Final Report Summary - ACIDIBACLIGHT (Impacts of Ocean Acidification on Bacterioplankton Functioning: Effects on Proteorhodopsin-containing Marine Bacteria)
1. FINAL PUBLISHABLE SUMMARY
The aim of the funded project was to evaluate the effects of the ocean acidification on marine bacterioplankton with a particular focus on bacteria that are able to use the light as an energy source using proteorhodopsin photosystems. During this project the researcher has been trained to successfully: 1) Accurately measure pH in seawater, 2) apply pH manipulation techniques using air with different concentrations of CO2gas on two different setups and 3) measure environmental levels of pH in the Ocean to directly correlate them with bacterial diversity and the presence of proteorhodopsin photosystems.
The project was divided into three approaches with different levels of control/manipulation; the results of each module are described bellow:
1) Highly-controlled/manipulated systems: study of the physiology of different bacterial strains in pure cultures. For this purpose, she used the strain Dokdonia donghaensis (MED134), which contains the proteorhodopsin gene and is known to use light to improve its growth in seawater. When exposed to pre-industrial CO2levels (190 ppm CO2), bacteria showed reduced growth rates within the first 24 hours as compared to the ones exposed to present CO2 concentrations (Fig 1, A and B). Between 24 and 48 hours incubation, bacterial growth in the light stopped but remained constant in the dark. After 72 hours, bacterial numbers in the light increased while decreased significantly in the dark. On the treatments exposed to present CO2 concentrations, bacterial numbers increased within the first 24 hours, most significantly in the light, then remained constant until 72 hours incubation to then increase in the light treatments only. At CO2 concentrations predicted for year 2100, bacterial numbers did not increase during the first 48 hours of incubation, after 72 hours numbers went up and then stabilized on both light and dark treatments. These encouraging results, however, appeared to be difficult to replicate because of vitamin contamination in the seawater media. However, the researcher is presently running the last tests to ensure the consistency of the results before the data are published. Our preliminary data shows that proteorhodopsins in Dokdonia donghaensis MED134 provide the greatest growth advantage at present CO2 concentrations (where bacteria grew more than 4 times better in light as compared to dark), followed by pre-industrial concentrations (almost 4 times better growth in the light) and could stop providing a growth advantage in the light at 750 ppm. Thus, ocean acidification could have a negative effect on PR-containing bacteria (Figure 1). More than 50% of marine bacteria in the oceans are known to contain proteorhodopsins and if these results hold true for most of all of those species, the results obtained here could have great impact on our future predictions of CO2 impacts in the environment. For this reason, this first experiments will be repeated and performed for longer periods of time, and also other PR-containing species will be included to provide with more solid evidence to this observations.
2) Relatively controlled/manipulated systems: use of mesocosm experiments where we can study the response of the bacterioplankton community as a whole to the specific pH manipulations. The experiment consisted of 8 incubations of 200 liters of seawater, bubbled with CO2 gas to reach present CO2 concentrations (380 ppm) and predicted concentrations for year 2100 (750 ppm). In addition to the CO2 disturbance, two replicates of each CO2 treatment were amended with nutrients (N, P and Si). The addition of nutrients was done to reduce possible nutrient limitation on phyto- and bacterioplankton.
A high number of parameters were sampled on this experiment (in cooperation with other researchers): bacteria, virus and phytoplankton abundance, bacterial production and enzymatic activities, bacterial diversity (through ARISA and 454 tag sequencing) and expression (metatranscriptomics), pH and alkalinity. Phytoplankton abundance inferred by chlorophyll a concentration showed that the treatments with both nutrients and high CO2 concentration were most stimulated, followed by the treatments with nutrients and present CO2 concentrations (Figure 2). This result could be expected since photosynthesis metabolism depends on CO2 acquisition and the higher concentration might favor the process. Bacterial abundance followed a similar trend, however the numbers dropped on the first two days of incubation and then increased on the nutrient treatments. Phyto- and bacterioplankton diversity are expected to give some hints on the specific species that are most affected by the CO2 increase. We are presently analyzing those diversity data as well as the retinal concentrations that will be used as a proxy for proteorhodopsin abundance.
3) Un-manipulated systems: in situ analyses of natural communities. The researcher participated the 6-week oceanographic cruise “Hotmix” in May-June 2014 where she analyzed the pH and bicarbonate concentrations and took retinal samples along with DNA for diversity analysis. This cruise consisted on a 29-stations transect that started in the east Mediterranean and ended in the Atlantic Ocean by the Canary Islands (Figure 3). Not only this transect followed an important nutrient and organic matter gradient that may also affect the use of phototrophy, but also allow samplings at different Ocean depths, which are physicochemically different. These priceless samples not only represent the first quantifications of proteorhodopsin in different environmental settings, but will also allow the detection of pH effects on the presents of these photosystems.
