Bacterial extracellular enzyme activity in a future ocean

<p>Heterotrophic bacteria are recognised as vital components in the cycling and regulation of inorganic and organic matter in the ocean. Research to date indicates that future changes in ocean conditions may influence bacterial extracellular enzyme hydrolysis rates, which could affect the strength of the microbial loop and consequently organic matter export. The aim of this thesis was to examine how changes in ocean acidification and warming predicted to occur by the end of the century will affect extracellular enzyme activities in the near-surface ocean and below the surface mixed layer in the South West Pacific. A series of small-scale seawater incubations were conducted under three different perturbed conditions: elevated temperature (ambient +3°C), low pH (pCO₂ 750 ppmv; pHт 7.8) and greenhouse conditions (elevated temperature and low pH), with responses compared to ambient control samples. In particular, the response of protease activity (leucine- and arginine-aminopeptidase) and glucosidase activity (β- and α-glucosidase) were examined, as these enzymes are known to degrade the two major components of organic matter in the ocean, namely proteins and carbohydrates. Bacterial secondary production rates (³H-TdR & ³H-Leu incorporation) were also examined as a proxy for carbon turnover. To investigate spatial variability, parameter responses from near-surface open ocean seawater consisting of different phytoplankton communities were compared with coastal seawater, as well as seawater collected from below the surface mixed layer. To determine temporal variability, both direct and indirect parameter responses were investigated. Finally, responses were determined from a shallow CO₂ vent that provided a natural low pH environment in coastal waters north of New Zealand. By comparing responses derived from vent water and artificially low pH water, vent plumes were also investigated for their utility as proxies for future low pH environments. Incubation results showed that protease activity increased in response to low pH conditions in each seawater environment tested. However, near-surface open ocean incubations showed variability in the response of protease and glucosidase activity and bacterial cell numbers between different phytoplankton communities and treatments, suggesting that parameter responses were determined by direct and indirect effects. Elevated temperature had an overall positive effect on bacterial secondary production rates between different phytoplankton communities in the near-surface open ocean. Surprisingly, although elevated temperature and low pH treatments showed independent effects, no clear additive or synergistic effect was detected in any parameter under greenhouse conditions. In contrast to the near-surface ocean, greenhouse conditions had an additive effect on protease activity in seawater collected from below the surface mixed layer (100 m depth). Bacterial secondary production rates and bacterial numbers varied in response to elevated temperature in the subsurface ocean, while bacterial secondary production rates declined under greenhouse conditions. Glucosidase and protease activities were highest in the coastal seawater, with both enzymes responding positively to low pH conditions. Coastal seawater also contained the highest bacterial secondary production rates and bacterial cell numbers, however these parameters were not significantly affected by low pH conditions. Variation in the direct response of enzyme activity to low pH between ocean environments could indicate the synthesis of different extracellular enzymes by surface and subsurface bacteria. Importantly, results from a naturally low pH vent plume indicated that pH was not the only factor influencing the response of extracellular enzymes. Other influential factors could include high concentrations of dissolved nutrients and trace metal ions. Natural low pH vents off Whale Island in the Bay of Plenty were determined not suitable as proxies for future low pH environments based on vent variability and differences in seawater biogeochemistry when compared to the ambient ocean. Overall, the incubation results show that under conditions predicted for the end of the century, protease activity will increase in open ocean and coastal waters which could accelerate and strengthen the heterotrophic microbial loop. Bacterial secondary production rates are expected to vary in the near-surface ocean, but decline in the subsurface. The resulting increase in surface ocean protease activity could increase heterotrophic metabolic respiration and reduce organic matter export, weaken the biological carbon pump and diminish long-term carbon sequestration. An increased turnover of proteins and amino acids in each environment tested could lead to nitrogen limitation and contribute to an expansion of oligotrophic waters. This future scenario may create a positive inorganic carbon feedback that would further exacerbate acidification of the surface ocean.</p>

