טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDesitti Chaitanyakumar
SubjectAtrazine Biodegradation without the Addition of External
Carbon Source and Potential Application
DepartmentDepartment of Civil and Environmental Engineering
Supervisors Professor Emeritus Michal Green
Dr. Sheldon Tarre
Full Thesis textFull thesis text - English Version


Abstract

Encapsulation of Pseudomonas sp. strain ADP (P. ADP) bacteria in durable co-electrospun microtubes for treatment of atrazine contaminated water without the addition of external carbon source was the main objective of this research. A microtube formulation resistant to biodegradation was investigated and consisted of polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) as the shell and polyvinylpyrrolidone (PVP) as the core solution carrying the bacteria. Viable P. ADP were successfully encapsulated in 4-6 µm diameter microtubes and atrazine biodegradation activity recovered after a number of consecutive batches in a buffer phosphate solution with atrazine only (BPA).

Long term atrazine biodegradation experiments using microtube encapsulated bacteria were carried out in consecutive batches with BPA. 248 consecutive batches were conducted in 892 days with an average atrazine biodegradation of 95.2%±6.8% observed. Even though no addition of external carbon source was given throughout the experiment, the microtube encapsulated bacteria did not require periodic regeneration in growth solution to maintain atrazine biodegradation activity. This is in contrast to literature reports that P. ADP is unable to use atrazine as a carbon and energy source. Mounting evidence for the presence of bacterial contamination able to use atrazine’s side chains as an energy source prompted microtube bacterial population analysis by PCR-DGGE. After 1.5 years, the original P. ADP inoculant was observed along with Chitinophagaceae, Variovorax, and Microbacterium. After 2.5 years, Variovorax, Cupriavidus, Comamonas, Xanthobacter, Microbacterium and Tsukamurella were observed, however, the microtube inoculant, P. ADP, was absent. Even though P. ADP was absent from the microbial population, the presence of pADP-1 plasmid responsible for atrazine degradation was confirmed and shown to be harboured in Variovorax.

Although microtubes were shown to be physically stable after 2.5 years, the inevitable growth of biofilm on the surface of the microtubes deters its use in long term bioremediation applications. Consequently, a batch fed fixed bed biofilm reactor was chosen to investigate the long term stability of the newly found heterogeneous atrazine degrading bacterial population under non sterile conditions without the addition of an external carbon source. A 1 liter lab scale reactor containing 300 ml of packed scoria media was operated for 66 consecutive batches with BPA over 230 days. 100% atrazine degradation was observed in all the batches. Ammonium, the end product of atrazine degradation, was observed in early batches but was replaced by full nitrification to nitrate till the end of reactor operation where DGGE analysis revealed bands belonging to the ammonia oxidizing archaea (AOA).

Next generation sequencing (NGS) analysis from DNA samples taken from the reactor confirmed a relative abundance of 97.8% bacteria dominated by Variovorax, Xanthobacter and Comamonas following reactor start-up with growth media. However, bacteria decreased to 69%, while archaea increased from 0% to 32.1% due to proliferation of the nitrifier Candidatus Nitrosophaera. The relative abundance of Variovorax was 17.3% at start-up and decreased dramatically to 0.8% where it remained stable at 1 to 2% till the end of the experiment. pADP-1 plasmid was present at the end of reactor operation, albeit in low concentrations.