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

Abstract

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 FeSO₄ 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 FeSO₄ 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.

Keywords: Cold temperatures; Enzymatic biodegradation; Fermenter; Mono-aromatic pollutants; Pilot; Sand tank.