerant of changes in pH that have occurred since the 1950s but bivalve larvae and pteropods have declined. An improved monitoring programme is required as ocean acidification may be occurring at a rate that will exceed the environmental niches of numerous planktonic taxa, testing their capacities for acclimation and genetic adaptation.

Marine fungi may benefit from ocean acidification

Recent studies have discussed the consequences of ocean acidification for bacterial processes and diversity. However, the decomposition of complex substrates in marine environments, a key part of the flow of energy in ecosystems, is largely mediated by marine fungi. Although marine fungi have frequently been reported to prefer low pH levels, this group has been neglected in ocean acidification research. We present the first investigation of direct pH effects on marine fungal abundance and community structure. In microcosm experiments repeated in 2 consecutive years, we incubated natural North Sea water for 4 wk at in situ seawater pH (8.10 and 8.26), pH 7.82 and pH 7.67. Fungal abundance was determined by colony forming unit (cfu) counts, and fungal community structure was investigated by the culture-independent fingerprint method Fungal Automated Ribosomal Intergenic Spacer Analysis (F-ARISA). Furthermore, pH at the study site was determined over a yearly cycle. Fungal cfu were on average 9 times higher at pH 7.82 and 34 times higher at pH 7.67 compared to in situ seawater pH, and we observed fungal community shifts predominantly at pH 7.67. Currently, surface seawater pH at Helgoland Roads remains >8.0 throughout the year; thus we cannot exclude that fungal responses may differ in regions regularly experiencing lower pH values. However, our results suggest that under realistic levels of ocean acidification, marine fungi will reach greater importance in marine biogeochemical cycles. The rise of this group of organisms will affect a variety of biotic interactions in the sea.

Deformities in larvae and juvenile European lobster (Homarus gammarus) exposed to lower pH at two different temperatures

Trends of increasing temperatures and ocean acidification are expected to influence benthic marine resources, especially calcifying organisms. The European lobster (Homarus gammarus) is among those species at risk. A project was initiated in 2011 aiming to investigate long-term synergistic effects of temperature and projected increases in ocean acidification on the life cycle of lobster. Larvae were exposed to pCO₂ levels of ambient water (water intake at 90 m depth, tentatively of 380 μatm pCO₂), 727 and 1217 μatm pCO₂, at temperatures 10 and 18 °C. Long-term exposure lasted until 5 months of age. Thereafter the surviving juveniles were transferred to ambient water at 14 °C. At 18 °C the development from Stage 1 to 4 lasted from 14 to 16 days, as predicted under normal pH values. Growth was very slow at 10 °C and resulted in only two larvae reaching Stage 4 in the ambient treatment. There were no significant differences in carapace length at the various larval stages between the different treatments, but there were differences in total length and dry weight at Stage 1 at 10 °C, Stage 2 at both temperatures, producing larvae slightly larger in size and lighter by dry weight in the exposed treatments. Stage 3 larvae raised in 18 °C and 1217 μatm pCO₂ were also larger in size and heavier by dry weight compared with 727 μatm. Unfortunate circumstances precluded a full comparison across stages and treatment. Deformities were however observed in both larvae and juveniles. At 10 °C, about 20% of the larvae exposed to elevated pCO₂were deformed, compared with 0% in larvae raised in pH above 8.0. At 18 °C and in high pCO₂ treatment, 31.5% of the larvae were deformed. Occurrence of deformities after 5 months of exposure was 33 and 44% in juveniles raised in ambient and low pCO₂, respectively, and 20% in juveniles exposed to high pCO₂. Some of the deformities will possibly affect the ability to find food, sexual partner (walking legs, claw and antenna), respiration (carapace), and ability to swim (tail-fan damages).

Consumers mediate the effects of experimental ocean acidification and warming on primary producers

It is well known that ocean acidification can have profound impacts on marine organisms. However, we know little about the direct and indirect effects of ocean acidification and also how these effects interact with other features of environmental change such as warming and declining consumer pressure. In this study, we tested whether the presence of consumers (invertebrate mesograzers) influenced the interactive effects of ocean acidification and warming on benthic microalgae in a seagrass community mesocosm experiment. Net effects of acidification and warming on benthic microalgal biomass and production, as assessed by analysis of variance, were relatively weak regardless of grazer presence. However, partitioning these net effects into direct and indirect effects using structural equation modeling revealed several strong relationships. In the absence of grazers, benthic microalgae were negatively and indirectly affected by sediment-associated microalgal grazers and macroalgal shading, but directly and positively affected by acidification and warming. Combining indirect and direct effects yielded no or weak net effects. In the presence of grazers, almost all direct and indirect climate effects were nonsignificant. Our analyses highlight that (i) indirect effects of climate change may be at least as strong as direct effects, (ii) grazers are crucial in mediating these effects, and (iii) effects of ocean acidification may be apparent only through indirect effects and in combination with other variables (e.g., warming). These findings highlight the importance of experimental designs and statistical analyses that allow us to separate and quantify the direct and indirect effects of multiple climate variables on natural communities.

A potential tool for high-resolution monitoring of ocean acidification

Current anthropogenic carbon dioxide emissions generate besides global warming unprecedented acidification rates of the oceans. Recent evidence indicates the possibility that ocean acidification and low oceanic pH may be a major reason for several mass extinctions in the past. However, a major bottleneck for research on ocean acidification is long-term monitoring and the collection of consistent high-resolution pH measurements. This study presents a low-power (< 1 W) small sample volume (25 μl) semiconductor based fluorescence method for real-time ship-board pH measurements at high temporal and spatial resolution (approximately 15 s and 100 m between samples). A 405 nm light emitting diode and the blue and green channels from a digital camera was used for swift detection of fluorescence from the pH sensitive dye 6,8-Dihydroxypyrene-1,3-disulfonic acid in real-time. Main principles were demonstrated by automated continuous measurements of pH in the surface water across the Baltic Sea and the Kattegat region with a large range in salinity (∼ 3–30) and temperature (∼ 0–25 °C). Ship-board precision of salinity and temperature adjusted pH measurements were estimated as low as 0.0001 pH units.

Biological impacts of enhanced alkalinity in Carcinus maenas

Further steps are needed to establish feasible alleviation strategies that are able to reduce the impacts of ocean acidification, whilst ensuring minimal biological side-effects in the process. Whilst there is a growing body of literature on the biological impacts of many other carbon dioxide reduction techniques, seemingly little is known about enhanced alkalinity. For this reason, we investigated the potential physiological impacts of using chemical sequestration as an alleviation strategy. In a controlled experiment, Carcinus maenas were acutely exposed to concentrations of Ca(OH)₂ that would be required to reverse the decline in ocean surface pH and return it to pre-industrial levels. Acute exposure significantly affected all individuals’ acid–base balance resulting in slight respiratory alkalosis and hyperkalemia, which was strongest in mature females. Although the trigger for both of these responses is currently unclear, this study has shown that alkalinity addition does alter acid–base balance in this comparatively robust crustacean species.