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)
