Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future.

In this paper, Quantitative Polymerase Chain Reaction (qPCR), Illumina MiSeq sequencing and 15N stable isotopic tracing methods were applied to study the effects of influent organic loading, BOD5/TN and NO2-N/NH4-N ratios on the combined nitrogen removal through denitrification and anammox. The results show that, the total nitrogen removal was above 90% at the optimum reaction condition: organic loading 0.04kg/m3·day, BOD5/TN 0.2, and NO2-N/NH4-N 1.0. Approximately 9.43% of the nitrogen removal occurred through anammox process when BOD5/TN was 0.1, and increased to 21.46% when BOD5/TN was 0.2. The anammox accounted for 10% when NO2-N/NH4-N was 0.5, and that increased to 20.72% when NO2-N/NH4-N was 1.0-1.5. hzsA was directly proportional to the influent BOD5/TN. A positive correlation between BOD5/TN and anammox microorganisms existed. Similarly, there was a positive correlation between NO2-N/NH4-N and denitrification microorganisms. Therefore, it is feasible to regulate the influent to improve nitrogen removal efficiency.

A methodology is developed to systematically design the membership functions of fuzzy-logic controllers for multivariable systems. The methodology consists of a systematic derivation of the critical points of the membership functions as a function of predefined control objectives. Several constrained optimization problems corresponding to different qualitative operation states of the system are defined and solved to identify, in a consistent manner, the critical points of the membership functions for the input variables. The consistently identified critical points, together with the linguistic rules, determine the long term reachability of the control objectives by the fuzzy logic controller. The methodology is highlighted using a single-stage side-stream partial nitritation/Anammox reactor as a case study. As a result, a new fuzzy-logic controller for high and stable total nitrogen removal efficiency is designed. Rigorous simulations are carried out to evaluate and benchmark the performance of the controller. The results demonstrate that the novel control strategy is capable of rejecting the long-term influent disturbances, and can achieve a stable and high TN removal efficiency. Additionally, the controller was tested, and showed robustness, against measurement noise levels typical for wastewater sensors. A feedforward-feedback configuration using the present controller would give even better performance. In comparison, a previously developed fuzzy-logic controller using merely expert and intuitive knowledge performed worse. This proved the importance of using a systematic methodology for the derivation of the membership functions for multivariable systems. These results are promising for future applications of the controller in real full-scale plants. Furthermore, the methodology can be used as a tool to help systematically design fuzzy logic control applications for other biological processes.

Anaerobic ammonium oxidizing (anammox) bacteria based technologies are widely applied for nitrogen removal from warm (25-40 °C) wastewater with high ammonium concentrations (∼1 gNH4-N L(-1)). Extension of the operational window of this energy and resource efficient process is restricted by the \"supposed\" low growth rate of the responsible microorganisms. Here we demonstrate that the maximum specific growth rate (μ(max)) of anammox bacteria can be increased to a μ(max) value of 0.33 d(-1) by applying a novel selection strategy based on the maximization of the electron transfer capacity in a membrane bioreactor. This value is four times higher than the highest previously reported value. The microbial community was strongly dominated by anammox bacteria closely related (99%) to Candidatus Brocadia sp.40 throughout the experiment. The results described here demonstrate the remarkable capacity of a phylogenetically stable anammox community to adjust its growth rate in response to a change in the cultivation conditions imposed.