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Vast amounts of carbon are stored forest soils, a product of decaying organic matter. Increased CO2 in the atmosphere is predicted to lead to increasing global temperatures, and more extreme moisture regimes. Such increases in mean temperature could accelerate the rate of organic matter decay in soils and lead to additional release of CO2 into the atmosphere, thus exacerbating climate change. However, due to its impact on plant metabolism, high atmospheric CO2 concentrations may also lead to greater condensed tannins (CT) and reduced nitrogen (N) content in leaf litter. This reduction in litter quality has the potential to slow decay of organic matter in soil and therefore offset the accelerated decay resulting from a warmer climate. My research aimed to quantify the effects of climate and litter chemistry, specifically CT and N, on litter decay, N mineralization and associated microbes in the field. Strings of litterbags were laid on the forest floor along climate transects of mature Douglas-fir stands of coastal British Columbia rain-shadow forests. In-situ climate was monitored alongside carbon and nitrogen loss over 3.58 years of decay along three transects located at different latitudes, each transect spanning the coastal Western Hemlock and Douglas-fir biogeoclimatic zones. Microbial communities in the decaying litter and in forest soils were also analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Microbial biogeography at field sites was partially influenced by climate, soil characteristics and spatial distance, but did not improve best fit decay models using climate and litter chemistry variables. Litter with greater initial CT and smaller N concentration slowed down early decay (0 - 0.58 yr) and net N mineralization. Warmer temperatures accelerated later decay (0.58 - 3.58 yr) and net N mineralization. Water-soluble CT were rapidly lost during decay, while other forms of CT were likely responsible for slower decay. The composition of fungal communities on decaying litter was affected by initial concentrations of CT and N. On a yearly basis, the slower decay of litter with high CT and reduced N content can offset accelerated rates of decay associated with warmer temperatures. Concurrent shifts in microbial communities and net N mineralization suggest potential benefits to trees. / Graduate / 2017-12-19

Deammonification is a widely used technology for side stream treatment with rich ammonium streams at relatively high temperatures, such as, the reject water coming from dewatering units in treatment of digested sludge and industrial wastewaters. The deammonification process has lower operational costs than conventional systems, consumes less energy, enables the increase of biogas production and it is easy to implement. However, this technology has not yet been applied in full- scale mainstream treatment due to its restrictions in coping with high C/N ratios, low temperatures, and the need for post-treatment processes. These conditions are allegedly negative to the growth and performance of anammox bacteria affecting the bacterial groups’ behavior in the process. This master thesis project aimed to evaluate the feasibility of using deammonification to remove nitrogen from mainstream wastewater, which was studied by monitoring the bacterial activity in a pilot scale reactor. The different bacterial groups involved (AOB, NOB, heterotrophs, and denitrifiers) were monitored by weekly measuring their activity in batch activity tests. The results allowed the evaluation of different operational scenarios and their impact by following up on the changes in the bacterial competition. The study was conducted for six months in a single-stage IFAS (integrated fixed-film activated sludge) pilot-scale reactor located in Stockholm and fed with pretreated (with a UASB) municipal wastewater. The different operational scenarios involved changes in temperature, aeration patterns, DO concentration, SRT, and HRT. The adjustment of these features was done in the interest of promoting AOB and anammox bacterial growth, leading to an improvement of the deammonification efficiency in future studies. However, the chosen operational conditions were to enhance bacterial competition and facilitate its visualization, not to maximize nitrogen removal. Thus, the most suitable scenario found during this study included DO concentration of 1.5 mg/L with 10 aeration-20 non-aeration pattern and ensured nitrogen removal rates within normal values while allowing the monitoring of all the bacterial groups. TN removal reached a value above 50% and NH4-N above 95%, whereas nitrogen Removal Rate (NRR) increased to 30g/N/m3-d and the system had an overall nitrogen removal efficiency of 75%. Nevertheless, it was proven that in the right environment, the necessary bacterial groups can be selectively accumulated and successfully perform deammonification and reduce nitrogen levels in mainstream wastewater. / Deammonifikation är en välanvänd teknik för rening av sidoströmmar med höga