UNDERSTANDING THE ROLE THAT BIOFILMS PLAY IN THE SPECIATION AND REMOVAL OF ARSENIC FROM WATER IN NON-AERATED AND AERATED CONSTRUCTED WETLAND MESOCOSMS

Abstract

A potentially cheap and effective way to remove the toxic metalloid, arsenic, from water may be to use constructed wetland systems. There are many interacting factors that influence arsenic removal in constructed wetlands. Factors previously assessed include pH, redox, the availability of nutrients, the types of substrates used and the type of microbial activity present in the wetlands. This thesis, while including and assessing previously studied aspects affecting arsenic removal, expanded the understanding of how novel Equisetum sp. (horsetails), biofilms and aeration contribute and drive arsenic removal in constructed wetland systems. In the first study, the ability of three species of horsetail plants (Equisetum hyemale, Equisetum fluviatile and Equisetum scirpoides) to take up arsenate under hydroponic conditions at 1 and 10 mg/L was assessed. This was done to to test how tolerant the plants were to arsenic and to select the species that would be best suited for planting in constructed wetlands treating arsenic. The speciation of arsenic in E. hyemale and E. fluviatile tissues using X-ray absorption spectroscopy (XAS) was used to understand the kinds of arsenic species found in the plants after exposure and gain a better understanding of how the plants physiologically responded to high concentrations of arsenic. All Equisetum sp. removed less arsenic from the hydroponic solution at the higher As(V) dose than at the lower As(V) dose (78%, 71% and 53% at 1 mg/L As(V) compared to 67%, 55% and 43% at 10 mg/L As(V) for E. fluviatile, E. hyemale and E. scirpoides respectively). Plant tissue arsenic concentrations increased with increasing arsenic dose, where arsenic concentrations were higher in the roots than the shoots for all Equisetum sp. The plant species that showed the highest arsenic root and shoot arsenic concentrations during growth at the highest As(V) dose was E. hyemale (Shoots: 162 mg/kg dw As, roots: 510 mg/kg dw As). There was a higher proportion of As(III) and As(III)-S species in the plant tissues of E. fluviatile, at the highest dose of As(V). As(III)-S, found in the roots of E. fluviatile, was hypothesized to be a thiolated arsenic species that restricted the movement of arsenic to the shoots. Most of the arsenic in E. hyemale remained as As(V) at both doses and may be the reason why E. hyemale was able to take up more arsenic in the shoots. To underpin the remaining studies described in this thesis, a total of 12 mesocosms (3 non-aerated planted, 3 non-aerated unplanted, 3 aerated planted and 3 aerated unplanted) were set up to test their ability to remove 1 mg/L As(V) of arsenic from water. The mesocosms were planted with E. hyemale. The experiment lasted 33 weeks. The mesocosms were fed a nutrient solution that supported the growth of plants and microbes on a weekly basis. Arsenic was added 1 day after fresh nutrients were added to the mesocosms. The mesocosms were extensively monitored by collecting data on water quality, nutrient cycling, microbial activity and plant growth. Porewater samples were also collected to determine arsenic concentrations and identify the species of arsenic present. Water samples were also filtered to collect biofilm samples to identify the prokaryotes in the porewater through next generation 16s rRNA sequencing. At the end of the 33 week experiment, the mesocosms were dismantled and biofilm and plant samples were collected. Biofilms were detached from gravels to determine the concentration of arsenic in the biofilms as well as the mineral composition through X-ray diffraction analysis (XRD) as well as the types of arsenic species found in the biofilms through X-ray absorption spectroscopy (XAS). Detached gravel and root biofilms were also collected for next generation 16s rRNA sequencing. The arsenic remaining in the biofilm detachment solutions were used to attempt to determine the amount of arsenic that was adsorbed onto the biofilms and substrates. Plant tissues were collected to determine the concentration of arsenic as well as the type of species found in the plant tissues through XAS (roots and shoots). In the second study, we examined the fate and removal of arsenic in the mesocosms. It was discovered that non-aerated mesocosms removed more arsenic (% removal of 70%) than aerated mesocosms (% removal of 40%) because the non-aerated biofilms (115 mg/kg dw As) contained more arsenic than the aerated biofilms (30 mg/kg dw As). Based on mass balance calculations, non-aerated mesocosms retained more arsenic in biofilms (19 - 37%) and through adsorption on to biofilms and substrates (18 - 24%) than aerated mesocosms (biofilms 4 - 6%, substrate/biofilm adsorbed (8 - 9%). Plants