title: Integrated fermentative production and downstream processing of 2,3-Butanediol from sucrose by non-pathogenic strains of Bacillus subtilis and Serratia plymuthica
Creators: name: Suttikul, Suthkamol
Publisher: name: University of Reading
Year: 2024

description: 2,3-Butanediol (2,3-BD) is an important platform chemical of industrial interest 
due to its extensive applications and high market value. Global warming, environmental 
impact and limitation of fossil fuel reserves are driving the development of eco-friendly 
and sustainable bio-based 2,3-BD. A global market of 2,3-BD is anticipated to reach 300 
million US dollars in 2030. 2,3-BD stereoisomers are linked to specific industrial 
applications, with pure chiral 2,3-BD isomers being highly sought after from a 
commercial point of view. The present study focused on the production of highly pure 
2,3-BD stereoisomers by non-pathogenic wild-type bacterial strains and the 
development of efficient downstream processing. The first task was the screening for 
non-pathogenic wild-type bacterial strains capable of producing 2,3-BD from sucrose-based media. Among the strains tested, Bacillus subtilis and Serratia plymuthica showed 
promising characteristics of producing notable concentrations of 2,3-BD in batch-flask 
cultures. Corn steep liquor (CSL) was proven as an alternative low-cost nitrogen source 
to costly nitrogen sources for 2,3-BD production. Furthermore, the impact of dissolved 
oxygen concentrations on 2,3-BD production was assessed in batch bioreactor process. 
Limited dissolved oxygen conditions suppressed butanediol dehydrogenase activity and 
supported maximum (R, R)-2,3-BD production at high purity (100%) in B. subtilis, 
whereas high dissolved oxygen conditions promoted bacterial growth and acetoin 
formation. S. plymuthica was reported for the first time as capable producer of highly
pure (95%) meso-2,3-BD. A fed-batch fermentation with optimum DO conditions and a 
constant residual sucrose feeding regime improved 2,3-BD production in both strains, 
reaching 42.31 g/L of 2,3-BD with a production yield of 0.52 g/g sucrose in B. subtilis,
v
and 26.23 g/L of 2,3-BD with a production yield of 0.34 g/g sucrose in S. plymuthica
fermentation. With regards to downstream processing, the yellowish-brown colour of 
two different fermentation broths (S. plymuthica and B. subtilis) were successfully 
reduced through treatment with activated charcoal. Subsequently, the ionic liquid (IL),
1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([C2mim][CF3SO3]) was 
investigated with inorganic salts for 2,3-BD separation. In the case of S. plymuthica
broth, 95.6% of 2,3-BD was successfully recovered from the fermentation broth with a 
partition coefficient of 24.6 by using a system composed of 30%[C2mim][CF3SO3] -
35%K2HPO4 (w/w). In addition, 35%[C2mim][CF3SO3] - 30%K2HPO4 provided a high 
yield for recovering 2,3-BD (97.3%) from B. subtilis fermentation broth, thus verifying 
the effectiveness of this separation method. Additionally, the study showed that IL could 
be effectively recovered (90% to 98%). Hence, non-pathogenic wild-type B. subtilis and
S. plymuthica could be potentially employed as promising strains for high chiral purity 
of 2,3-BD stereoisomer production. Also, IL-based salting out method enables the 
development of a novel separation process for efficient recovery of 2,3-BD from diverse 
fermentation broths.