Development of engineered Escherichia coli whole-cell biocatalysts for high-level conversion of L-lysine into cadaverine.

A whole-cell biocatalytic system for the production of cadaverine from L-lysine has been developed. Among the investigated lysine decarboxylases from different microorganisms, Escherichia coli LdcC showed the best performance on cadaverine synthesis when E. coli XL1-Blue was used as the host strain. Six different strains of E. coli expressing E. coli LdcC were investigated and recombinant E. coli XL1-Blue, BL21(DE3) and W were chosen for further investigation since they showed higher conversion yield of lysine into cadaverine. The effects of substrate pH, substrate concentrations, buffering conditions, and biocatalyst concentrations have been investigated. Finally, recombinant E. coli XL1-Blue concentrated to an OD(600) of 50, converted 192.6 g/L (1317 mM) of crude lysine solution, obtained from an actual lysine manufacturing process, to 133.7 g/L (1308 mM) of cadaverine with a molar yield of 99.90 %. The whole-cell biocatalytic system described herein is expected to be applicable to the development of industrial bionylon production process.

Valorization of sugarcane bagasse with in situ grown MoS2 for continuous pollutant remediation and microbial decontamination.

In this study, sugarcane bagasse (SB) was strategically subjected to a delignification process followed by the in situ growth of multi-layered molybdenum disulfide (MoS2) nanosheets with hexagonal phase (2H-phase) crystal structure via hydrothermal treatment. The MoS2 nanosheets underwent self-assembly to form nanoflower-like structures in the aligned cellulose inter-channels of delignified sugarcane bagasse (DSB), the mechanism of which was understood through FTIR and XPS spectroscopic studies. DSB, due to its porous morphology and abundant hydroxyl groups, shows remediation capabilities of methylene blue (MB) dye through physio-sorption but shows a low adsorption capacity of 80.21 mg/g. To improve the removal capacity, DSB after in situ growth of MoS2 (DSB-MoS2) shows enhanced dye degradation to 114.3 mg/g (in the dark) which further improved to 158.74 mg/g during photodegradation, due to catalytically active MoS2. Interestingly, DSB-MoS2 was capable of continuous dye degradation with recyclability for three cycles, reaching an efficiency of > 83%, along with a strong antibacterial response against Gram-positive Staphylococcus aureus (S.aureus) and Gram-negative Escherichia coli (E. coli). The present study introduces a unique strategy for the up-conversion of agricultural biomass into value-added bio-adsorbents, which can effectively and economically address the remediation of dyes with simultaneous microbial decontamination from polluted wastewater streams.

A halophilic Chromohalobacter species from estuarine coastal waters as a detoxifier of manganese, as well as a novel bio-catalyst for synthesis of n-butyl acetate.

Anthropogenic pollution due to ferro-manganese ore transport by barges through the Mandovi estuary in Goa, India is a major environmental concern. In this study a manganese (Mn) tolerant, moderately halophilic Chromohalobacter sp. belonging to the family Halomonadaceae was isolated from the sediments of a solar saltern adjacent to this Mandovi estuary. Using techniques of Atomic absorption spectroscopy, Scanning electron microscopy-Energy dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy and Atomic Force Microscopy, the Chromohalobacter sp. was explored for its ability to tolerate and immobilize Mn in amended and unamended media with 20% natural salt concentration (w/v). In aqueous media supplemented with 0.1 mM Mn, the Chromohalobacter sp. was capable of sequestering up to 76% Mn with an average immobilization rate of 8 mg Mn /g /day. Growth rate kinetic analysis using Gompertz mathematical functions was found to model the experimental data well. The model inferred that the maximum growth rate of Chromohalobacter sp. was at 10% natural salt concentration (w/v). The Chromohalobacter sp. was further found to be multimetal tolerant showing high tolerance to Iron (Fe), Nickel (Ni) and Cobalt (Co), (each at 4 mM), and tolerated Manganese (Mn) up to 6 mM. Morphologically, the Chromohalobacter sp. was a non-spore forming, Gram negative motile rod (0.726 µ× 1.33 µ). The adaptative mechanism of Chromohalobacter sp. to elevated Mn concentrations (1 mM) resulted in the reduction of its cell size to 0.339 µ× 0.997 µ and the synthesis of an extracellular slime, immobilizing Mn from the liquid phase forming Manganese oxide, as confirmed by Scanning Electron Microscopy. The expression of Mnx genes for manganese oxidation further substantiated the finding. This bacterial synthesized manganese oxide also displayed catalytic activity (∼50% conversion) for the esterification of butan-1-ol with CH3COOH to yield n-butyl acetate. This Chromohalobacter sp. being indigenous to marine salterns, has adapted to high concentrations of heavy metals and high salinities and can withstand this extremely stressed environment, and thus holds a tremendous potential as an environmentally friendly "green bioremediator" of Mn from euryhaline environments. The study also adds to the limited knowledge about metal-microbe interactions in extreme environments. Further, since Chromohalobacter sp. exhibits commendable catalytic activity for the synthesis of n-butyl acetate, it would have several potential industrial applications.

