Design and construction of an artificial labor-division consortium for phenanthrene degradation with three-functional modules.

Polycyclic Aromatic Hydrocarbons (PAHs) are highly toxic organic pollutants. Phenanthrene often serves as a model compound for studying PAHs biodegradation. In this work, we firstly engineered Escherichia coli M01 containing seven phenanthrene degradation genes and combined it with existing engineered strains E. coli M2 and M3 to form an artificial three-bacteria consortium, named M0123, which exhibited a degradation ratio of 64.66% for 100 mg/L of phenanthrene over 8 days. Subsequently, we constructed engineered Pseudomonas putida KTRL02 which could produce 928.49 mg/L rhamnolipids and integrated it with M0123, forming a four-bacteria consortium with an impressive 81.62% phenanthrene degradation ratio. Assessment of extracellular adenosine levels during the degradation process indicated high cellular energy demand in the four-bacteria consortium. Then, we introduced Bacillus subtilis RH33, a riboflavin-producing strain, as an energy-supplying bacterium, to create a five-bacteria consortium, which exhibited an 88.19% degradation ratio for phenanthrene. The NADH/NAD ratio in the five-bacteria consortium during the degradation process was monitored, which was consistently higher than that of the four-bacteria consortium over the eight-day period, indicating a higher overall intracellular reduction capacity. Furthermore, the five-bacteria consortium displayed good tolerance to phenanthrene, even achieving a degradation ratio of 79.38% for 500 mg/L of phenanthrene. This study demonstrates that designing and constructing artificial consortia from the functional perspective and various angles can effectively enhance the degradation of phenanthrene after the addition of the energy-supplying bacterium. This study demonstrates that designing and constructing artificial labor-division consortia from the functional perspective and various angles can effectively enhance the degradation of phenanthrene.