PhD Defense | Isabelle Van Dyck | Application of Lemna minor in site remediation strategies

Application of Lemna minor in site remediation strategies

Pollutants such as radionuclides and heavy metals are present in the environment due to the exploitation of nuclear installations, mining and milling processes, industrial activities (e.g. involving naturally occurring radionuclides), nuclear accidents and the dumping of radioactive waste. Polluted surface waters can become an environmental problem for both aquatic and terrestrial organisms and therefore remediation is needed. Phytoremediation uses the biological processes of living plants (and their associated microbiome) to remove, degrade or stabilise pollutants from their surrounding environment. Phytoremediation is recognised as an efficient site remediation technology for various types of pollutants and can be used as a complementary waste water treatment technique for specific scenarios. Within this PhD project, phytoremediation of water, polluted with radionuclides and metals, using the macrophyte Lemna minor has been systematically studied.

L. minor is one of the 36 Lemnaceae species that can absorb and accumulate various pollutants in its biomass, making it a candidate plant species for phytoremediation purposes. L. minor plants are used and studied for wastewater evaluation and treatment due to their fast growth, ability to form extended surface mats on the water, high removal capacity and sensitivity towards different kinds of stress (e.g. heavy metals, radionuclides). Due to their easy growth in a wide variety of laboratory conditions, L. minor is often used as a model organism for environmental studies and fundamental plant research. Therefore, L. minor is suitable for studying toxicity of wastewater and for investigating in detail its applicability for phytoremediation purposes.

Commercial phytoremediation technologies appear to be globally underutilised notwithstanding their effectiveness in field applications and the academic research studies available in support of this methodology. More in-depth research is required to demonstrate that phytoremediation can be a valuable and eco-friendly solution for the treatment of polluted environmental or industrial water. L. minor can be used to remove radioactive or heavy metal pollutants from water but can also be affected by the pollutants that are present in the water, hence this study covers also pollutant interactions with L. minor.

We started this study by providing a brief introduction on environmental pollution, the concept of phytoremediation and the test organism L. minor (Chapter 1). Subsequently, the scope and objectives are described in Chapter 2.

L. minor growth was studied under different optimal and sub-optimal environmental conditions (temperature, light irradiation and variable nutrient concentrations), while varying one environmental parameter per experiment. By combining experimental and mathematical modelling approaches, it was possible to develop a L. minor growth model as a function of biomass, temperature, light irradiation and variable nutrient concentrations, suitable for application in phytoremediation of radionuclides and other water pollutants (Chapter 3). A previously described L. minor growth model was used as a starting point for developing an extended and more process-based growth model. Important changes were made to this model, such as modification of the crowding term to a logistic biomass function and addition of a light irradiance function with variable photoperiod.

We made growth curves for each experimental condition and fitted the model parameters to the experimental data using ModelMaker 3. Equations for the associated environmental conditions were tested and adapted where necessary (e.g. new equation for temperature and light irradiance function, changes to the nutrients equations). A sensitivity analysis indicated that parameters related to growth (r_opt, r_max,L, r_max,N, r_max,P and r), optimum and experimental temperature (T_opt and T_exp) and maximum biomass allowed by the system on the used surface area (h_B) are the most sensitive parameters of the model. Finally, after an overall parameter optimisation of the model, it was clear that our improved L. minor growth model can predict L. minor growth under different environmental conditions. It is an improvement on the way to establish a full remediation model capable of evaluating different remediation options in real case scenarios.

Not only growth of L. minor is affected by changing environmental conditions, but also starch and soluble sugars contents are affected (Chapter 4). Accumulation of starch can either be connected with typical stress related responses (e.g. salinity, nutrient deprivation, low or high temperatures, pollutants) or related to increased growth rates when more substrates are needed for metabolism and growth. By performing experiments under different environmental conditions (temperature, light irradiance and nutrients) it was possible to distinguish between these processes. Non-optimal temperatures and low nitrogen concentrations resulted in decreased growth rates and higher starch and soluble sugars accumulation, which are typical stress associated responses. Results suggest that lower temperatures are more stressful to L. minor compared to higher temperatures. We also demonstrated that higher light intensities and longer photoperiods led to increased growth rates and more starch formation. These results contribute to a better understanding of L. minor growth, starch and soluble sugar content under different environmental conditions and form a basis for optimising plant cultivation conditions to improve the suitability of L. minor biomass for practical applications (e.g. biofuels, biomaterials, animal feed and even human nutrition).

Next, we studied the removal and effects of pollutants (Co, ⁶⁰Co, Cs, ¹³⁷Cs, Mn, Ni and Zn) by and on L. minor. Information related to the uptake mechanisms of some radionuclides and metals by L. minor (biosorption versus bioaccumulation) and possible effects on L. minor’s physiological and biochemical functions is not readily available. However, this knowledge is needed to generally better understand uptake and effects on L. minor when exposed to multiple pollutants, to assess the possibility of using L. minor in phytoremediation applications and to improve environmental impact assessments.

