Development of a Novel Lyophilization Method for the Production of Bacterial Strain Powders to Enhance the Cleanup Efficiency of Petroleum Hydrocarbon–Polluted Soils

Abstract

A novel lyophilization (freeze-drying) technique has been developed to produce the petroleum hydrocarbon (PH)–degrading bacterial strain powders for the enhancement of bioremediation of PH-polluted soils. Four different PH-degrading strains (Paenarthrobacter ureafaciens, Klebsiella oxytoca, Pseudomonas citronellolis, and Enterobacter cloacae) isolated from a PH-polluted site were used for bacterial strain powder production. The modified lyophilization buffer contained skimmed milk powder, trehalose, sucrose, and glycerin. The water content, bacterial activity, bacterial survival factor, glass transition temperature, and surface characteristics were analyzed to determine the properties of the produced bacterial strain powders. Addition of 20% skimmed milk powder as an excipient and 15% trehalose as a cryoprotectant could significantly enhance the stability and survival factor of the bacterial strains. The incorporation of 0.75% sucrose and 0.05% glycerol into the basic formula of skimmed milk powder and trehalose, respectively, could allow the bacterial strain powder to be stored at ambient temperature due to the effective environmental isolation and continuous nutrient supplement capabilities of the two agents. Up to 95% of the bacterial survival could be obtained when 24 h of prefreezing at −80°C was applied as the first stage of the modified lyophilization process followed by the second stage of freeze-drying for 24 h. With this process, the depreciation rate was less than 1% after storing the powder for 6-month at 25°C. The glass transition temperature of the strain powder fell within the range of 10°C to 25°C, indicating that the optimized formulation for bacterial strain powder production could facilitate the strain storage at room temperature and produce diverse powder variants. Through the application of novel lyophilization process, more stable bacterial strain powder could be produced with a higher survival factor and a longer storage period. Up to 83% of total PH removal could be obtained in microcosms with the application of strain bacterial powders.