Whey valorization for sustainable polyhydroxyalkanoate production by Bacillus megaterium: Production, characterization and in vitro biocompatibility evaluation.

Polyhydroxyalkanoates (PHAs) are biodegradable biopolymers acclaimed as an eco-friendly substitute of hazardously polluting petrochemical plastics. Using industrial by-products as PHA feedstocks could improve its process economics and market implementation. Valorizing the plenteous, nutritive pollutant whey as PHA production feedstock would be an excellent whey management strategy. This study aimed at whole/crude whey valorization for value-added PHA production using B. megaterium Ti3 innate protease, alleviating pretreatments. Response surface methodology (RSM) media optimization ascertained whey (%) as the key influential factor (p < 0.05). The optimized and validated RSM model (R2, 0.991; desirability, 1) facilitated 12.2, 11.5 folds increased PHA yield (2.20 ± 0.11 g/L) and productivity (0.05 gPHA/L/h). A positive correlation (r2, 0.95 and 0.87) was observed amid the innate enzymes (protease and lipase) and PHA production. The PHA was characterized by 1H and 13C NMR, GPC, TGA, and was identified as poly (3-hydroxybutyrate) (P3HB) by NMR. A significantly reduced roughness (110 ± 5.6 nm); increased hydrophilicity (8.6 ± 0.3 and 8.7 ± 0.5%), protein adsorption (68.75 ± 2.55 µg/cm2) and 1.6 folds higher biocompatibility achieved on poly (ethylene glycol) (PEG) blending compared to neat P3HB films. This is the first report on B. megaterium innate enzyme based whey valorization to PHAs also demonstrating its biomedical applicability.

Polyhydroxyalkanoate (PHA) biosynthesis from directly valorized ragi husk and sesame oil cake by Bacillus megaterium strain Ti3: Statistical optimization and characterization.

Polyhydroxyalkanoates (PHAs) signify the most promising biological substitute to petrochemical plastics. Renewable and inexpensive agro-industrial by-products can be used as potent fermentation feedstocks for sustainable PHA biosynthesis. This study aimed at using a wild type B. megaterium strain Ti3 innate hydrolytic enzyme/s for eco-friendly valorization of 16 lignocellulosic agrowastes to PHA without pretreatments. Initial hydrolytic screening PHA concentration of (0.04-0.17 g/L), highlighted the strain's metabolic versatility. Pareto ranking of Taguchi orthogonal array (TOA) established ragi husk (RH), sesame oil cake (SOC) and KH2PO4 as the most influential factors (p < 0.05). The optimized and validated Response surface methodology (RSM) model (R2, 0.979; desirability, 1) resulted in 3.8 and 3.6 fold increased PHA production, 4.3 and 3.25 fold increased PHA productivity. A positive correlation (r2, 0.5-0.97) was observed amid the producer innate hydrolytic enzymes (lipase, amylase and cellulase) and PHA production. The PHA was characterized by 1H and 13C NMR, GPC, TGA. The polymer was identified as a scl-mcl copolyester with 92% 3HB (3-hydroxybutyrate) and 8% 3HHp (3-hydroxyheptanoate) monomers by NMR. This the first report on B. megaterium self-enzyme reliant non-food agrowastes bioconversion to PHA with 3HHp (3-hydroxyheptanoate) monomers excluding precursor addition, commercial enzymes, pure carbon and nitrogen sources.

Biolytic extraction of poly(3-hydroxybutyrate) from Bacillus megaterium Ti3 using the lytic enzyme of Streptomyces albus Tia1.

The applicability of Streptomyces sp. cell lytic enzymes for devising a simple and competent biological polyhydroxyalkanoate (PHA) recovery approach from Bacillus megaterium cells was investigated. B. megaterium strain Ti3 produced 50% (w/w) PHA using glucose as carbon source. The intracellular PHA was recovered employing a non-PHA accumulating actinomycetes (Tia1) identified as Streptomyces albus, having potent lytic activity against living and heat inactivated B. megaterium. Interestingly, maximum biomass (2.53 ±â€¯0.6 g/L by 24 h) of the lytic actinomycete was obtained in PHA production medium itself thus circumventing the prior actinomycete acclimatization just by co-inoculation with B. megaterium as an inducer. Maximum lytic activity was observed at pH 6.0, 40 °C, 220 mg of biomass and 33.3 mL of concentrated culture filtrate in a 100 mL reaction mixture. Preliminary biochemical investigations confirmed the proteolytic and caseinolytic nature of the lytic enzyme. PHA yield of 0.55 g/g by co-inoculation extraction approach was comparable with the conventional sodium hypochlorite based extraction method. Interestingly, S. albus also demonstrated a broad spectrum lytic potential against varied Gram-negative and Gram-positive PHA producers highlighting the extensive applicability of this biolytic PHA recovery approach. The lytic enzyme retained almost 100% relative activity on storage at -20 °C upto two months. 1H Nuclear magnetic resonance analysis of the extracted polymer confirmed it as a homopolymer composed of 3-hydroxybutyrate monomeric units. This is the first report on Streptomyces sp. based biological and eco-friendly, intracellular PHA recovery from Bacillus spp.

Interaction of vitamin D receptor with HLA DRB1 0301 in type 1 diabetes patients from North India.

BACKGROUND: Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where interaction and integration of immune response genes along with environmental factors play a role in autoimmune destruction of the insulin producing Pancreatic Beta cells. METHODOLOGY/PRINCIPAL FINDINGS: We have studied four single nucleotide polymorphisms (FokI site in Exon 2, BsmI and ApaI sites in Intron 8 and TaqI site in exon 9) in the vitamin D receptor (VDR) gene using PCR-RFLP and HLA-DRB1 alleles using PCR and hybridization with sequence specific oligonucleotide probes and studied their interaction using LD based statistics for non-linked loci followed by sequence analysis of the vitamin D response element (VDRE) present in the promoter region of HLA-DRB1 0301. Haplotypes, constructed using SHEsis program for four single nucleotide polymorphisms in the VDR gene, were studied for their interaction with HLA-DRB1 alleles in 233 T1D patients and 191 healthy controls from North India. A significant increase of haplotypes FBAt and fBAT (p<0.02, OR = 1.44 and p<0.002, OR = 3.23 respectively) was observed in the patients. Both the haplotypes FBAt and fBAT were significantly increased in male patients with age at onset less than 18 years; however, fBAT was significantly increased in female patients irrespective of their age at onset. LD based statistics showed significant interaction between the high producer F and T alleles with HLA-DRB1 0301. F and T alleles of VDR have been shown to contribute to VDR mRNA independently. The promoter sequence analysis of HLA-DRB1 0301 showed presence of VDRE involved in higher expression of HLA-DRB1 030, which was confirmed by flow cytometry and real time PCR analysis. CONCLUSIONS/SIGNIFICANCE: These data suggest that the interaction between VDR and HLA alleles is mediated by VDRE present in the promoter region of HLA-DRB1 0301 allele, which may be detrimental for the manifestation of T1D in the absence of 1,25-(OH)(2)D(3) in early childhood due to poor expression of DRB1 0301 in the thymus resulting in autoimmunity.