Shashi Kant Bhatia

Biosynthesis of polyesters and polyamide building blocks using microbial fermentation and biotransformation

Biopolymers can be a green alternative to fossil-based polymers and can contribute to environmental protection because they are produced using renewable raw materials. Biopolymers are composed of various small subunits (building blocks) that are the intermediates or end products of major metabolic pathways. Most building blocks are secreted directly outside of cells, making downstream processes easier and more economic. These molecules can be extracted from fermentation broth and polymerized to produce a variety of biopolymers, e.g., polybutylene terephthalate, polyethylene terephthalate, polytrimethylene terephthalate, nylon-5,4 and nylon-4,6, with applications in medicine, pharmaceuticals, and textiles. Microbes are unable to naturally produce these types of polymers; thus, the production of building blocks and their polymerization is a fascinating approach for the production of these polymers. In comparison to naturally occurring biopolymers, synthesized polymers have improved and controlled structures and higher purity. The production of monomer units provides a new direction for polymer science because new classes of polymers with unique properties that were not previously possible can be prepared. Furthermore, the engineering of microbes for building-block production is an easy process compared to engineering an entire biopolymer synthesis pathway in a single microbe. Polyesters and polyamide polymers have become an important part of human life, and their demand is increasing daily. In this review, recent approaches and technology are discussed for the production of polyester/polyamide building blocks, i.e., 2-hydroxyisobutyric acid, 3-hydroxypropionic acid, mandelic acid, itaconic acid, adipic acid, terephthalic acid, succinic acid, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, 1,3-butanediol, cadaverine, and putrescine.

Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source.

In this study, a biosugar obtained from empty fruit bunch (EFB) of oil palm by hot water treatment and subsequent enzymatic saccharification was used for undecylprodigiosin production, using Streptomyces coelicolor. Furfural is a major inhibitor present in EFB hydrolysate (EFBH), having a minimum inhibitory concentration (MIC) of 1.9mM, and it reduces utilization of glucose (27%), xylose (59%), inhibits mycelium formation, and affects antibiotic production. Interestingly, furfural was found to be a good activator of undecylprodigiosin production in S. coelicolor, which enhanced undecylprodigiosin production by up to 52%. Optimization by mixture analysis resulted in a synthetic medium containing glucose:furfural:ACN:DMSO (1%, 2mM, 0.2% and 0.3%, respectively). Finally, S. coelicolor was cultured in a fermenter in minimal medium with EFBH as a carbon source and addition of the components described above. This yielded 4.2μg/mgdcw undecylprodigiosin, which was 3.2-fold higher compared to that in un-optimized medium.

Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01

The effect of various biomass derived inhibitors (i.e. furfural, hydroxymethylfurfural (HMF), vanillin, 4-hydroxy benzaldehyde (4-HB) and acetate) was investigated for fatty acid accumulation in Rhodococcus sp. YHY 01. Rhodococcus sp. YHY01 was able to utilize acetate, vanillin, and 4-HB for biomass production and fatty acid accumulation. The IC50 value for furfural (3.1mM), HMF (3.2mM), vanillin (2.0mM), 4-HB (2.7mM) and acetate (3.7mM) was calculated. HMF and vanillin affect fatty acid composition and increase saturated fatty acid content. Rhodococcus sp. YHY 01 cultured with empty fruit bunch hydrolysate (EFBH) as the main carbon source resulted in enhanced biomass (20%) and fatty acid productivity (37%), in compression to glucose as a carbon source. Overall, this study showed the beneficial effects of inhibitory molecules on growth and fatty acid production, and support the idea of biomass hydrolysate utilization for biodiesel production by avoiding complex efforts to remove inhibitory compounds.

Microbial production of volatile fatty acids: current status and future perspectives

Volatile fatty acids (VFAs) are used as building blocks to synthesize a wide range of commercially-important chemicals. Microbially produced VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid) can be considered as a replacement for petroleum-based VFAs due to their renewability, degradability, and sustainability. The main objective of this review is to summarize research and development of VFA production methods via microbial routes, their downstream processes, current applications, and main challenges. Various fermentation processes have been developed to produce of VFAs starting from commercially-available sugars and other raw materials such as lignocellulose, whey, and waste sludge. Only few microbes have been explored for their potential to produce VFAs, and very little genomic information data is available at the present time. There is a need to use metabolic engineering, systematic biology, evolutionary engineering, and bioinformatics to discover VFA biosynthesis routes since the pathways for isobutyric acid and isovaleric acids are still not well understood.

Overexpression of succinyl‐CoA synthase for poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) production in engineered Escherichia coli BL21(DE3)

This study aims to increase the 3‐hydroxyvalerate (3HV) fraction in poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) [P(HB‐co‐HV)] using succinyl‐CoA synthase.

