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A novel downstream process for highly pure 1,3-propanediol from an efficient fed-batch fermentation of raw glycerol by Clostridium pasteurianum.

ABSTRACT: An efficient downstream process without prior desalination was developed for recovering 1,3-propanediol (1,3-PDO) with high purity and yield from broth of a highly productive fed-batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3-PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3-PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3-PDO (< 80%). In order to improve the efficiency of final 1,3-PDO rectification, we examined alkaline hydrolysis to eliminate the ester impurities. By the use of 20% (w/w) water and 2% (w/w) sodium hydroxide, > 99% reduction of 1,3-PDO esters was achieved. This step conveniently provided free 1,3-PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3-PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3-PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high-salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3-PDO esters are crucial for the efficient separation of highly pure 1,3-PDO with acceptable yield from fermentation broth of raw glycerol.

SUBMITTER: Zhang C

PROVIDER: S-EPMC8182277 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundA fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation.ResultsA putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent.ConclusionsThis study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

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A novel downstream process for highly pure 1,3-propanediol from an efficient fed-batch fermentation of raw glycerol by Clostridium pasteurianum.

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