K. pneumoniae BLh-1 strain was genetically modified aiming at obtaining high ethanol productivity in cultivations using residual glycerol from biodiesel synthesis as substrate. The recombinant strain K. pneumoniae Kp17 was obtained by inserting the multicopy plasmid pTOPOBL17 containing the AdhE gene, and its own promoter, from K. pneumoniae BLh-1. Influence of Fe²⁺ supplementation and initial glycerol concentration on culture conditions were analyzed, both in rotatory shaker and in batch bioreactors. In the bioreactor cultures, K. pneumoniae Kp17 strain produced 4.5 g L⁻¹ of ethanol (productivity of 0.50 g L⁻¹ h⁻¹ and yields of 0.15 g g⁻¹) after 24-h cultivation, corresponding to an increase of approximately 40% in ethanol concentration compared to wild strain, K. pneumoniae BLh-1. Best conditions were then applied in exponential fed-batch bioreactors, with final ethanol concentration of 17.30 g L⁻¹ (productivity of 0.59 g L⁻¹ h⁻¹ and yields of 0.16 g g⁻¹) after 30 h of feeding, representing 11.5% of increment in titer of ethanol compared to the wild strain. Mutant cells kept 92.5% of the plasmids under batch in 24 h, and 71.9% under fed-batch after 27 h of exponential feeding. The findings in this work show the possibility of using a simple approach to genetically modify K. pneumoniae to be employed this versatile bacterium for the bioconversion of residual glycerol into ethanol.

肺炎克雷伯菌BLh-1 菌株经过基因改造，旨在使用生物柴油合成中的残留甘油作为底物在培养中获得高乙醇生产率。通过插入来自肺炎克雷伯菌BLh-1 的含有 AdhE 基因及其自身启动子的多拷贝质粒 pTOPOBL17 获得重组菌株肺炎克雷伯菌Kp17。在旋转摇床和分批生物反应器中分析了 Fe ²⁺补充和初始甘油浓度对培养条件的影响。在生物反应器培养物中，肺炎克雷伯氏菌Kp17 菌株产生 4.5 g L ^-1乙醇（0.50 g L ^-1  h ^{-1 的}生产力和 0.15 g g^-1 ) 培养 24 小时后，对应于与野生菌株肺炎克雷伯菌BLh-1相比乙醇浓度增加约 40% 。然后在指数补料分批生物反应器中应用最佳条件，进料 30 小时后乙醇最终浓度为 17.30 g L ^-1（生产率为 0.59 g L ^-1  h ^-1，产量为 0.16 g g ^-1），占 11.5%与野生菌株相比，乙醇滴度的增加。突变细胞在 24 小时内分批保留了 92.5% 的质粒，在指数补料 27 小时后分批补料下保留了 71.9%。这项工作的发现表明使用简单方法对肺炎克雷伯菌进行基因改造的可能性 将使用这种多功能细菌将残留的甘油生物转化为乙醇。