Beijing, China / 21-27 August 2017. Searching for nitrifying bacteria from sewage sludge assisted by metagenomic and metatranscriptomic analyses for optimisation of the wet oxidation process. Julien Crovadore1, Vice Soljan2, Alain Mercier2, Véronique Rosset and François Lefort1, 1 Plants and pathogens group, Institute Land Nature Landscape, hepia Geneva Institute for Technology Architecture and Landscape, HES-SO University of Applied Sciences and Arts Western Switzerland, 150 route de Presinge, 1254 Jussy, Switzerland, E-mail: francois.lefort@hesge.ch 2 Granit Technologies S.A., 1350 Orbe, Switzerland. Abstract Due to increasing costs of municipal sludge management (dewatering, storage, and transport to landfill), further disposal of sludge is becoming an escalating problem. Sludge treatments represent 50% of current operating costs of wastewater treatment and therefore a major source of energy savings. Anaerobic digestion is the most commonly used disposal method, reducing the amount of final sludge solids for disposal and enabling biogas production processes. Wet oxidation is one of the methods used to improve anaerobic digestion, by maximizing the conversion of carbon into methane. This process allows for the elimination of organic components in the liquid phase by oxidation at high temperature and pressure. It can be used as a sludge pre-treatment before entering into anaerobic digestion or as additional treatment of anaerobic treatment effluent in order to destroy biologically hard degradable fractions. This process is efficient for waste sludge treatment and also important for environment protection as an alternative to landfilling or sewage sludge dispersion in farm fields, practices banned from many countries. It is also an economic alternative to incineration. Wet oxidation would thus contribute strongly to energy savings in sludge treatments and transportation. Though highly efficient for degrading complex organic compounds, wet oxidation of sewage sludge produces effluents, which contain high ammonia concentrations. Ammonia removal is thus the crucial issue, since wet oxidation improves anaerobic digestion, but returns high concentration of ammonia which in turn inhibits the production of methane, since ammonia inhibits the activity of methanogens. Our project aimed to develop a novel biological process, using aerobic granules in order to remove high ammonia concentration from the wet oxidation effluent. This new process would address the major drawbacks of existing technologies, such as: price, selectivity, stability, sensitivity and process efficiency. In a first experiment, in order to maximize the genes diversity involved in the denitrification pathway in highly polluted environment, the selection of cultivable, high ammonia concentration tolerant and denitrifying bacteria was carried out from diverse samples, such as: existing aerobic granules, activated sludge from coke plant and formaldehyde production plant and sediments from wet oxidation. Isolated bacteria have been genetically identified and some of them have been evaluated in vivo in 5L experimental bioreactors using adapted activated sludge in order to confirm denitrification activity, as well as other interesting characteristics like flocculation capacities. Finally metagenomic and metatranscriptomic analyses were carried out on the samples from experimental bioreactors in order to characterize the evolution of respective microbial population structures as well as gene expression typology, with a focus on nitrogen removal. These analyses showed that bacterial communities adapted to the wet oxidation effluent, by moving towards an increased nitrogen metabolism, confirm that biology could be an economic alternative for ammonia elimination, which would allow to reduce chemical use and energy consumption in sewage plants. The present study, combining metagenomic and metatranscriptomic to assess microbial community structure and gene expression of AS, is the second of this type and the first with such a strong sequencing depth (From 4.17 to 7.6 Gb compare to 2.4 for the study of Yu and Zhang (2012)), which really increased the results power. The present study is also the second to reveal the abundance and expression levels of genes involved in nitrification, denitrification, ammonification, DNRA and nitrogen fixation processes in AS, and the first comparing 2 different bioreactors experiments along an experimental time. Methodology and Results Metatranscriptomic allowed on one hand for an accurate characterization of whole living communities, and on the other hand for monitoring all specific functional adaptations of these communities to their environment. As shown in the fig. 4 and fig. 5 (metagenomics), nitrogen metabolism genes are present at low level in all microorganisms genomes (only 1%) compared to carbohydrates (13 %) or amino acids (11%) metabolism. But metatranscriptomic showed that nitrogen metabolism appeared as the most relative expressed activity and increased dramatically over a month in our bioreactor conditions, especially concerning denitrification (Fig. 8). Metagenomic analysis of high ammonia adapted Activated Sludge during bioreactor experiment over a month 1 2 3 9 10 Metagenomic analysis of the activated sludge (AS) showed a dominating bacteria domain yielding 98% organisms (Fig. 1). Highest genus and species abundances are dominated by microorganisms involved in nitrogen metabolism such as Nitrosomonas, Chitinophaga or Nitrobacter genus (Fig. 2 and 3). 4 5 The pie charts on the left (Fig. 4 and 5) illustrate the distribution of functional genes categories for the initial activated sludge sample at highest expression level and for nitrogen metabolism, respectively. Each slice indicates the percentage of reads with predicted protein functions annotated to each category . Metatranscriptomic analysis of high ammonia adapted Activated Sludge during bioreactor experiment over a month 6 7 As shown in the fig. 9, 145 different bacterial genus containing sequences of at least one of the coding sequences of these nitrogen metabolism enzymes have been identified (against 37 for Yu and Zhang study, at the same thresholds of selection). Huge diversity involved in the nitrogen metabolism. hit reads from cDNA dataset were relatively lower (genes expression Vs genes presence (DNA)) Nitrosomonas and Nitrosospira had the only abundances of ammonia monooxygenase. Ammonia monooxygenase presents the highest expression level compare to other enzymes, which supports the idea of a strong nitrification activity. 11 As shown in the fig. 10, The addition of 200 ml of aerobic granules solution in the bioreactor 2 clearly modified the composition of the adapted activated sludge bacterial flora, with 56% (51/91) common active bacterial genus in both bioreactors, 23% (21/91) active bacterial genus specific to bioreactor 1 “Activated Sludge”, and 21% (19/91) specific to bioreactor 2 “Activated Sludge + Aerobic Granules”. We can also notice that the 2 bioreactors nitrogen metabolism profile look very close at the first sight, which is not the case, especially regarding the processes intensity, as we can notice in fig. 11. As shown in the fig. 6 and fig. 7 above, the relative level activity of Nitrosomonas genus based on metatranscriptomic analysis increases from 6 to 25 % (5 to 30% in metagenomic analysis) which highlights the evolution and specialization of the living flora of activated sludge microorganisms in response to the experimental conditions in the bioreactor over a month (under high ammonia concentration). 8 Conclusion Nitrification is clearly the strongest process of the nitrogen metabolism in our both bioreactors conditions, followed by denitrification and ammonification. The addition of 200 ml of adapted aerobic granules (AG) solution in the activated sludge (bioreactor 2) clearly modified the nitrogen processes activity. We can note a stronger nitrification at the end of the experiment and a stabilization of the ammonification activity. Both bioreactors are characterized by an increasing denitrification rate over the experimental time. Both experimental conditions prove that AS and AS+AG microorganisms flora well adapted over the experimental month to their life media (leachate substrate, high ammonia rate, settling frequency) and both enhance their nitrification and denitrification processes capacity, with a noticeable advantage to the AS+AG. Figure 8 : Heatmaps describing the abundance of major functional categories (on the left) and closer at the nitrogen metabolism (on the right) in the two assembly metatranscriptomes datasets 4572418.3 and 4572368.3, respectively Initial and final activated sludge RNA. The color coding represents each category of function in hierarchy and the mRNA abundances are indicated by the color scale. Corresponding contigs abundance is represented on a scale from 0 to 1. INTECOL 2017 Beijing, China / 21-27 August 2017.