The aim of the funded project was to evaluate the effects of the ocean acidification on marine bacterioplankton with a particular focus on bacteria that are able to use the light as an energy source using proteorhodopsin photosystems. During this project the researcher has been trained to successfully: 1) Accurately measure pH in seawater, 2) apply pH manipulation techniques using air with different concentrations of CO2gas on two different setups and 3) measure environmental levels of pH in the Ocean to directly correlate them with bacterial diversity and the presence of proteorhodopsin photosystems.
The project was divided into three approaches with different levels of control/manipulation; the results of each module are described bellow:
1) Highly-controlled/manipulated systems: study of the physiology of different bacterial strains in pure cultures. For this purpose, she used the strain Dokdonia donghaensis (MED134), which contains the proteorhodopsin gene and is known to use light to improve its growth in seawater. When exposed to pre-industrial CO2levels (190 ppm CO2), bacteria showed reduced growth rates within the first 24 hours as compared to the ones exposed to present CO2 concentrations (Fig 1, A and B). Between 24 and 48 hours incubation, bacterial growth in the light stopped but remained constant in the dark. After 72 hours, bacterial numbers in the light increased while decreased significantly in the dark. On the treatments exposed to present CO2 concentrations, bacterial numbers increased within the first 24 hours, most significantly in the light, then remained constant until 72 hours incubation to then increase in the light treatments only. At CO2 concentrations predicted for year 2100, bacterial numbers did not increase during the first 48 hours of incubation, after 72 hours numbers went up and then stabilized on both light and dark treatments. These encouraging results, however, appeared to be difficult to replicate because of vitamin contamination in the seawater media. However, the researcher is presently running the last tests to ensure the consistency of the results before the data are published. Our preliminary data shows that proteorhodopsins in Dokdonia donghaensis MED134 provide the greatest growth advantage at present CO2 concentrations (where bacteria grew more than 4 times better in light as compared to dark), followed by pre-industrial concentrations (almost 4 times better growth in the light) and could stop providing a growth advantage in the light at 750 ppm. Thus, ocean acidification could have a negative effect on PR-containing bacteria (Figure 1). More than 50% of marine bacteria in the oceans are known to contain proteorhodopsins and if these results hold true for most of all of those species, the results obtained here could have great impact on our future predictions of CO2 impacts in the environment. For this reason, this first experiments will be repeated and performed for longer periods of time, and also other PR-containing species will be included to provide with more solid evidence to this observations.
2) Relatively controlled/manipulated systems: use of mesocosm experiments where we can study the response of the bacterioplankton community as a whole to the specific pH manipulations. The experiment consisted of 8 incubations of 200 liters of seawater, bubbled with CO2 gas to reach present CO2 concentrations (380 ppm) and predicted concentrations for year 2100 (750 ppm). In addition to the CO2 disturbance, two replicates of each CO2 treatment were amended with nutrients (N, P and Si). The addition of nutrients was done to reduce possible nutrient limitation on phyto- and bacterioplankton.
A high number of parameters were sampled on this experiment (in cooperation with other researchers): bacteria, virus and phytoplankton abundance, bacterial production and enzymatic activities, bacterial diversity (through ARISA and 454 tag sequencing) and expression (metatranscriptomics), pH and alkalinity. Phytoplankton abundance inferred by chlorophyll a concentration showed that the treatments with both nutrients and high CO2 concentration were most stimulated, followed by the treatments with nutrients and present CO2 concentrations (Figure 2). This result could be expected since photosynthesis metabolism depends on CO2 acquisition and the higher concentration might favor the process. Bacterial abundance followed a similar trend, however the numbers dropped on the first two days of incubation and then increased on the nutrient treatments. Phyto- and bacterioplankton diversity are expected to give some hints on the specific species that are most affected by the CO2 increase. We are presently analyzing those diversity data as well as the retinal concentrations that will be used as a proxy for proteorhodopsin abundance.
3) Un-manipulated systems: in situ analyses of natural communities. The researcher participated the 6-week oceanographic cruise “Hotmix” in May-June 2014 where she analyzed the pH and bicarbonate concentrations and took retinal samples along with DNA for diversity analysis. This cruise consisted on a 29-stations transect that started in the east Mediterranean and ended in the Atlantic Ocean by the Canary Islands (Figure 3). Not only this transect followed an important nutrient and organic matter gradient that may also affect the use of phototrophy, but also allow samplings at different Ocean depths, which are physicochemically different. These priceless samples not only represent the first quantifications of proteorhodopsin in different environmental settings, but will also allow the detection of pH effects on the presents of these photosystems.