accounted for <0.4% of the total arsenic retained in the mesocosms in all planted mesocosms. Further examination of the mineral composition of the biofilms from the mesocosms revealed that non-aerated mesocosms may have retained arsenic through the biogenic formation of pyrite (2 - 15 wt%) that can adsorb/co-precipitate arsenic with iron oxyhydroxides. This may have occurred due to the activity of sulfate reducing and fermentative bacteria. The sulfate reducing bacteria likely produced hydrogen sulfide and the fermentative bacteria lowered the pH through the release of organic acids and carbon dioxide that caused the dissolution of ankerite and release of iron(II). The reaction of iron(II) and hydrogen sulfide may have led to the formation of pyrite. The abiotic formation of As-S species such as orpiment (As2S3) in lower quantities was not ruled out. In the third study, arsenic speciation was examined in the porewater, detached gravel biofilms and plants in order to further explain removal in both types of mesocosms. Although we know that non-aerated mesocosms removed more arsenic than aerated mesocosms from the previous investigation, porewater samples taken 20 min, 1 day and 6 days after addition on weeks 1, 6 and 14 revealed that non-aerated mesocosms rapidly removed the maximum amount of arsenic 1 day after addition whereas aerated mesocosms required more than 1 day to hit maximal removal. Most of the arsenic in the porewater from oxidizing aerated mesocosms remained as As(V) by day 6 for weeks 1, 6 and 14. There was a higher proportion of DMA(V) and MMA(V) in the reducing non-aerated porewaters. As(III) found in the non-aerated gravel biofilms was likely released and converted into the methylated arsenic by porewater bacteria. Non-aerated gravel biofilms also contained a high proportion of As-S species, indicating the potential formation of abiotic orpiment. Biofilms from aerated mesocosms showed no arsenic transformations. There was no discernable speciation pattern between plant tissues from non-aerated and aerated mesocosms, as most of the arsenic was found as As(V) and As(III) with some samples showing the formation of As-S species hypothesized to be arsenic bound to thiols/phytochelatins, mostly in roots, similar to our hydroponic As(V) uptake study in Chapter 3. Finally, in the last study of the thesis we examined the identity, microbial activity and ecophysiological role of the prokaryotes found in non-aerated and aerated porewaters and detached gravel and root biofilms. It was found that although the microbial activity of porewater prokaryotes from non-aerated mesocosms remained higher than that of prokaryotes from aerated mesocosms throughout the exposure period, the microbial activity of prokaryotes in non-aerated mesocosms decreased over the exposure period while the activity in aerated mesocosms remained the same. This may have been due to the production of more toxic arsenic species in the non-aerated mesocosms. We identified the most common and abundant prokaryotic genera in porewaters, gravel and root biofilms from our mesocosms through 16s rRNA next generation sequencing. There was a higher richness and diversity of prokaryotes in biofilms than in porewaters for all mesocosms. There was a higher diversity of prokaryotes in porewaters from aerated mesocosms compared to those from non-aerated porewaters. Root biofilms in non-aerated planted mesocosms had a higher prokaryote diversity than in non-aerated porewater. Both non-aerated porewaters and biofilms are characterised by having prokaryotes that are capable of fermentation, sulfide reduction and sulfide oxidation irrespective of planting status. Prokaryotes found in porewater and biofilms from non-aerated mesocosms were largely dominated by highly versatile facultative and strict anaerobic prokaryotes capable of fermentation (Candidatus Omnitrophus, Endomicrobium, Candidatus Competibacter) , nitrate reduction/denitrification (Candidatus Omnitrophus, Candidatus Competibacter, Thauera), sulfate reduction (Desulfomonile) and sulfide oxidation (Thiovirga, Sulfuricurvum, Thauera) with the capability of switching metabolisms depending on the availability of carbon, nitrogen and sulfur. These organisms led to the removal of nitrate and carbon within 24 hrs of nutrient addition. The presence of fermentative and sulfate reducing prokaryote may have also led to the formation of pyrite prior to addition of arsenic. In contrast, prokaryotes found in porewater and biofilms from aerated mesocosms were dominated by aerobic prokaryotes capable of aerobic respiration and assimilating nitrogen and sulfate for cell growth but not removal of these nutrients (TM7a, Nakamurella, Pir4 lineage, Chryseobacterium, Mycobacterium, Micropruina, Nocardioides, Chryseobacterium, Comamonas, Candidatus Xiphinematobacter as well as others).

