Comprehensive evaluation of microalgal based dairy effluent treatment process for clean water generation and other value added products.

The current study demonstrates a comprehensive investigation on clean water generation from raw dairy wastewater (RDW) using a robust microalgal strain, Ascochloris sp. ADW007 and its growth, biomass, and lipid productivities in outdoor conditions. Microalgal treatment studies were conducted in column photobioreactor (CPB) and flat-pate photobioreactor (FPB), where the volumetric algal biomass productivity in RDW was significantly increased in both CPB (0.284 ± 0.0017 g/L/d) and FPB (0.292 ± 0.0121 g/L/d) as compared to synthetic mediums viz., BG11 and TAP, respectively, with enhanced lipid content. Maximum lipid accumulation of 33.40% was obtained within 7 d growth. The volumetric and areal lipid productivities in CPB and FPB were 94 mg/L/d and 5.597 g/m2/d, and 98 mg/L/d and 9.754 g/m2/d, respectively. Chemiflocculation, filtration, and centrifugation techniques were employed for harvesting microalgal biomass. Among the flocculants, 0.08% (w/v) FeCl3 harvested >99% of algal cells within 5 min, while 0.03% (w/v) cetyl trimethyl ammonium bromide and 0.125% (w/v) sodium hydroxide harvested >96% of the cells in 30 and 60 min. After microalgal treatment, >80% of clean and odorless water was obtained with reduction in 94-96% of COD, 72-80% of nitrate and 80-97% of total phosphate, respectively. Highlights Utilization of 100% raw dairy wastewater without any treatment. Production of clean and odorless water for recycle and reuse. COD, nitrate and total phosphate reduction by 94-96%, 72-80%, and 80-97% after treatment. Microalgal treatment studies in simple column and flat-plate photobioreactors. Biomass and lipid production as other value added by-products.

Toward a mechanistic and physiological understanding of a ferredoxin:disulfide reductase from the domains Archaea and Bacteria.

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster. UV-visible, EPR, and Mössbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR, advancing the physiological understanding of FDR's role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show that FDR homologs are widespread in diverse microbes from the domain Bacteria.

Structural and Biochemical Characterizations of Methanoredoxin from Methanosarcina acetivorans, a Glutaredoxin-Like Enzyme with Coenzyme M-Dependent Protein Disulfide Reductase Activity.

Glutaredoxins (GRXs) are thiol-disulfide oxidoreductases abundant in prokaryotes, although little is understood of these enzymes from the domain Archaea. The numerous characterized GRXs from the domain Bacteria utilize a diversity of low-molecular-weight thiols in addition to glutathione as reductants. We report here the biochemical and structural properties of a GRX-like protein named methanoredoxin (MRX) from Methanosarcina acetivorans of the domain Archaea. MRX utilizes coenzyme M (CoMSH) as reductant for insulin disulfide reductase activity, which adds to the diversity of thiol protectants in prokaryotes. Cell-free extracts of M. acetivorans displayed CoMS-SCoM reductase activity that complements the CoMSH-dependent activity of MRX. The crystal structure exhibits a classic thioredoxin-glutaredoxin fold comprising three α-helices surrounding four antiparallel ß-sheets. A pocket on the surface contains a CVWC motif, identifying the active site with architecture similar to GRXs. Although it is a monomer in solution, the crystal lattice has four monomers in a dimer of dimers arrangement. A cadmium ion is found within the active site of each monomer. Two such ions stabilize the N-terminal tails and dimer interfaces. Our modeling studies indicate that CoMSH and glutathione (GSH) bind to the active site of MRX similar to the binding of GSH in GRXs, although there are differences in the amino acid composition of the binding motifs. The results, combined with our bioinformatic analyses, show that MRX represents a class of GRX-like enzymes present in a diversity of methane-producing Archaea.

Natural deep eutectic solvent mediated pretreatment of rice straw: bioanalytical characterization of lignin extract and enzymatic hydrolysis of pretreated biomass residue.