Novel bio-catalytic degradation of endocrine disrupting compounds in wastewater.

Against the backdrop of towering ecological health implications of estrogen pollution and the inefficacies associated with cost-intensive treatment techniques, this study recorded the earliest attempt of developing an inexpensive bacterial laccase-based biocatalysts for biodegradation of EDCs (Endocrine disrupting compounds), particularly estrogens. First, a central composite design was used to investigate the interactive effects of pH (6.0-8.0), inoculum size (100-500 U/mL), and copper (Cu) (25-75 mg/L) on laccase activity and estrogen degradation respectively. Thereafter, biocatalysts was synthesized comprising laccase and glass beads or silver impregnated clay granules (SICG), which was further used to treat estrogen infused aquatic matrices under different reaction conditions. Maximum laccase activities and estrogen removal for the two tested laccases were 620 U/mL (85.8-92.9%) and 689.8 U/mL (86.8-94.6%) for Lysinibacillus sp. BP1 and Lysinibacillus sp. BP2, respectively, within 72 h, under conditions of optimal inoculum size and/or Cu concentration. Apart from a higher estrogen removal rate compared to free laccased, the biocatalysts were more resistant to temperature, pH and other environmental perturbations, and had enhanced storage ability and reusability. In comparison to clay, beads had a higher potential for recyclability and were more stable under certain experimental factors such as pH, reuse, and temperature, as well as storage conditions. Immobilized enzymes were able to remove 100% of E2, as well as over 90% of E1 and EE2, in 24 h, indicating that they could be scaled up to benchtop bioreactor levels.

Effectiveness of a bio-catalytic agent used in the bioremediation of crude oil-polluted seawater.

Oil spillage contamination has been one of the most common and challenging problems in marine ecosystems over the years due to frequent petroleum exploitation, washing, and transportation activities. The use of nature-derived surfactants has become an attractive approach to restore the sites affected by oil spillage. Several studies have demonstrated that nutrient addition is an efficient strategy to enhance oil biodegradation since microorganisms can use petroleum hydrocarbons as their carbon and energy source, thus favoring and increasing the hydrocarbons degradation rate. This study aimed to assess the effectiveness of a commercial bio-catalytic agent used in the biological remediation of crude oil-contaminated sites through the qualitative analysis of its properties. The tests applied to this bio-catalyst showed excellent results. For instance, the emulsification (E24) and critical micellar concentration (CMC) assays displayed average values of 74.47% and 40 mg L-1, respectively. A significant reduction of Chemical Oxygen Demand (COD), turbidity, and Total Petroleum Hydrocarbon Content (TPHC) were observed in all the samples with bio-catalytic agent solution and aeration system. The best water quality was achieved by the sample with the highest concentration (10000 ppm) of bio-catalytic agent solution. It displayed a Total Petroleum Hydrocarbon removal efficiency (RTPH) of 81.537% after 30 days of the remediation time.

Application of the immobilized enzyme on magnetic graphene oxide nano-carrier as a versatile bi-functional tool for efficient removal of dye from water.

Herein, we report bi-functional applications of a novel immobilized enzyme on the modified magnetic graphene oxide (GO) for effective removal of dyes from water. The amine functionalized GO nano-carrier was covalently attached to a model enzyme (PersiManXyn1). The enzyme assays showed that the specific activities of the free and immobilized enzyme were 856.05 and 1141.1 µmolmin-1mg-1, respectively. While the free enzyme showed only 5% of its maximum activity, the immobilized PersiManXyn1 preserved more than 35% of its activity, at 90 °C. After four weeks storage, the free enzyme has been deactivated, but the immobilized enzyme retained 54% of its initial activity. The immobilized PersiManXyn1 was proficiently applied for dye removal from water using two strategies. While only pristine nano-carrier and free enzyme showed no considerable catalytic ability, the immobilized PersiManXyn1 could catalytically reduce the concentrated dye solutions within 150 s with superior reusability (94% dye removal after 15th cycle). Proficient treatment of a real textile effluent by the immobilized PersiManXyn1 approved its practical applications in the water remediation.

Engineering and optimization of phosphate-responsive phytase expression in Pichia pastoris yeast for phytate hydrolysis.