We studied the uptake and effects of stable Co and Cs, together with radiation effects of ⁶⁰Co and ¹³⁷Cs on L. minor’s physiological and biochemical functions, in order to differentiate between chemo- and radiotoxicity (Chapter 5). Regarding effects of ⁶⁰Co and ¹³⁷Cs, some studies have investigated the effects of external gamma radiation on L. minor, but studies that investigated the internal radiation effects or chemical effects are scarce. Since the usage of high concentrations of ⁶⁰Co and ¹³⁷Cs in solution was not practically feasible in our laboratory (due to safety restrictions related to working with open radioactive sources), we used an innovative approach that combined the external irradiation (from a ⁶⁰Co and ¹³⁷Cs source) together with the direct uptake of a corresponding amount of stable Co or Cs. By doing this, the radiological and chemical components of ⁶⁰Co and ¹³⁷Cs are combined and the uptake by and effects on L. minor when applying higher activity concentrations of ⁶⁰Co and ¹³⁷Cs can be imitated. Experiments were performed by exposing L. minor plants to different concentrations of stable Co and Cs, different external dose rates of ⁶⁰Co and ¹³⁷Cs and the combined exposure experiments. Dose-response curves, based on percentages of growth inhibition, were established for each exposure condition and (radio)element. The derived EDR₅₀ values indicated that the combined exposure caused a higher toxicity compared to (a) external irradiation applied as a single stressor or (b) exposure of L. minor to the stable isotope. The highest toxicity was observed for ¹³⁷Cs. In addition, exposure ⁶⁰Co or ¹³⁷Cs caused a dose rate-dependent increase of the starch content.

Since we considered gamma radiation and chemical pollutants as stressors that probably affect different physiological systems of an organism, a combined effect assessment was performed. We used the two reference models/approaches originally developed for assessing the toxicity of mixtures of chemicals, namely independent action and concentration addition. Both approaches resulted in a relatively good fit between our predicted growth curve and the experimental data points from the ⁶⁰Co/Co and ¹³⁷Cs/Cs combined exposure experiments, but independent action led to a slight overestimation. Therefore, this is the most conservative approach regarding assessing the combination of metal exposure and gamma radiation. Our method is a practical tool for predicting the effects on L. minor growth caused by a mixture of stable elements and gamma radiation.

Besides stable Co and Cs, also the uptake of Mn, Ni and Zn on L. minor was extensively studied as function of pollutant concentration and time (Chapter 6). L. minor plants were exposed to different concentrations of Mn, Ni and Zn, in order to produce dose-response curves. From the derived EC₅₀ values, an ordering was made based on the toxicity of the elements on L. minor: Ni > Co > Zn > Cs = Mn. Pollutant removal parameters were also determined; a similar but reversed order was obtained as for the toxicity (Mn = Cs > Zn > Ni > Co). Generally, the higher the initial exposure concentration, the lower the specific growth rate, removal percentage and bioconcentration factor of L. minor for the elements. Elements with a high toxicity (low EC₅₀ values) demonstrated lower removal per L. minor dry mass. For essential elements, such as Mn, L. minor showed a high removal capacity. The removal capacity for Cs was also high, most likely resulting from the high similarity with the essential element K. Our detailed study on the influence of K on Cs uptake by L. minor confirms that the presence of high K concentrations results in more Cs-K competition, less Cs uptake and eventually less toxicity.

Our previously developed L. minor growth model was further extended by adding all uptake and release time series data of the five elements (Co, Cs, Mn, Ni and Zn) leading to an experimentally-based mathematical single pollutant uptake model for L. minor, based on the concepts of biokinetic modelling (first order kinetics) and population modelling. All key uptake and release parameters were numerically optimised and it can be concluded that they depend on the applied pollutant. Our L. minor uptake and release model makes it possible to predict plant growth and pollutant removal for single elements present in water, opening the door to predicting the concentrations of pollutants in L. minor in real-life remediation applications.

Lastly, we focussed on a broad study about the possible effects of pollutants (Co, Cs, Mn, Ni and Zn) on L. minor’s physiological and biochemical functions (Chapter 7). We also compared uptake mechanisms (biosorption and bioaccumulation), but mainly focused on effects of single pollutants on growth, photosynthetic pigments, photosynthesis, starch and soluble sugars content. When applying increased concentrations of Co, Cs, Mn, Ni and Zn, toxic effects got visible as decreases in photosynthetic pigments (Chl a, Chl b and carotenoids) and an increase in starch content in a concentration-dependent manner. Soluble sugars contents were also higher for all exposure conditions. Photosynthesis only displayed pronounced effects during exposure to toxic concentrations of Mn and Zn. Too high Zn concentrations caused damage to the light harvesting capacity of PSII and by consequence a smaller fraction of the incoming energy could be used for photosynthesis trough PSII. In contrast, the light harvesting capacity of PSII remained intact during exposure to Mn and we even observed an enhanced capability to perform photosynthesis. Meanwhile, Mn exposure also negatively affected the photosynthetic pigment concentrations which led to a reduced light absorption.

By performing this study, a broad overview of the effect of metals and radionuclides on L. minor’s uptake, physiological and biochemical functions was obtained. Together with the L. minor growth study under different environmental conditions, the possibility is there to use L. minor for remediation purposes due to our quantification of the relevant processes. This work provides also essential information about how the remediation ability of L. minor is influenced by the physico-chemical and biological characteristics together with environmental factors such as cations, anions, radionuclide concentration and speciation, metal concentration, growing conditions (e.g. light, nutrients, temperature) and set-up (e.g. biomass/water ratio, contact time).

 

Promotor:

• Jaco Vangronsveld (UHASSELT)

SCK CEN mentors:

• Nathalie Vanhoudt (SCK CEN)

• Jordi Vives i Batlle (SCK CEN)

 

Click here for a list of obtained PhD degrees.