ammoniumkoncentrationer vid relativt hög temperatur, som till exempel rejektvatten från avvattning av rötslam eller industriellt avloppsvatten. Deammonifikationsprocessen har lägre driftkostnad än konventionella reningsprocesser, förbrukar mindre energi samt möjliggör högre biogasproduktion samtidigt som processen är enkel att implementera. Reningstekniken har dock ännu inte tillämpats i fullskala för rening av huvudströmmen på grund av den höga C/N-kvoten och de låga vattentemperaturerna i kommunalt avloppsvatten samt behovet av efterbehandling. Detta anses ha en negativ inverkan på anammoxbakteriernas tillväxthastighet och funktion vilket påverkar bakteriegruppens beteende i processen. Syftet med detta examensarbete var att utvärdera om det är praktiskt genomförbart att använda deammonifikation för att rena kväve från kommunalt avloppsvatten, vilket följdes upp genom att studera bakterieaktiviteten i en pilotskalereaktor. De involverade bakteriegrupperna (AOB, NOB, heterotrofer och denitrifierare) övervakades genom att mäta den mikrobiella aktiviteten varje vecka med hjälp av batch-tester. Resultaten användes till att utvärdera olika driftstrategier och deras effekt genom att följa förändringarna i mikrobiell aktivitet hos de konkurrerande bakteriegrupperna. Studien genomfördes i Stockholm under sex månader i en enstegs-IFAS-pilotskalereaktor (integrerad process med biofilm på fast bärarmaterial och aktivslam) som matades med kommunalt avloppsvatten som förbehandlats i en UASB-reaktor. De olika driftstrategierna omfattade olika temperaturer, luftningsstrategier, syrekoncentrationer, slamåldrar och hydrauliska uppehållstider. Syftet med driftstrategierna var att främja AOB- och anammoxbakteriers tillväxt för att i framtida studier kunna erhålla en förbättrad deammonifikationsprocess. Syftet i denna studie var dock i första hand att förbättra den bakteriella konkurrensen och göra den lättare att mäta, inte att uppnå bästa möjliga kväverening. Den driftstrategi som gav bäst resultat i denna studie innebar att hålla en syrehalt på 1,5 mg/l med 10 minuter luftning följt av 20 minuter utan luftning vilket säkerställde en normal kväveavskiljning och samtidigt möjliggjorde övervakning av samtliga fyra bakteriegrupper. Totalkväveavskiljningen var över 50 % och ammoniumavskiljningen över 95 % medan kvävereningsaktiviteten ökade till 30 g N/m3-d och systemet hade en övergripande effektivitet på 75 %. Studien visade att under rätt förutsättningar kan de nödvändiga bakteriegrupperna selekteras fram och deammonifikation av kommunalt avloppsvatten kan utföras på ett framgångsrikt sätt.

Protecting the quality and quantity of our water resources requires advanced treatment technologies capable of removing nutrients from wastewater. This research work investigated the capability of one such technology, a hollow-fiber membrane-aerated biofilm reactor (HFMBR), to achieve completely autotrophic nitrogen removal from a wastewater with high nitrogen content. Because the extent of oxygenation is a key parameter for controlling the metabolic processes that occur in a wastewater treatment system, the first part of the research investigated oxygen transfer characteristics of the HFMBR in clean water conditions and with actively growing biofilm. A mechanistic model for oxygen concentration and flux as a function of length along the non-porous membrane fibers that comprise the HFMBR was developed based on material properties and physical dimensions. This model reflects the diffusion mechanism of non-porous membranes; namely that oxygen follows a sorption-dissolution-diffusion mechanism. This is in contrast to microporous membranes in which oxygen is in the gas phase in the fiber pores up to the membrane surface, resulting in higher biofilm pore liquid dissolved oxygen concentrations. Compared to offgas oxygen analysis from the HFMBR while in operation with biofilm growing, the model overpredicted mass transfer by a factor of approximately 1.3. This was in contrast to empirical mass transfer coefficient-based methods, which were determined using either bulk aqueous phase dissolved oxygen (DO) concentration or the DO concentration at the membrane-liquid interface, measured with oxygen microsensors. The mass transfer coefficient determined with the DO measured at the interface was the best predictor of actual oxygen transfer under biofilm conditions, while the bulk liquid coefficient underpredicted by a factor of 3. The mechanistic model exhibited sensitivity to parameters such as the initial lumen oxygen concentration (at the entry to the fiber) and the diffusion coefficient and partitioning coefficients of oxygen in the silicone membrane material. The mechanistic model has several advantages over empirical-based methods. Namely, it does not require experimental determination of KL, it is relatively simple to solve without the use of advanced mathematical software, and it is based upon selection of the membrane-biofilm interfacial DO concentration. The last of these is of particular importance when designing and operating HFMBR systems with redox (aerobic/anoxic/anaerobic) stratification, because the DO concentration will determine the nature of the microenvironments, the microorganisms present, and the metabolisms that occur. During the second phase of the research, the coupling of two autotrophic metabolisms, partial nitrification to nitrite (nitritation) and anaerobic ammonium oxidation, was demonstrated in a single HFMBR. The system successfully treated a high-strength nitrogen wastewater intended to mimic a urine stream from such sources as extended space missions. For the last 250 days of operation, operating with an average oxygen to ammonia flux (J_O₂/J_NH₄⁺) of 3.0 resulted in an average nitrogen removal of 74%, with no external organic carbon added. Control of nitrite-oxidizing bacteria (NOB) presented a challenge that was addressed by maintaining the J_O₂/J_NH₄⁺ below the stoichiometric threshold for complete nitrification to nitrate (4.57 g O₂ / g NH₄⁺). The DO-limiting condition resulted in formation of harmful gaseous emissions of nitrogen oxides (NO, N2O), which could not be prevented by short-term control strategies. Controlling JO2/JNH4+ prevented NOB proliferation long enough to allow an anaerobic ammoniaoxidizing bacteria (AnaerAOB) population to develop and be retained for >250 days. Addition of a supplemental nutrient solution may have contributed to the growth of AnaerAOB by overcoming a possible micronutrient deficiency. Disappearance of the gaseous nitrogen oxide emissions coincided with the onset of anaerobic ammonium oxidation, demonstrating a benefit of coupling these two autotrophic metabolisms in one reactor. Obvious differences in biofilm density were evident across the biofilm depth, with a region of low density in the middle of the biofilm, suggesting that low cell density or exocellular polymeric substances were primarily present in this region, Microbial community analysis using fluorescence in situ hybridization (FISH) did not reveal consistent trends with respect to length along the fibers, but radial stratification of aerobic ammonia-oxidizing bacteria (AerAOB), NOB, and AnaerAOB were visible in biofilm section samples. AerAOB were largely found in the first 25% of the biofilm near the membrane, AnaerAOB were found in the outer 30%, and NOB were found most often in the mid-depth region of the biofilm. This community structure demonstrates the importance of oxygen availability as a determinant of how microbial groups spatially distribute within an HFMBR biofilm. The combination of these two aspects of the research, predictive oxygen transfer capability and the effect of oxygen control on performance and populations, provides a foundation for future application of HFMBR technology to a broad range of wastewaters and treatment scenarios. / Ph. D.

Ecosystems currently face widespread biodiversity losses and other environmental disturbances, such as climate warming, related to increased anthropogenic activities. Within this context, scientists consider the effects of such changes on the biodiversity, and hence on the activity, of soil microorganisms. Indeed, soil microorganisms mediate a wide range of soil processes. Currently, knowledge on soil microbial diversity is still limited, partially due to technical limitations. The advent of molecular-based analyses now allows studying the soil microbial diversity. These advances in the study of soil microbial communities have lead to a growing evidence of the critical role played by the microbial community in ecosystem functioning. This relationship is supposed to be relevant for narrow processes, regulated by a restricted group of microorganisms, such as the nitrification process. This PhD thesis aimed at studying ammonia oxidizing bacteria (AOB) community structure and richness as an integrated part of soil functioning. This research aimed at investigating the effect of aboveground plant diversity on ammonia oxidizing bacteria diversity and function in forest and grassland soils with focus on the influence of (a) functional group identity of grassland plants (legumes, grasses, forbs), (b) grassland plant species richness and (c) tree species, on AOB diversity and function. Another objective of this research was to study the effect of a 3°C increase in air temperature on AOB diversity and function. The link between AOB diversity and function (potential nitrification) is also investigated. For grassland ecosystems, a microcosm experiment was realized. An experimental platform containing 288 assembled grassland communities was established in Wilrijk (Belgium). Grassland species were grown in 12 sunlit, climate controlled chambers. Each chamber contained 24 communities of variable species richness (S) (9 S=1, 9 S=3 and 6 S=9).The grassland species belonged to three functional groups: three species of each grasses (Dactylis glomerata L., Festuca arundinacea SCHREB., Lolium perenne L.), forbs (non-N-fixing dicots; Bellis perennis L., Rumex acetosa L., Plantagolanceolata L.), and legumes (N-fixing dicots; Trifolium repens L., Medicago sativa L., Lotus corniculatus L.). Half