PSG College of Technology

Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant Ananas comosus. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) Hac1 on folding and secretion of recombinant stem bromelain in Pichia pastoris. Stem bromelain gene (BL) from Ananas comosus was codon optimized and expressed in the Pichia pastoris X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, Hac1p was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when HAC1 was co-expressed with stem bromelain (BL). Fed batch studies were performed for both pGAPαBL1 and pGAPBLHAC1 clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones like Kar2p, Sec 63, Pdi, Cne1. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies Pichia pastoris as a robust platform to produce stem bromelain for various industrial applications.

This study evaluates the techno-economic feasibility of using DWSIM Pro, an extension to the open-source process simulation software DWSIM, for modelling a bio-refinery co-producing bio-ethanol and biodiesel from genetically modified lipid-producing sorghum. The primary aim is to compare its performance and economic analysis capabilities with the Aspen Plus.

A detailed flowsheet was designed in DWSIM to simulate key unit operations. Material and energy balances were conducted to evaluate process efficiency. Economic assessments involved estimating capital and operational cost and comparing Minimum Ethanol Selling Prices (MESP) in DWSIM Pro. Sensitivity analyses were performed to assess the impact of varying parameters like feed flow rate and ethanol production volume on economic indicators.

DWSIM provided comparable results to Aspen Plus for material and energy balances, with differences typically within 2-10 %. The capital costs, operating costs and minimum ethanol selling price calculated by DWSIM Pro were lower by USD 240.3 M, USD 12.6 M/year and USD 3/gal, respectively. Sensitivity analysis revealed that increasing feed flow rates improved ethanol production efficiency and lowered MESP, indicating the scalability of the process.

DWSIM Pro is a cost-effective alternative to Aspen Plus for conducting techno-economic analyses of bio-fuel production processes.

This study highlights the potential of DWSIM Pro as an accessible and cost-effective tool for bioprocess simulation. It brings out the software's flexibility in modeling biorefinery processes while identifying key areas for further optimization, particularly in energy recovery and enzyme utilization.

Azo dye contamination poses significant environmental challenges due to its persistence and toxicity. Plant-microbe integrated systems offer a sustainable solution for dye bioremediation, yet the functional roles of microbial communities and their interactions within community and with host plants during bioremediation remain underexplored. This study presents an integrated, multi omics approach to dissect the microbial diversity, functional potential, and plant-microbe interactions within a plant-microbe integrated bioremediation system for model azo dye, methyl red degradation. The microbial diversity of various organisms enriched under different treatment conditions for effective azo dye treatment was explored. A read-based approach using HUMAnN 3 pipeline was adopted to extract metabolic information from the shotgun metagenomic reads. Diversity analysis showed the enrichment of microorganisms capable of growing in the presence of the pollutant methyl red in an oligotrophic condition. The metabolic potential of the enriched organisms in dye removal was studied. Based on the enzymatic abundance, a pathway for the degradation of methyl red is proposed. Endophytic bacteria such as Klebsiella pneumoniae and Klebsiella varicola were responsible for encoding major dye-degrading enzymes in plant-integrated systems. In the plant-microbe integrated system both endophytic and intestinal microorganisms such as Kluyvera intestini and Escherichia coli are among the top 5 contributors of genes encoding downstream aromatic compound degradation enzymes. Notably Enterococcus casseliflavus showed highest enzyme abundance for azobenzene reductase in plant -microbe integrated strategy with 11.5-fold greater abundance than the treatment system containing only microbial inoculum. Metabolomics data from root exudates experiment revealed the role of root exudates in selective recruitment of microbial community. The role of biofilm and quorum sensing pathways in enhancing the bioremediation potential of the microbiome and the potential microbe-microbe and plant-microbe interaction was analysed. Deciphering the metabolic contribution of each microorganism and the microbiome as a whole is crucial to design engineered bioremediation systems. ENVIRONMENTAL IMPLICATIONS: Understanding the potential of microorganisms, their enrichments, and survival will help in designing specific consortia for effective degradation of pollutants. Metagenomic analysis reveal that the functional complementation in the microbiome is responsible for the pollutant degradation and the presence of plants through the root exudates, provide the nutrients lacking in the oligotrophic conditions observed in many waste streams, thereby enriching suitable microorganisms. This metagenomic study along with the metabolomics component, provides the justification for the efficiency of the plant microbe treatment of model dye methyl red and this could be exploited in real time situations.