Exopolysaccharide from psychrotrophic Arctic glacier soil bacterium Flavobacterium sp. ASB 3-3 and its potential applications

A novel exopolysaccharide (EPS) producing psychrotrophic bacterium Flavobacterium sp. ASB 3-3 was isolated from Arctic glacier soil and identified. The optimum fermentation conditions for EPS production were an initial medium pH of 7.2 and an initial inoculum size of 5% (v/v). The maximum yield of EPS (7.25 ± 0.26 g L−1) was obtained after cultivation at 25 °C for 120 h with glycerol as the sole carbon source. The EPS was purified and its structural characteristics were analyzed by 1H and 13C NMR. The predominant repeating units of this EPS are (α, β) D-glucose and D-galactose and it is different from the structure of EPSs produced by other Arctic and Antarctic bacteria, which have mannose units. In addition, EPS has demonstrated a comparable emulsifying property than SDS and flocculating properties with kaolinite, suggesting their potential applications in various industries. The EPS also significantly improved the tolerance of Flavobacterium sp. and Escherichia coli from freeze–thaw cycles, suggesting that it might be used to survive in polar regions and it can have possible usage as microbial cryoprotectants.

Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli

To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes. By comparing noxE- and fdh-harbouring strains, the form of NADH, rather than NAD+, was the major effector of furfural tolerance. Overall, this study is the application of the salvage pathway to isobutanol production in the presence of furfural, and this system seems to be applicable to alleviate furfural toxicity in the production of other biochemical.

Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase ( cadA ) genes in Escherichia coli

Itaconate, a C5 unsaturated dicarboxylic acid, is an important chemical building block that is used in manufacturing high-value products, such as latex and superabsorbent polymers. Itaconate is produced by fermentation of sugars by the filamentous fungus Aspergillus terreus. However, fermentation by A. terreus involves a long fermentation period and the formation of various byproducts, resulting in high production costs. E. coli has been developed as an alternative for producing itaconate. However, fermentation of glucose gives low conversion yields and low productivity. Here, we report the whole-cell bioconversion of citrate to itaconate with enhanced aconitase and cis-aconitate decarboxylase activities by controlling the expression of multiple cadA genes. In addition, this bioconversion system does not require the use of buffers, which reduces the production cost and the byproducts released during purification. Using this whole-cell bioconversion system, we were able to catalyze the conversion of 319.8 mM of itaconate (41.6 g/L) from 500 mM citrate without any buffer system or additional cofactors, with 64.0% conversion in 19 h and a productivity of 2.19 g/L/h. Our bioconversion system suggests very high productivity for itaconate production.

Metal removal and reduction potential of an exopolysaccharide produced by Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620

An exopolysaccharide (EPS) was produced from psychrotrophic Arctic glacier fore-field soil bacterium Pseudomonas sp. PAMC 28620 using glycerol enriched medium and the maximum productivity 7.24 ± 0.31 g L−1 of EPS was obtained after 168 h of fermentation. The EPS was purified and analysed by HPLC, GC-MS, FT-IR, 1H and 13C NMR. The EPS obtained from Arctic strain PAMC 28620 exhibits a distinctive structural composition and the constituent sugar monomers are rhamnose, galactose, glucose, fucose, mannose and ribose. The purified EPS has shown excellent flocculating and emulsification capacities with promising biotechnological and ecological implications. From the metal removal experiments, the EPS exhibited remarkable metal adsorption (99%) potential adopting the order Fe2+ > Cu2+ > Mg2+ > Zn2+ > Mn2+ > Ca2+. FE-SEM combined with EDX analysis has shown that the metal ions were complexed or immobilized onto the EPS matrix and further reduced to nanoparticles (150–950 nm). This study is significant in terms of metal removal and reduction potential of Arctic bacterial EPS and the possible ecological roles of the EPS in Arctic environment.

Production and characterization of medium-chain-length polyhydroxyalkanoate copolymer from Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620

Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620 was found to produce a distinctive medium-chain-length polyhydroxyalkanoate (MCL-PHA) copolymer when grown on structurally unrelated carbon sources including glycerol. The maximum MCL-PHA copolymer yield was obtained about 52.18±4.12% from 7.95±0.66g/L of biomass at 144h of fermentation when 3% glycerol was used as sole carbon and energy source during the laboratory-scale bioreactor process. Characterization of the copolymer was carried out using fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), proton (1H) and carbon (13C) nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), differential scanning calorimeter (DSC) and thermo-gravimetric analysis (TGA). The copolymer produced by Pseudomonas sp. PAMC 28620 consisting of four PHA monomers and identified as 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxytetradecanoate (3HTD). An average molecular weight of the copolymer was found approximately 30.244kDa with polydispersity index (PDI) value of 2.05. Thermal analysis showed the produced MCL-PHA copolymer to be low-crystalline (43.73%) polymer with great thermal stability, having the thermal decomposition temperature of 230°C-280°C, endothermic melting temperature (Tm) of 172.84°C, glass transition (Tg) temperature of 3.99°C, and apparent melting enthalpy fusion (ΔHm) about 63.85Jg-1.