Toluene biodegradation by Pseudomonas putida F1: targeting culture stability in long-term operation

Abstract

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h(-1)). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO2 produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.

Response of Pseudomonas putida F1 cultures to fluctuating toluene loads and operational failures in suspended growth bioreactors

“…The lower respiration rate of 3-MC when compared to toluene respiration at comparable concentrations suggests the potential accumulation of 3-MC during the long term run. However, accumulation of 3-MC in P. putida F1 cultures was recorded neither in this study nor in previous experiments carried out in our lab (Diaz et al 2008). In addition, it must be highlighted that both the occurrence of severe pH shifts and pH mediated effects was totally unexpected based on the high stability shown by P. putida F1 in a similar experimental set-up during…”

“…In order to elucidate the source of the deterioration in bacterial activity a NH 4 + enriched MSM was supplied after the 3rd process collapse. The pernicious effect of nitrogen deprivation on toluene biodegradation at concentrations up 20 g m -3 (Diaz et al 2008).…”

“…The influence of fluctuations and operational failures on process performance was investigated under sterile conditions in a magnetically stirred 1-l glass bioreactor (Afora S.A., Spain) operated as a chemostat (Diaz et al 2008). In all tests, the bioreactor was filled with 900 ml of sterile MSM and inoculated with 40 ml of P. putida F1 to attain initial biomass concentrations ranging from 14 to 38 mg Dry Weight l -1 (from now on mg DW l -1 ).…”

“…In addition, Dissolved Oxygen Concentration (DOC) and Temperature (T) were monitored on line. In the following, process stability was mainly evaluated based on process removal efficiency (RE), elimination capacity (EC), carbon dioxide production, and specific ATP content as defined in Diaz et al (2008).…”

Isolation and characterization of naphthalene biodegrading Methylobacterium radiotolerans bacterium from the eastern coastline of the Kingdom of Saudi Arabia

“…For instance, P. aeruginosa can grow in the presence of a variety of aliphatic and aromatic compounds such as lactate (Gao et aromatic fl uoranthene, phenanthrene (Zhang et al 2011), hexadecane, benzene, toluene (S Mukherjee et al 2010), paracetamol (Hu et al 2013), and 4-chlorobenzoate (Hoskeri et al 2011). Similar wide range of assimilation of substrates has been reported with other species of the same genus, such as Pseudomanas putida with biodegradation of aromatic compounds (Diaz et al 2008, Ebrahimi and Plettner 2013, El-Naas et al 2009, Fernandez et al 2012, Hwang et al 2009, Li et al 2011, Q Lin and Jianlong 2010, Phale et al 2013, Takeo et al 2006, You et al 2013) and alkane derivatives (Dunn et al 2005, Johnson and Hyman 2006, Smith and Hyman 2004. In line with previous reports, Pseudomonas aeruginosa strain N7B1 had a wide range of substrate specifi city, thus is an important bacterium that could be used in bioremediation strategies.…”