L'utilisation de terres humides artificielles représente une méthode potentiellement économique et efficace pour éliminer l'arsenic, un métalloïde toxique, de l'eau. Plusieurs facteurs interagissent et influencent l'élimination de l'arsenic dans ces milieux humides. Parmi les facteurs déjà évalués figurent le pH, le potentiel redox, la disponibilité des nutriments, les types de substrats utilisés et l'activité microbienne présente. Cette thèse, tout en intégrant et en évaluant les aspects précédemment étudiés concernant l'élimination de l'arsenic, approfondit la compréhension du rôle de nouvelles espèces d'Equisetum (prêles), des biofilms et de l'aération dans ce processus au sein des zones humides artificielles. Dans un premier temps, la capacité de trois espèces de prêles (Equisetum hyemale, Equisetum fluviatile et Equisetum scirpoides) à absorber l'arséniate en culture hydroponique à des concentrations de 1 et 10 mg/L a été évaluée. L'objectif était de tester la tolérance des plantes à l'arsenic et de sélectionner les espèces les plus adaptées à la plantation dans des milieux humides artificiels pour le traitement de l'arsenic. La spéciation de l'arsenic dans les tissus d'Equisetum hyemale et d'Equisetum fluviatile, analysée par spectroscopie d'absorption des rayons X (XAS), a permis d'identifier les différentes espèces d'arsenic présentes dans les plantes après exposition et de mieux comprendre leur réponse physiologique à de fortes concentrations d'arsenic. Toutes les espèces d'Equisetum ont éliminé moins d'arsenic de la solution hydroponique à forte dose d'As(V) qu'à faible dose (78 %, 71 % et 53 % à 1 mg/L d'As(V), contre 67 %, 55 % et 43 % à 10 mg/L d'As(V) pour E. fluviatile, E. hyemale et E. scirpoides, respectivement). Les concentrations d'arsenic dans les tissus végétaux ont augmenté avec la dose d'arsenic, et étaient plus élevées dans les racines que dans les parties aériennes pour toutes les espèces d'Equisetum. L'espèce végétale présentant les concentrations les plus élevées d'arsenic dans les racines et les parties aériennes lors de sa croissance à la dose la plus élevée d'As(V) était E. hyemale (parties aériennes : 162 mg/kg de matière sèche As, racines : 510 mg/kg de matière sèche As). On a observé une proportion plus élevée d'espèces As(III) et As(III)-S dans les tissus végétaux d'E. fluviatile, à la dose la plus élevée d'As(V). L'As(III)-S, présent dans les racines d'E. fluviatile, serait une espèce d'arsenic thiolée limitant la diffusion de l'arsenic vers les parties aériennes. La majeure partie de l'arsenic présent chez E. hyemale est demeurée sous forme d'As(V) aux deux doses, ce qui pourrait expliquer sa capacité d'absorption plus importante dans les parties aériennes. Douze mésocosmes (3 non aérés et plantés, 3 non aérés et non plantés, 3 aérés et plantés, et 3 aérés et non plantés) ont été mis en place pour tester leur capacité à éliminer 1 mg/L d'arsenic As(V) de l'eau. Les mésocosmes plantés contenaient de l'Eucalyptus hyemale. L'expérience a duré 33 semaines. Une solution nutritive favorisant la croissance des plantes et des micro-organismes a été ajoutée chaque semaine aux mésocosmes. L'arsenic a été ajouté un jour après chaque apport de solution nutritive. Les mésocosmes ont fait l'objet d'un suivi rigoureux, avec la collecte de données sur la qualité de l'eau, le cycle des nutriments, l'activité microbienne et la croissance des plantes. Des échantillons d'eau interstitielle ont également été prélevés pour déterminer les concentrations d'arsenic et identifier les espèces présentes. Des échantillons d'eau ont été filtrés afin de recueillir des échantillons de biofilm pour identifier les procaryotes présents dans l'eau interstitielle par séquençage de nouvelle génération de l'ARNr 16S. À la fin de l'expérience de 33 semaines, les mésocosmes ont été démantelés et des échantillons de biofilm et de plantes ont été prélevés. Les biofilms ont été détachés des graviers pour déterminer la concentration d'arsenic et la composition minérale par diffraction des rayons X (DRX), ainsi que les espèces d'arsenic présentes par spectroscopie d'absorption des rayons X (XAS). Des biofilms détachés des graviers et des racines ont également été prélevés pour un séquençage de nouvelle génération de l'ARNr 16S. L'arsenic restant dans les solutions demdétachement des biofilms a été utilisé pour tenter de déterminer la quantité d'arsenic adsorbée sur les biofilms et les substrats. Des tissus végétaux ont été prélevés afin de déterminer la concentration d'arsenic ainsi que les espèces présentes par spectroscopie d'absorption des rayons X (XAS) (racines et parties aériennes). Dans une deuxième étude, nous avons examiné le devenir et l'élimination de l'arsenic dans les mésocosmes. Nous avons constaté que les mésocosmes non aérés éliminaient plus d'arsenic (élimination de 70 %) que les mésocosmes aérés (élimination de 40 %), car les biofilms non aérés (115 mg/kg de matière sèche As) contenaient plus d'arsenic que les biofilms aérés (30 mg/kg de matière sèche As). Selon les calculs de bilan massique, les mésocosmes non aérés ont retenu davantage d'arsenic dans les biofilms (19 à 37 %) et par adsorption sur les biofilms et les substrats (18 à 24 %) que les mésocosmes aérés (biofilms : 4 à 6 %, adsorption sur substrat/biofilm : 8 à 9 %). Les plantes représentaient moins de 0,4 % de l'arsenic total retenu dans les mésocosmes plantés. L'analyse de la composition minérale des biofilms a révélé que les mésocosmes non aérés pourraient avoir retenu l'arsenic par la formation biogénique de pyrite (2 à 15 % en poids), capable d'adsorber/coprécipiter l'arsenic.