of these chambers were exposed to ambient temperature and the other half were exposed to (ambient +3°C) temperature. One ambient and one (ambient+3°C) chambers were destructively harvested 4, 16 and 28 months after the start of the experiment. The influence of plant functional group identity on the nitrification process and on AOB community structure and richness (AOB diversity) was assessed in soils collected from the first two destructive amplings (chapter 2). The effect of plant species richness on AOB diversity and function was considered for soils sampled after 16 and 28 months (chapter 3). AOB function was determined by potential nitrification. AOB community structure and richness were assessed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) and sequencing of excised DGGE bands. I found that functional group identity can affect AOB community structure. In particular, the presence of legumes, both in monoculture or in mixture with forbs and grasses, lead to AOB community composition changes towards AOB clusters tolerating higher ammonium concentrations. This change in AOB community structure was only linked to increased potential nitrification under monocultures of legumes, when ammonium was supposed to be not limiting. This study revealed that physiological attributes of AOB and resource availability may be important factors in controlling the nitrification process. This research showed that the impact of plant species richness on the nitrification process could be mediated by the interactions between plants and AOB, through competition for substrate. A 3°C increase in air temperature did not affect AOB community structure, richness or function. In forest ecosystems, we studied the effect of tree species in forest sites located in Belgian and in the Grand-Duchy of Luxembourg covered each by several deciduous or coniferous tree species (Fagus sylvatica L., Quercus petraea (Mattuschka) Lieblein, Picea abies (L.) Karst, Pseudotsuga menziesii (Mirbel) Franco). We investigated the influence of these tree species on microbial processes (chapter 5) related to C and N cycling, particularly with emphasize on the nitrification process and on the diversity of AOB (chapter 6). The results showed that the effect of tree species on net N mineralization was likely to be mediated through their effect on soil microbial biomass, reflecting their influence on organic matter content and carbon availability. Influence of tree species on nitrification (potential and relative) might be related to the presence of ground vegetation through its influence on soil ammonium and labile C availability. AOB community structure was more site-specific than tree specific. However, within sites, AOB community structure under broadleaved trees differed from the one under coniferous trees. The effect on tree species on AOB was likely to be driven by the influence of tree species on net N mineralization, which regulates the substrate availability for AOB. The results also demonstrated that the relationship between AOB diversity and function might be related both to AOB abundance and AOB community structure and richness. This thesis showed no clear relationship between AOB community structure or richness and AOB function. However, we revealed that aboveground grassland plant richness, grassland plant functional groups and tree species influence AOB community structure and richness. Actuellement, les écosystèmes sont soumis à dimportantes pressions anthropiques et environnementales, pouvant aboutir à des pertes massives de biodiversité. Les scientifiques sinterrogent sur limpact de ces perturbations sur la diversité et, par conséquent, sur lactivité des microorganismes du sol. En effet, ceux-ci régulent de nombreux processus du sol. Actuellement, de nombreuses lacunes subsistent dans la connaissance de la diversité microbienne du sol. Celles-ci peuvent être partiellement attribuées aux difficultés méthodologiques associées à l'étude des micro-organismes du sol. Lavènement des techniques moléculaires nous permet de combler ces lacunes. Les avancées réalisées dans l'étude des communautés microbiennes du sol ont mis en évidence le rôle crucial joué par les communautés microbiennes dans le fonctionnement des écosystèmes. De plus, il semblerait que les processus régulés par un groupe restreint dorganismes, tel le processus de nitrification, soient plus sensibles à toute altération de la communauté. Lobjectif de cette thèse était détudier la structure de la communauté ainsi que la richesse (nombre de bandes DGGE) des bactéries oxydant lammoniac (AOB) comme une partie intégrante du fonctionnement des sols. Notre étude se focalisait sur linfluence de (a) différents groupes fonctionnels de plantes (graminées, légumineuses, dicotylédones), (b) communautés de plantes présentant une richesse spécifique croissante et (c) différentes essences forestières, sur la diversité (structure de la communauté et richesse des AOB) et la fonction des AOB. Cette recherche étudiait également limpact dune augmentation de température de 3°C sur ces paramètres. Létablissement