The present investigation demonstrated pretreatment of lignocellulosic biomass rice straw using natural deep eutectic solvents (NADESs), and separation of high-quality lignin and holocellulose in a single step. Qualitative analysis of the NADES extract showed that the extracted lignin was of high purity (>90 %), and quantitative analysis showed that nearly 60 ± 5 % (w/w) of total lignin was separated from the lignocellulosic biomass. Addition of 5.0 % (v/v) water during pretreatment significantly enhanced the total lignin extraction, and nearly 22 ± 3 % more lignin was released from the residual biomass into the NADES extract. X-ray diffraction studies of the untreated and pretreated rice straw biomass showed that the crystallinity index ratio was marginally decreased from 46.4 to 44.3 %, indicating subtle structural alterations in the crystalline and amorphous regions of the cellulosic fractions. Thermogravimetric analysis of the pretreated biomass residue revealed a slightly higher T dcp (295 °C) compared to the T dcp (285 °C) of untreated biomass. Among the tested NADES reagents, lactic acid/choline chloride at molar ratio of 5:1 extracted maximum lignin of 68 ± 4 mg g(-1) from the rice straw biomass, and subsequent enzymatic hydrolysis of the residual holocellulose enriched biomass showed maximum reducing sugars of 333 ± 11 mg g(-1) with a saccharification efficiency of 36.0 ± 3.2 % in 24 h at 10 % solids loading.

Structural and Biochemical Characterization of a Ferredoxin:Thioredoxin Reductase-like Enzyme from Methanosarcina acetivorans.

Bioinformatics analyses predict the distribution in nature of several classes of diverse disulfide reductases that evolved from an ancestral plant-type ferredoxin:thioredoxin reductase (FTR) catalytic subunit to meet a variety of ecological needs. Methanosarcina acetivorans is a methane-producing species from the domain Archaea predicted to encode an FTR-like enzyme with two domains, one resembling the FTR catalytic subunit and the other containing a rubredoxin-like domain replacing the variable subunit of present-day FTR enzymes. M. acetivorans is of special interest as it was recently proposed to have evolved at the time of the end-Permian extinction and to be largely responsible for the most severe biotic crisis in the fossil record by converting acetate to methane. The crystal structure and biochemical characteristics were determined for the FTR-like enzyme from M. acetivorans, here named FDR (ferredoxin disulfide reductase). The results support a role for the rubredoxin-like center of FDR in transfer of electrons from ferredoxin to the active-site [Fe4S4] cluster adjacent to a pair of redox-active cysteines participating in reduction of disulfide substrates. A mechanism is proposed for disulfide reduction similar to one of two mechanisms previously proposed for the plant-type FTR. Overall, the results advance the biochemical and evolutionary understanding of the FTR-like family of enzymes and the conversion of acetate to methane that is an essential link in the global carbon cycle and presently accounts for most of this greenhouse gas that is biologically generated.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a novel plant-type ferredoxin/thioredoxin reductase-like protein from Methanosarcina acetivorans.

The genome of Methanosarcina acetivorans contains a gene (ma1659) that is predicted to encode an uncharacterized chimeric protein containing a plant-type ferredoxin/thioredoxin reductase-like catalytic domain in the N-terminal region and a bacterial-like rubredoxin domain in the C-terminal region. To understand the structural and functional properties of the protein, the ma1659 gene was cloned and overexpressed in Escherichia coli. Crystals of the MA1659 protein were grown by the sitting-drop method using 2 M ammonium sulfate, 0.1 M HEPES buffer pH 7.5 and 0.1 M urea. Diffraction data were collected to 2.8 Šresolution using the remote data-collection feature of the Advanced Light Source, Lawrence Berkeley National Laboratory. The crystal belonged to the primitive cubic space group P23 or P2(1)3, with unit-cell parameters a=b=c=92.72 Å. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient (VM) of 3.55 Å3 Da(-1), corresponding to a solvent content of 65%.