Construction of Recombinant Klebsiella pneumoniae to Increase Ethanol Production on Residual Glycerol Fed-Batch Cultivat
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Construction of Recombinant Klebsiella pneumoniae to Increase Ethanol Production on Residual Glycerol Fed-Batch Cultivations Vanessa Zimmer da Silva 1 & Laura Jensen Ourique 1 Marco Antonio Zachia Ayub 1
& Cíntia de David
1
&
Received: 15 May 2020 / Accepted: 16 July 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
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. Keywords AdhE . Glycerol . Klebsiella pneumoniae . Ethanol . Exponential fed-batch culture
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003397-5) contains supplementary material, which is available to authorized users.
* Marco Antonio Zachia Ayub [email protected]
1
Biotechnology & Biochemical Engineering Laboratory (BiotecLab), Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
Applied Biochemistry and Biotechnology
Introduction In view of the current political, economic, and environmental problems associated with the use of fossil fuels, production of biofuels from renewable sources has been increasing over the last decades [1–4]. In particular, biodiesel production has gained appeal as an alternative to petroleum-based diesel [1, 3, 5–7]. Biodiesel is obtained by the transesterification of natural triglycerides, such as animal fats or vegetable oils, generating as main residue large amounts of glycerol, representing up to 10% (mass fraction) of the products of this reaction [4, 5, 8–10]. When compared to pure glycerol, residual glycerol is a cheaper feedstock, but its applications in pharmaceutical industries require expensive purification processes, compromising its economic feasibility [1,
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