dun lien éventuel entre la diversité et la fonction (nitrification potentielle) des AOB a aussi été envisagé. Concernant les écosystèmes prairiaux, nous avons réalisé une étude en microcosmes. Une plateforme expérimentale comprenant 288 communautés artificielles de plantes a été établie à Wilrijk (Belgique). Cette plateforme consistait en 12 chambres, dont une moitié était à température ambiante et la seconde était à température ambiante augmentée de 3°C. Chaque chambre contient 24 communautés de plantes de richesse spécifique variable (9 S=1, 9 S=3 et 6 S=9). Les communautés de plantes sont créées avec 9 espèces de plantes appartenant à trois groupes fonctionnels : 3 espèces de graminées (Dactylis glomerata L., Festuca arundinacea SCHREB., Lolium perenne L.), de légumineuses (dicotylédones fixatrices dazote ;Trifolim repens L., Medicago sativa L., Lotus corniculatus L.), et de dicotylédones non fixatrices dazote (Bellis perennis L., Rumex acetosa L., Plantago lanceolata L.). Les sols issus dune chambre à température ambiante et dune chambre à température ambiante augmentée de 3°C ont été échantillonnés, respectivement, 4, 16 et 28 mois après le début de lexpérimentation. Linfluence des groupes fonctionnels de plantes sur le processus de nitrification ainsi que sur la structure de la communauté et la richesse des AOB a été mesuré sur les sols issus des deux premiers échantillonnages (chapitre 2). Nous avons mesuré leffet de la richesse croissante en plantes sur la diversité et lactivité des AOB sur les sols échantillonnés après 16 et 28 mois dexpérimentation (chapitre 3). La structure de la communauté ainsi que la richesse des AOB ont été évaluées à laide dune amplification spécifique par réaction de polymérisation en chaîne (PCR) de lADN génomique extrait du sol suivie par une séparation par électrophorèse sur gel dacrylamide en présence dun gradient dénaturant (DGGE). Nous avons identifié les différentes AOB présentes par séquençage des bandes DGGE excisées. Nos résultats ont montré que les différents groupes fonctionnels peuvent affecter la structure de la communauté des AOB. En particulier, la présence de légumineuses, aussi bien en monoculture quen mélange avec des graminées ou des dicotylédones non fixatrices dazote, provoque des changements au sein de la structure de la communauté des AOB, favorisant la présence de clusters tolérants des concentrations en ammonium plus élevées. Ces changements de la structure de la communauté des AOB sont liés à des augmentations de la production potentielle de nitrates (nitrification potentielle) quand lammonium est supposé être non limitant. Cette étude révèle que la physiologie des AOB ainsi que la disponibilité en substrat peuvent être des facteurs majeurs intervenant dans le contrôle du processus de nitrification. Cette recherche montre que linfluence de la richesse spécifique des plantes sur la nitrification pourrait dépendre des interactions entre les plantes et les AOB via la compétition pour le substrat. Une augmentation de la température de lair de 3°C na pas influencé les richesse, structure de la communauté ou les fonctions des AOB. Pour les écosystèmes forestiers, nous aborderons leffet de différentes essences forestières (Picea abies (L.) KARST, Fagus sylvatica L., Quercus petraea LIEBLEIN ; Pseudotsuga menziezii (MIRB.) FRANCO) dans différents peuplements au Grand Duché du Luxembourg et en Belgique. Nous avons étudié l'influence de ces essences forestières sur les processus microbiens (chapitre 5) liés aux cycles du carbone et de lazote, en particulier leur effet sur le processus de nitrification et la diversité des AOB (chapitre 6). Notre étude révèle que linfluence des essences forestières sur la minéralisation nette de lazote pourrait être attribuable à leur effet sur la biomasse microbienne, reflétant ainsi leur effet sur la teneur en matière organique et la disponibilité en carbone. Limpact des essences forestières sur la nitrification (à la fois sur la nitrification relative et sur la nitrification potentielle) serait conditionné par la présence de végétation au sol, en raison de linfluence de celle-ci sur la disponibilité en ammonium et en carbone labile. Nous avons observé que la structure de la communauté des AOB était plus spécifique aux sites quaux essences forestières. Cependant, au sein dun site, elle différait sous feuillus et sous conifères. Les essences forestières influenceraient la structure de la communauté des AOB au travers de limpact quelles ont sur la minéralisation nette de lazote qui régule, quant à elle, la disponibilité en ammonium. Cette recherche démontre que le lien observé entre la diversité et la fonction dépendrait la fois de labondance, de la structure de la communauté et de la richesse des AOB. Cette thèse na révélé aucune relation claire entre la structure de la communauté ou la richesse des AOB et leur fonction. Par contre, nous avons observé que la richesse spécifique et les groupes fonctionnels de plantes prairiales et les essences forestières affectent la structure de la communauté et la richesse des AOB.