Periodic Reporting for period 1 - RESPIMMOX (UnRaveling the molEcular baSis Promoting Candidatus BrocadIa as the doMinant anaMOX genus in wastewater treatment plants)
Período documentado: 2021-03-01 hasta 2023-02-28
The global water demands have triggered a vast generation of wastewater that can end up contaminating water bodies. Water pollution is already disrupting economies and people’s life worldwide. Nowadays, nitrogen removal from wastewater is based on energy demanding processes. However, the finding of anammox bacteria brought up the possibility of achieving energy-self-sufficient wastewater treatment plants. When anammox processes are implemented, the aeration costs are reduced because of the lower oxygen requirements of the process compared to conventional systems and biogas production can be increased since most of the organic matter can be destined to an anaerobic digestion process. Nevertheless, despite their ubiquity and key denitrifying role in nature, and the manifest interest of implementing anammox-based processes by industry, the widespread technological application of anammox processes is still under research and there is a critical absence of molecular studies to understand how the anammox bacteria make a living. One of the current challenges of anammox bacteria research is to understand niche differentiation. The overall objective of the RESPIMMOX project is to understand the biomolecular characteristics of anammox Ca. Brocadia that trigger their niche differentiation as the main anammox genus present in wastewater treatment plants. The results from this proposal will entail a step forward for understanding the dominance of Ca. Brocadia in bioreactors (key to improve efficiency and stability) and will contribute to unravel the puzzle that anammox bacteria pose.
This report comprises the work performed and the results obtained during the development of the project during the first 10 months. This work corresponds to two specific objectives of the project: SO1: Achieving a highly enriched Ca. Brocadia culture; and SO2: Training the researcher in biochemical methods to characterize anaerobic microbial processes.
Cultivation of Brocadia-species and characterization of the active culture:
Two different configurations of bioreactors were installed: SBR and CSTR. The SBR allowed to obtain a stable anammox culture at high cellular concentrations with granular biomass. The SBR and the side equipment were set up and a stable operation and high anammox activity were achieved with nitrogen loading rates of 0.4 mgN/L/d. A high enrichment was obtained with the anammox species corresponding to Ca. Brocadia fulgida. The SBR showed operation stability and reactor robustness to confront changing loading rates, starving conditions, stop and restart operations and oxygen disturbances. The CSTR allows to obtain an active culture with dispersed cells, facilitating the proposed biochemical studies on anammox bacteria. The CSTR and the side equipment were set up, and the programming and control strategy were designed to favor the operation of interest. Monitoring and control of headspace pressure, dissolved oxygen, pH, temperature, and substrates concentrations were performed, but an active in the long-term planktonic culture was not sustained during the reported period. Different strategies for planktonic cultivation were assessed and the basis for further investigations were set. Also, several strategies of inoculation were applied with dispersed anammox cells from serum bottles and with biomass from the SBR added in different states of aggregation. Although the achievement of an anammox planktonic culture enriched in Ca. Brocadia was expected to be completed by month 12, this task has been delayed. Both the aggregated culture and also the dispersed cells obtained during the cultivation trials in CSTR were characterized according to the microbial population composition by 16S-rRNA gene amplicon sequencing, Denaturing Gradient Gel Electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). The anammox species predominant in our cultures was Ca. Brocadia fulgida.
Introduction to biochemical methods to characterize respiratory complexes using the respiration with halogenated aromatic compounds:
The researcher was trained on experimental techniques of identification and characterization of proteins involved in anaerobic microbial respiration. More specifically, the work was focused on the study of the multi-protein reductive dehalogenase complex of D. mccartyi strain CBDB1 and the differences observed when using different electron acceptors during cultivation. Investigation of the complex was focused on the composition, rather than the distribution of subunits. Shotgun proteomics approach was followed to identify the proteins of interest and co-migration of the subunits of the complex was observed when using the BN-PAGE approach. Most of the subunits were detected in our analyses, although further research must be done for improve the detection of the membrane-bound integrated proteins by nLC-MS/MS. New information on the complex structure was not obtained with our complexome analyses, but the aim of training the researcher on this methodology was greatly fulfilled. The study of the reductive dehalogenase complex was performed with samples from batch cultures and from a culture growing on a continuous bioreactor, using different organohalides as electron acceptors for cultivation: bromophenol blue (BPB), dibromotyrosine (DBT) and hexachlorobenzene (HCB). On one hand, several lines of batch cultures were cultivated with HCB and BPB. On the other hand, an experimental setup of a CSTR was installed and a continuous cultivation was operated by feeding the reactor with 3,5-DBT. The dehalogenation processes were followed by measuring the concentrations of the dehalogenation products and the cell density of the cultures. There was no evidence of variation of the total of subunits conforming the complex but there was evidence of variation of the expression of different RdhA when using BPB, HCB or DBT. The implementation of the CSTR technology for the cultivation of D. mccartyi strain CBDB1 was key to contribute to the results of the RESPIMMOX project but also to the research on the dehalogenation process in the host lab.