Bacterial extracellular enzyme activity in a future ocean

<p>Heterotrophic bacteria are recognised as vital components in the cycling and regulation of inorganic and organic matter in the ocean. Research to date indicates that future changes in ocean conditions may influence bacterial extracellular enzyme hydrolysis rates, which could affect the strength of the microbial loop and consequently organic matter export. The aim of this thesis was to examine how changes in ocean acidification and warming predicted to occur by the end of the century will affect extracellular enzyme activities in the near-surface ocean and below the surface mixed layer in the South West Pacific. A series of small-scale seawater incubations were conducted under three different perturbed conditions: elevated temperature (ambient +3°C), low pH (pCO₂ 750 ppmv; pHт 7.8) and greenhouse conditions (elevated temperature and low pH), with responses compared to ambient control samples. In particular, the response of protease activity (leucine- and arginine-aminopeptidase) and glucosidase activity (β- and α-glucosidase) were examined, as these enzymes are known to degrade the two major components of organic matter in the ocean, namely proteins and carbohydrates. Bacterial secondary production rates (³H-TdR & ³H-Leu incorporation) were also examined as a proxy for carbon turnover. To investigate spatial variability, parameter responses from near-surface open ocean seawater consisting of different phytoplankton communities were compared with coastal seawater, as well as seawater collected from below the surface mixed layer. To determine temporal variability, both direct and indirect parameter responses were investigated. Finally, responses were determined from a shallow CO₂ vent that provided a natural low pH environment in coastal waters north of New Zealand. By comparing responses derived from vent water and artificially low pH water, vent plumes were also investigated for their utility as proxies for future low pH environments. Incubation results showed that protease activity increased in response to low pH conditions in each seawater environment tested. However, near-surface open ocean incubations showed variability in the response of protease and glucosidase activity and bacterial cell numbers between different phytoplankton communities and treatments, suggesting that parameter responses were determined by direct and indirect effects. Elevated temperature had an overall positive effect on bacterial secondary production rates between different phytoplankton communities in the near-surface open ocean. Surprisingly, although elevated temperature and low pH treatments showed independent effects, no clear additive or synergistic effect was detected in any parameter under greenhouse conditions. In contrast to the near-surface ocean, greenhouse conditions had an additive effect on protease activity in seawater collected from below the surface mixed layer (100 m depth). Bacterial secondary production rates and bacterial numbers varied in response to elevated temperature in the subsurface ocean, while bacterial secondary production rates declined under greenhouse conditions. Glucosidase and protease activities were highest in the coastal seawater, with both enzymes responding positively to low pH conditions. Coastal seawater also contained the highest bacterial secondary production rates and bacterial cell numbers, however these parameters were not significantly affected by low pH conditions. Variation in the direct response of enzyme activity to low pH between ocean environments could indicate the synthesis of different extracellular enzymes by surface and subsurface bacteria. Importantly, results from a naturally low pH vent plume indicated that pH was not the only factor influencing the response of extracellular enzymes. Other influential factors could include high concentrations of dissolved nutrients and trace metal ions. Natural low pH vents off Whale Island in the Bay of Plenty were determined not suitable as proxies for future low pH environments based on vent variability and differences in seawater biogeochemistry when compared to the ambient ocean. Overall, the incubation results show that under conditions predicted for the end of the century, protease activity will increase in open ocean and coastal waters which could accelerate and strengthen the heterotrophic microbial loop. Bacterial secondary production rates are expected to vary in the near-surface ocean, but decline in the subsurface. The resulting increase in surface ocean protease activity could increase heterotrophic metabolic respiration and reduce organic matter export, weaken the biological carbon pump and diminish long-term carbon sequestration. An increased turnover of proteins and amino acids in each environment tested could lead to nitrogen limitation and contribute to an expansion of oligotrophic waters. This future scenario may create a positive inorganic carbon feedback that would further exacerbate acidification of the surface ocean.</p>

Coomassie Blue G250 for Visualization of Active Bacteria from Lake Environment and Culture

Bacteria play a fundamental role in the cycling of nutrients in aquatic environments. A precise distinction between active and inactive bacteria is crucial for the description of this process. We have evaluated the usefulness of Coomassie Blue G250 for fluorescent staining of protein containing potentially highly active bacteria. We found that the G250 solution has excitation and emission properties appropriate for direct epifluorescence microscopy observations. It enables fast and effective fluorescent visualization of living, protein-rich bacteria, both in freshwater environment and culture. Our results revealed that the number of G250-stained bacteria from eutrophic lake was positively correlated with other standard bacterial activity markers, like number of bacteria containing 16S rRNA, bacterial secondary production or maximal potential leucine-aminopeptidase activity. In case of the E. coli culture, the percentage of bacteria visualized with G250 was similar to that of bacteria which accumulated tetracycline. Compared to other common methods utilizing fluorogenic substances for bacteria staining, the approach we evaluated is inexpensive and less hazardous (for example mutagenic) to the environment and researchers. It can be regarded as an additional or alternative method for protein rich, active bacteria staining.

Primary Productivity and heterotrophic activity in an enclosed marine area of central Patagonia (Puyuhuapi channel; 44° S, 73° W)

We assessed temporal variability in phytoplankton biomass, Chlorophyll a, nutrient availability, Gross Primary Production (GPP), community respiration (CR), and bacterial secondary production (BSP) over a year of monthly observations (October 2007 to October 2008) at a fixed station in the Puyuhuapi fjord, Chilean Patagonia (44° S, 73° W). A set of in situ observations gathered over two consecutive spring-summer seasons, and one autumn-winter season in the middle, has made it possible to connect the two-phase (i.e. productive season/non-productive season) pattern of Chlorophyll a (Chl a) variability shown by satellite data with a two-phase cycle in GPP, CR, and the composition of phytoplankton assemblages. Estimates of annual GPP and CR, integrated over the top 20 meters of the water column, were 533 and 537 g C m−2 yr−1, respectively. Low values of pCO2 were measured in mixed layer autotrophic waters (GPP/CR > 1) while high pCO2 levels were measured in mixed layer heterotrophic waters (GPP/CR < 1). Bacterial Secondary Production (BSP) was significantly and positively correlated with GPP (r = 0.6, p < 0.05, n = 24) and Chl a (r = 0.4, p < 0.05, n = 24) on an annual cycle basis. The winter drop in bacterioplankton (both bacteria and archea) activity (from 0.9 ± 0.6 g C m−2 d−1 to 0.6 ± 0.3 g C m−2 d−1) was not as pronounced as the winter drop in phytoplankton activity (from 1.1 ± 1.12 g C m−2 d−1 to 0.1 ± 0.09 g C m−2 d−1). It is hypothesized that dissolved organic matter (DOM) of terrestrial origin plays an important role (especially in winter) supporting bacterial activity in the Puyuhuapi fjord.

Bacteria and phytoplankton production rates in eight river stretches of the middle Rio Doce hidrographic basin (southeast Brazil)

Measurements of bacterial secondary production (BSP), together with primary phytoplanktonic production (PPP) were conducted during dry and rainy seasons, in eight rivers of different orders submitted to different degrees of human impacts (different trophic degree). We aimed to determine and compare the importance of BSP and PPP in carbon fixation in these different lotic ecosystems. Our results showed that the Ipanema River was the most modified system by anthropogenic effluents inputs. These inputs altered the trophic degree and BSP rates of these streams and rivers.