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2,3-Butanediol (2,3-BD), a platform chemical is estimated to have a worldwide market of 32 million tons/annum valued at approximately USD 43 billion in sales. 2,3-BD has its applications in manufacturing of synthetic rubber, printing ink, antifreeze, flavoring agent, pharmaceuticals and cosmetics. The current industrial production is done through the naptha cracking process at 800-900°C generating a huge amount of greenhouse gases (GHGs). Alternatively, microbial fermentation is a greener method for sustainable chemical manufacturing with minimal impact on environment. Also, 2,3-BD is biodegradable, making it a green platform chemical having many industrial applications.
The present work was undertaken to harness the potential of microorganism to produce 2,3-butanediol as major metabolite. Further, attempts were undertaken to maximize the production of 2,3-BD through process optimization. Fed-batch fermentation was also done by adopting methods for an economical scale up. Subsequently, downstream processing of 2,3-BD from the fermentation broth was investigated. A holistic approach with step-wise customized parameters were implemented for the selected strain to provide a cost-effective method of 2,3-BD production and downstream processing.
Out of total 21 strains screened from hydrocarbon-contaminated sites, thirteen strains showed potential to produce 2,3-BD. Aerobic condition promoted 2,3-BD accumulation compared to anaerobic condition. Among the potential strains, E. cloacae TERI BD 18 was found to assimilate most of the substrates showing maximum 2,3-BD production. Glucose was found to be the cheapest and showed maximum 2,3-BD production with a yield of 0.49 g/g in shake-flask condition. Among the different wastes E.cloacae TERI BD 18 showed the utilization of all the substrates for growth (rice straw hydrolysate, waste glycerol, dairy waste, molasses, distillery waste and syngas. However, significant 2,3-BD yield was from rice straw hydrolysate (0.40 g/g) and waste glycerol (0.41 g/g). Waste glycerol did not require any pre-treatment, hence was considered for feasibility study.
The best physiological conditions showing maximum 2,3-BD production was found with initial glucose concentration 30 g/l, initial pH 7.5, temperature 37 °C and agitation speed of 200 rpm. Similarly, the best conditions with waste glycerol as a carbon source was with initial waste glycerol of 30 g/l (actual glycerol with 7-9 g/l), initial pH 7.0, temperature 37 °C and agitation speed of 350 rpm for maximum 2,3-BD production by selected bacterial strain.
The optimized parameters helped to enhance 2,3-BD production in batch fermentation (3L). Maximum of 14.61g/l total 2,3-BD production (2,3-BD+ acetoin) with yield of 0.48 g/g was obtained with glucose in batch fermentation Similarly with waste glycerol, the 2,3-BD yield of 0.48 g/g was achieved in batch fermentation. Interim fed-batch and residual fed batch methods were adopted to enhance 2,3-BD production with glucose. Enhanced 2,3-BD production was observed with residual fed-batch mode in 3L bioreactor showing the total 2,3-BD production of 67.7 g/l with a yield of 0.45 g/g of glucose.
Residual fed-batch with secondary pH and secondary agitation strategy was optimized to further enhance 2,3-BD production. 2,3-BD fermentation from waste glycerol showed less production, however a forced pH change showed improvement in 2,3-BD production to 40.74 g/l with waste glycerol as carbon source.
Although encouraging results has been achieved in laboratory with waste glycerol, microbial conversion of 2,3-BD from glycerol is still a very slow process (0.21 g/l/h). The results obtained from glucose showed better productivity (1 g/l/h), hence commercial grade glucose was taken up for scale up studies.
By using the residual fed-batch with secondary pH and agitation change strategy the final 2,3-BD productivity was achieved up to 1.73 g/l/h with a yield of 0.480 g/g of 2,3-BD per gram of glucose in 150L bioreactor, which was 8% higher than the best result controlled by single pH and agitation speed and accounts to 96% of the theoretical value of 0.500 g 2,3-BD/g glucose.
Among the different downstream methodology (membrane separation, sugaring out, salting out) adopted for 2,3-BD separation from the fermentation broth, the salting out effect was found to be efficient in the present study. Maximum recovery of 86% was observed using salting out method. However, the separation with the used salts and recycled solvents showed to recover the remaining 2,3-BD (99% recovery). NMR also confirmed the purity of the product. The reuse of materials for multiple times for fermentation, or repeated downstream separation provides a solution to reduce the waste generation and make over all process more sustainable and efficient.
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