PRODUCTION OF 3-HYDROXYPROPIONIC ACID (3HP) USING ADVANCED BIOPROCESSING TECHNIQUES

Abstract

Conversion of low-value substrates to value-added chemicals has attracted significant attention of scientific community. This work focuses on novel approaches for production of value-added chemicals, 3-hydroxypropionic acid (3HP) and lactic acid (LA), from glycerol. 3 hydroxypropionic acid (3HP) is one of the top value-added chemicals of industrial importance produced by chemical catalysis and biological methods. Among several strains employed for bioconversion of glycerol, Lactobacillus reuteri attracts significant attention, because it does not require exogenous coenzyme-B12 and the extracellular secretion of 3HP facilitates downstream processing. Initially, the starter culture of L. reuteri was utilized to produce as much as 43 gl-1 of LA. For free cell culture, a maximum productivity of 2.4 gl-1h-1 was attained in 18 h. The highest LA production was achieved at the supplementation of 40 gl-1 glycerol to MRS media which is equivalent to substrate molar ratio of 0.27 (glucose to glycerol molar ratio). Further the study was extended to LA production from alginate immobilized L. reuteri. The immobilized cells were able to produce 38.3 gl-1 of LA with productivity of 2.1 gl-1h-1. The immobilized cells were able to produce LA for seven consecutive cycles with nearly constant production effciency. This reuseability feature of immobilized cells overtakes the lower production efficiency of immobilized cells as compared to free cells. The immobilized cell cultures also enabled easier downstream processing of the LA which may further improve the economics of the process. Further, the starter culture of L. reuteri was employed as whole cell biocatalysts for 3HP production in co-culture with Azospirilum brasilense cells. The co-culture exhibited significant benefits over the mono-culture of L. reuteri in terms of decrease in production time and increase in 3HP titre. This could be attributed to the synergistic functioning of the two microorganisms for efficient conversion of glycerol to 3HP. The biocatalysis process parameters were optimized using Response Surface Methodology (RSM) for maximum production of 3HP. The process parameters that were optimized are the amount of biomass of each of the individual strains, the concentration of glycerol supplementation, biocatalysis temperature and time. Under the optimized conditions, 4.9 gl-1 of 3HP was produced from 37 gl-1 of glycerol supplementation within 2 h at 37 ⁰C. The specific productivity and productivity for 3HP were found to be 0.3 g.gCDW-1l-1h-1 and 2.18 gl-1h-1, respectively. The potential of whole cell biocatalysts for reusability was examined by checking the viability of the mixed cells after 9 h of biocatalysis. Qualitative and quantitative profiling of the metabolites during biocatalysis was conducted using NMR spectroscopy and HPLC, respectively. To the best of our knowledge, this study is the first of its kind that integrates the whole cell biocatalysis with microbial co-cultures to enhance the production of industrially important bio-based chemicals, 3HP and LA. In order to reduce the labour and cost of the process, a recombinant strain of L. reuteri was developed which was able to show heterologous production of alpha- ketoglutraldehyde semialdehyde dehydrogenase (KGSADH) enzyme. KGSADH gene (GenBank: AB241137.1) of A. brasilence was cloned into pGST-TEV vector and transformed into L. reuteri by electroporation. The potential of induced recombinant L. reuteri was examined for production of 3HP. Under shake-flask conditions, the whole cell biocatalysts of recombinant L. reuteri showed enhanced production of 3HP upto 8.6 gl-1 within 2 h of incubation at 37 ⁰C. The recombinant strain was found to be more efficient in production of value-added chemical 3HP compared to native L. reuteri strain in terms of production levels and time of conversion.