Phytate is the major storage form of phosphorus in plants. It is present in cereals and raw materials of vegetable origin used in animal and human diets. However, non-ruminant animals have little phytase activity in their guts and, therefore, cannot digest phytate. As a result, almost all dietary phytate is discharged into the environment, causing phosphorus pollution. Phytate is also considered as an "antinutrient" for its ability to form insoluble and stable complexes with metal ions, thus reducing dietary absorption of essential minerals. It is a dire need to develop sustainable approaches for environmentally-friendly utilization for this valuable and abundant natural resource. To this end, we engineered Pichia pastoris to express and secrete phytase in a "made-to-order" fashion in response to external level of inorganic phosphate (Pi). Responsiveness to external Pi level was achieved by generating a Pi-responsive promoter library using directed evolution. The resultant yeast strains were proven to liberate Pi from wheat-based meal in a simulated in vitro digestion model. These yeast-based whole cell biocatalysts may serve as platform hosts with potential applications in food processing industry and animal waste treatment.

Performance of a yeast-mediated biological fuel cell.

Saccharomyces cerevisiae present in common Baker's yeast was used in a microbial fuel cell in which glucose was the carbon source. Methylene blue was used as the electronophore in the anode compartment, while potassium ferricyanide and methylene blue were tested as electron acceptors in the cathode compartment. Microbes in a mediator-free environment were used as the control. The experiment was performed in both open and closed circuit configurations under different loads ranging from 100 kOmega to 400Omega. The eukaryotic S. cerevisiae-based fuel cell showed improved performance when methylene blue and ferricyanide were used as electron mediators, rendering a maximum power generation of 146.71+/-7.7 mW/m(3). The fuel cell generated a maximum open circuit voltage of 383.6+/-1.5 mV and recorded a maximum efficiency of 28+/-1.8 % under 100 kOmega of external load.

Synergistic effect of natural chickpea leaf exudates acids in heterocyclization: a greener protocol for benzopyran synthesis.

Without using any toxic or hazardous reagent, ligand, acid, transition metal catalyst, additives/promoters and organic solvent, green Knoevenagel condensation and tandem Knoevenagel-Michael reactions have been successfully carried out by using chickpea leaf exudates as a naturally sourced Bronsted acid type bio-catalyst. The reaction proceeds in neat chickpea leaf exudates at room temperature in aqueous conditions in very short reaction times, and therefore, it is an evergreen and environmentally sound alternative to the existing protocols for benzopyran synthesis. In comparison to the conventional methods, this synthetic pathway complies with several key requirements of green chemistry principles such as the utilization of biodegradable catalyst obtained from renewable feedstock, auxiliary aqueous conditions, along with waste prevention. The same protocol was also extended to the synthesis of 2H-xanthene-1,8-diones by condensation of aromatic aldehydes with dimedone achieving excellent yields. Thus, the reported protocol offers an attractive option because of its ecological safety, environmental acceptance, sustainability, low-cost straightforward work-up procedure and with excellent values of green chemistry metrics as compared with other reported methods.

Magnetically Modified Agricultural and Food Waste: Preparation and Application.

The annual food and agricultural waste production reaches enormous numbers. Therefore, an increasing need to valorize produced wastes arises. Waste materials originating from the food and agricultural industry can be considered as functional materials with interesting properties and broad application potential. Moreover, using an appropriate magnetic modification, smart materials exhibiting a rapid response to an external magnetic field can be obtained. Such materials can be easily and selectively separated from desired environments. Magnetically responsive waste derivatives of biological origins have already been prepared and used as efficient biosorbents for the isolation and removal of both biologically active compounds and organic and inorganic pollutants and radionuclides, as biocompatible carriers for the immobilization of diverse types of (bio)molecules, cells, nano- and microparticles, or (bio)catalysts. Potential bactericidal, algicidal, or anti-biofilm properties of magnetic waste composites have also been tested. Furthermore, low cost and availability of waste biomaterials in larger amounts predetermine their utilization in large-scale processes.

Microbial engineering strategies to improve cell viability for biochemical production.

Efficient production of biochemicals using engineered microbes as whole-cell biocatalysts requires robust cell viability. Robust viability leads to high productivity and improved bioprocesses by allowing repeated cell recycling. However, cell viability is negatively affected by a plethora of stresses, namely chemical toxicity and metabolic imbalances, primarily resulting from bio-synthesis pathways. Chemical toxicity is caused by substrates, intermediates, products, and/or by-products, and these compounds often interfere with important metabolic processes and damage cellular infrastructures such as cell membrane, leading to poor cell viability. Further, stresses on engineered cells are accentuated by metabolic imbalances, which are generated by heavy metabolic resource consumption due to enzyme overexpression, redistribution of metabolic fluxes, and impaired intracellular redox state by co-factor imbalance. To address these challenges, herein, we discuss a range of key microbial engineering strategies, substantiated by recent advances, to improve cell viability for commercially sustainable production of biochemicals from renewable resources.