Dissemination of the project results to the international scientific community:
- 3rd International Conference on Anaerobic Biological Dehalogenation (DehaloCon III). Oral Presentation.
- One scientific article on the Dehalococcoides spp. growing on brominated phenolic compounds as electron acceptors (Under preparation)
Cultivation of Brocadia-species and characterization of the active culture:
Two different configurations of bioreactors were installed: SBR and CSTR. The SBR allowed to obtain a stable anammox culture at high cellular concentrations with granular biomass. The SBR and the side equipment were set up and a stable operation and high anammox activity were achieved with nitrogen loading rates of 0.4 mgN/L/d. A high enrichment was obtained with the anammox species corresponding to Ca. Brocadia fulgida. The SBR showed operation stability and reactor robustness to confront changing loading rates, starving conditions, stop and restart operations and oxygen disturbances. The CSTR allows to obtain an active culture with dispersed cells, facilitating the proposed biochemical studies on anammox bacteria. The CSTR and the side equipment were set up, and the programming and control strategy were designed to favor the operation of interest. Monitoring and control of headspace pressure, dissolved oxygen, pH, temperature, and substrates concentrations were performed, but an active in the long-term planktonic culture was not sustained during the reported period. Different strategies for planktonic cultivation were assessed and the basis for further investigations were set. Also, several strategies of inoculation were applied with dispersed anammox cells from serum bottles and with biomass from the SBR added in different states of aggregation. Although the achievement of an anammox planktonic culture enriched in Ca. Brocadia was expected to be completed by month 12, this task has been delayed. Both the aggregated culture and also the dispersed cells obtained during the cultivation trials in CSTR were characterized according to the microbial population composition by 16S-rRNA gene amplicon sequencing, Denaturing Gradient Gel Electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). The anammox species predominant in our cultures was Ca. Brocadia fulgida.
Introduction to biochemical methods to characterize respiratory complexes using the respiration with halogenated aromatic compounds:
The researcher was trained on experimental techniques of identification and characterization of proteins involved in anaerobic microbial respiration. More specifically, the work was focused on the study of the multi-protein reductive dehalogenase complex of D. mccartyi strain CBDB1 and the differences observed when using different electron acceptors during cultivation. Investigation of the complex was focused on the composition, rather than the distribution of subunits. Shotgun proteomics approach was followed to identify the proteins of interest and co-migration of the subunits of the complex was observed when using the BN-PAGE approach. Most of the subunits were detected in our analyses, although further research must be done for improve the detection of the membrane-bound integrated proteins by nLC-MS/MS. New information on the complex structure was not obtained with our complexome analyses, but the aim of training the researcher on this methodology was greatly fulfilled. The study of the reductive dehalogenase complex was performed with samples from batch cultures and from a culture growing on a continuous bioreactor, using different organohalides as electron acceptors for cultivation: bromophenol blue (BPB), dibromotyrosine (DBT) and hexachlorobenzene (HCB). On one hand, several lines of batch cultures were cultivated with HCB and BPB. On the other hand, an experimental setup of a CSTR was installed and a continuous cultivation was operated by feeding the reactor with 3,5-DBT. The dehalogenation processes were followed by measuring the concentrations of the dehalogenation products and the cell density of the cultures. There was no evidence of variation of the total of subunits conforming the complex but there was evidence of variation of the expression of different RdhA when using BPB, HCB or DBT. The implementation of the CSTR technology for the cultivation of D. mccartyi strain CBDB1 was key to contribute to the results of the RESPIMMOX project but also to the research on the dehalogenation process in the host lab.
Dissemination of the project results to the international scientific community:
- 3rd International Conference on Anaerobic Biological Dehalogenation (DehaloCon III). Oral Presentation.
- One scientific article on the Dehalococcoides spp. growing on brominated phenolic compounds as electron acceptors (Under preparation)
The RESPIMMOX project aims to understand the ecological niche of Ca. Brocadia as an abundant anammox genus in bioreactors for wastewater treatment. By knowing the biomolecular differences between anammox species, their cultivation could be optimized and, consequently, the operation of anammox bioreactors for wastewater treatment. In addition, the RESPIMMOX project is not only focused on anammox bacteria, but also on the study of dehalogenation process performed by of Dehalococcoides mccartyi strain CBDB1. Hence, direct project results were to gain understanding of the anammox energy metabolism and of the OHR complex of D. mccartyi strain CBDB1. Regarding indirect project outcomes, the RESPIMMOX project contributed to the generation of technological know-how of the bacteria responsible for processes which can solve current energetic issues of wastewater nitrogen removal and treatment issues of water bodies contaminated with organohalides.