TDU MFC
5.1 Introduction
Chapter 5
Effects of oxidative stress on the
production of bromoform by two marine diatom species
This study examines the role of nutrient limitation on the production of bromoform in two axenic diatom species. The cultures were grown in laboratory conditions and nutrient limitation was achieved by the addition of sodium hydroxide or starvation. The bromoform per cell concentrations increased during the exponential growth phase, but displayed significant decreases during the limited phases of the experiments.
The work presented here was a collaborative effort with the diatom culturing being performed by Mariam Nguvava, as part of her MSc (Nguvava, 2012).
of bromoform in the agar plates did not seem to affect the grazing by the snail (Gschwend et al., 1985).
No conclusions can be drawn from this single study. Although there is little evidence to support the theory, it has been suggested that bacterial attack might elicit a bromoform release response from macro- and microalgae (Quack and Wallace, 2003; Wever et al., 1991; Moore et al., 1996; Küpper et al., 2008;
Palmer et al., 2005). In this regard, it is therefore unlikely that bromoform is released as a defence mechanism against grazing or bacterial attack. Numerous studies have demonstrated increased bromoform release from micro- and macroalgae placed under various different oxidative stresses including increased irradiance, increased concentrations of ozone, oligoguluronates and hydrogen peroxide (Palmer et al., 2005; Pedersén et al., 1996; Manley and Barbero, 2001).
The mechanisms of formation of bromoform in macroalgae are not clearly understood (Quack et al., 2007; Palmer et al., 2005; Weinberger et al., 2007). While an abiotic formation process has been proposed, such as the irradiance of seawater with UV, the main formation process suggested centres around the oxidation of bromine atoms by the bromoperoxidase enzyme (BrPO) and the subsequent reaction with dissolved organic matter‘(Quack and Wallace, 2003; Class and Ballschmiter, 1987). The partial decay of the brominated dissolved organic matter via the haloform reaction results in the formation of bromoform (Wever et al., 1991; Pedersén et al., 1996; Paul and Pohnert, 2011; Manley and Barbero, 2001; Wever and Van der Horst, 2013; Palmer et al., 2005; Hill and Manley, 2009). While the presence of the bromoperoxidase enzyme has long been known in macroalgae (Wever et al., 1991), it has only more recently been shown in microalgae (Tokarczyk and Moore, 1994; Moore et al., 1996). Bromoperoxidase catalytically oxidises halide anions (Br–) in the presence of hydrogen peroxide (H2O2) which results in the formation of hypobromous acid (HOBr). The HOBr rapidly reacts with dissolved organic matter (DOM) near the ocean surface, producing unstable brominated intermediaries. The intermediaries decay via the haloform reaction, which results in the formation of bromoform (Reactions 5.1 – 5.3; Hill and Manley, 2009; Wever and Van der Horst, 2013; Paul and Pohnert, 2011). The rate of reaction between HOBr and DOM is of the order of milliseconds. In many species of marine algae BrPO enzymes have been shown to exist both intra- and extracellularly, proving a mechanism to cope with intra- and extracellular hydrogen peroxide (Wever and Van der Horst, 2013; Paul and Pohnert, 2011).
H2O2+H++Br−−−−−→BrP O HOBr+H2O (5.1)
HOBr+DOM →DOM(Br) (5.2)
DOM(Br)→CHBr3+DOM (5.3)
Increased light intensities have often been linked with elevated rates of bromoform production (e.g., Hill and Manley, 2009; Mtolera et al., 1996; Wever and Van der Horst, 2013; Carpenter et al., 2000; Pedersén et al., 1996; Collén et al., 1994; Palmer et al., 2005). During daylight hours hydrogen peroxide is produced during photosynthesis by means of the Mehler reaction. Mitochondrial respiration at night produces hydrogen peroxide, though at a much lower rate than photosynthesis (Pedersén et al., 1996; Collén et al.,
5.1. Introduction 117 1994; Hill and Manley, 2009). The intra- and extracellular H2O2, which is poisonous to algae (Pedersén et al., 1996; Collén et al., 1994), results in oxidative stress within the algae (Hill and Manley, 2009).
