Evaluation of scale-up effect on cold-active enzyme production and biodegradation tests using pilot-scale bioreactors and a 3D soil tank.

Despite recent attention being paid to the biodegradation of petroleum hydrocarbons in cold environments, scale-up studies of biodegradation are lacking. Herein, the effect of scale-up on the enzymatic biodegradation of highly contaminated soil at low temperatures was studied. A novel cold-adapted bacteria (Arthrobacter sp. S2TR-06) was isolated that could produce cold-active degradative enzymes (xylene monooxygenase (XMO) and catechol 2,3-dioxygenase (C2,3D)). Enzyme production was investigated on 4 different scales (lab to pilot scale). The results showed a shorter fermentation time, and the highest production of enzymes and biomass (107 g/L for biomass, 109 U/mL, and 203 U/mL for XMO and C2,3D after 24 h) was achieved in the 150-L bioreactor due to enhanced oxygenation. Multi-pulse injection of p-xylene into the production medium was needed every 6 h. The stability of membrane-bound enzymes can be increased up to 3-fold by adding FeSO at 0.1% (w/v) before extraction. Soil tests also showed that biodegradation is scale-dependent. The maximum biodegradation rate decreased from 100% at lab-scale to 36% in the 300-L sand tank tests due to limited access of enzymes to trapped p-xylene in soil pores, low dissolved oxygen in the water-saturated zone, soil heterogeneity, and the presence of the free phase of p-xylene. The result demonstrated that formulation of enzyme mixture with FeSO and direct injection of enzyme mixture (third scenario) can increase the efficiency of bioremediation in heterogeneous soil. In this study, it was demonstrated that cold-active degradative enzyme production can be scaled up to an industrial scale and enzymatic treatment can be used to effectively bioremediate p-xylene contaminated sites. This study could provide key scale-up guidance for the enzymatic bioremediation of mono-aromatic pollutants in water-saturated soil under cold conditions.