It is suggested that the formation of H2O2results in the bromoform diurnal cycle described previously (Chapter 4.1.3). Increased light intensities have also been suggested to result in the decay of algal tissue during the summer period. This decay has been linked with a peak in measured bromoform mixing ratios during the summer period (Goodwin et al., 1997; Quack and Wallace, 2003). Carbon limitation has been shown to result in an oxidative stress similar to that induced by sunlight (Mtolera et al., 1996 and references therein). Some laboratory studies have shown a relationship between bromoform concentration and pH (Mtolera et al., 1996). The decreasing acidity as pH increases might favour the formation of bromoform through one of the steps of formation (Reactions 5.1 – 5.3).
The releases of reactive oxygen species (ROS, hydrogen peroxide (H2O2), hydroxyl radical (OH), superoxide (O–2)) is a well documented defence trigger in macro- and microalgae (Küpper et al., 2008;
Palmer et al., 2005). It is suggested that the ROS may be rapidly released from the plant in an oxidative burst (Küpper et al., 2008). The sudden influx of oxidative species may result in the rapid formation of bromoform. The bromoform would be produced as a by-product of the BrPO enzyme scavenging the ROS and then reacts with DOM and is reduced via the haloform reaction as discussed. Although not necessarily allelopathic, the formation of bromoform may therefore be the by-product of algal defence against grazing (Küpper et al., 2008).
Seasonal variations in the rate of production of bromoform have been observed in multiple loca- tions (Laturnus, 1996; Carpenter et al., 2000; Zhou et al., 2008). The seasonal variation might be influenced by changes in sunlight, DOM concentrations or ocean temperature associated with the different seasons (Laturnus, 1996; Itoh and Shinya, 1994; Carpenter et al., 2000). The rate of photosynthesis in algae would be affected by changes in light intensity with the seasons, resulting in a maximum in summer and minimum in winter. In theory the decrease in photosynthetic rate would result in less H2O2 being formed, thus producing less bromoform. The local concentrations of DOM have been reported to vary through the seasons with a maxium in summer (Quack and Wallace, 2003). Variations in the DOM would affect the formation of bromoform by limiting the amount of matter that can react with HOBr to form intermediaries (Reaction 5.2). The rate of efficiency of the BrPO enzyme has been shown to be inversely related to sea surface temperature (Itoh and Shinya, 1994; Laturnus, 1996). Variation of the efficiency of the BrPO enzyme might limit the formation of HOBr, resulting in a limitation of bromoform (Reaction 5.1).
The effect of oxidative stress in the form of nutrient limitation on bromoform production from two sub-tropical marine diatoms, cultured in axenic conditions (i.e. with no bacteria present), was explored in this project. Because bacteria have also been shown to produce bromoform (Manley et al., 1992;
Quack and Wallace, 2003), this creates a complication of attributing the production of bromoform to the microalgae or bacteria present in phytoplankton laboratory cultures contaminated by bacteria and in mixed community environmental samples (Tokarczyk and Moore, 1994). Mixed community environmental
samples are also rarely done so this necessitates the need for laboratory microalgal culture experiments to be run under axenic conditions (Tokarczyk and Moore, 1994). After being grown under continuous light, the diatom species here were first subjected to carbon (CO2) limitation, which has been shown to induce oxidative stress in microalgae (Vardi et al., 1999; Sunda et al., 2002). It is thought that an increased amount of bromoform will be produced as a result of a natural allelopathic mechanism of the microalgae.
In a second experiment, one of the diatom species was grown under nitrate limitation. Increased upper ocean stratification and temperature, as a result of climate change, are reported to result in the decrease in ocean productivity in the tropics (Behrenfeld et al., 2006; Carpenter et al., 2011). This stratification limits the nutrient availability, placing increasing strain on micro- and macroalgae found in these regions.
The increased stress induced by the natural changes in CO2 and nutrients may result in an increase in bromoform production, despite an overall decrease in productivity. The increased bromoform atmospheric mixing ratios might have a significant impact on ozone concentrations and lead to positive climate change feedbacks. It is of great importance to understand the mechanism of variability within microalgae as they may be the largest global bromoform producers (Quack and Wallace, 2003).