Treatment for purification water of biodiesel using electrofloculation

Biodiesel was created as a solution for a great economic and enviromental problem of petroleum, a resource with sustainable characteristics. But its production still needs optimization, because it uses a lot of water and generates a large volume of this residue, which appears improper to disposal without correct treatment. This work propose an economic, viable and efficient way to treat the Biodiesel purification water, not only aiming at a proper disposal, but the reuse as input in the process, generating a large industrial economy, and greater environmental progress. For both treatment uses the electrofloculation technique.

Preparative separation and purification of bufadienolides from ChanSu by high-speed counter-current chromatography combined with preparative HPLC

Eight bufadienolides were successfully isolated and purified from ChanSu by high-speed counter-current chromatography (HSCCC) combined with preparative HPLC (prep-HPLC). First, a stepwise elution mode of HSCCC with the solvent system composed of petroleum ether - ethyl acetate - methanol - water (4:6:4:6, 4:6:5:5, v/v) was employed and four bufadienolides, two partially purified fractions were obtained from 200 mg of crude extract. The partially purified fractions III and VI were then further separated by prep-HPLC, respectively, and another four bufadienolides were recovered. Their structures were confirmed by ESI-MS and ¹H-NMR spectra.

Separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography

An effective method for the rapid separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography (HSCCC) was successfully established. In the present study, a two-phase solvent system composed of chloroform-n-butanol-methanol-water (4:1:4:2, v/v/v/v) was used for HSCCC separation. A one-step separation in 4 h from 150 mg of crude extract produced 26.3 mg of trans-resveratrol-3-O-glucoside, 42.0 mg of pieceid-2"-O-gallate, and 17.9 mg of trans-resveratrol with purities of 99.1%, 97.8%, and 99.4%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these compounds were identified by nuclear magnetic resonance (NMR) spectroscopy.

Selection of microorganisms producer of lipase for fat removal from biodiesel purification water

The objective this study has been the selection of lipase productor microorganism, for removal of oils and grease, in the pre-treatment of biodiesel wastewater washing. For this, analyses of the physicist-chemistries characteristics had been made with the wastewater of the biodiesel washing, and then it had been isolated and chosen, by means of determinations of the lipase activity. Following, it was made a test of fat biodegradation, in the conditions: pH (5.95), temperature (35 ºC), rotation (180 rpm) and ammonium sulfate as nitrogen source (3 g L-1) and establishing as variable the two microorganism preselected and the time (24; 48; 72; 96 and 120 h). The biodiesel purification wastewater had presented high potential of environmental impact, presenting a concentration of O of 6.76 g L-1. From the six isolated microbiological cultures, two microorganisms (A and B) had been selected, with enzymatic index of 0.56 and 0.57, respectively. The treatment of the wastewater using the isolated microorganism (Klebsiella oxytoca) had 80% of the fatty removal in 48 h.

Anaerobic co-digestion of crude glycerin and starch industry effluent

The Brazil's Biodiesel Production and Use Program introduces biodiesel in the Brazilian energy matrix, bringing along the perspective of a growth of the glycerin offer, co-product generated in the proportion of 10 kg for each 100 L of biodiesel. The aim of this study was to evaluate the addition of crude glycerin in the anaerobic digestion of cassava starch industry effluent (cassava wastewater), in a horizontal semi-continuous flow reactor of one phase in laboratory scale. It was used a reactor with a 8.77 L of useful volume, a medium support for corrugated conduit of polyvinyl chloride (PVC), temperature of 261 ºC, fed with cassava wastewater and glycerin, with hydraulic detention times of 4 and 5 days and increasing volumetric organic load of 3.05; 9.32; 14.83 and 13.59 g COD L-1 d-1, obtained with the addition of glycerin at 0; 2; 3 and 2% (v/v), respectively. The average removal efficiencies of TS and TVS were decreasing from the addition of glycerin to the cassava wastewater, averaging 81.19 to 55.58% for TS and 90.21 to 61.45% for TVS. The addition of glycerin at 2% increased the biogas production compared to the control treatment, reaching 1.979 L L-1 d-1. The biogas production as a function of the consumed COD was higher for the control treatment than for the treatments with addition of glycerin, which indicates lower conversion of organic matter into biogas.

Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.

A lectin present in the marine red alga Pterocladiella capillacea was purified and characterised by extraction of soluble proteins (crude extract) in 20 mM Tris-HCl buffer, pH 7.5. Among the analysed erythrocytes (human blood group A, B and O and the animals ox, goat, chicken and rabbit) the lectin agglutinated specifically rabbit erythrocytes. The hemagglutinating activity assay showed that the lectin was not dependent on divalent cations and was shown to be inhibited by the glycoproteins avidin and mucin. The purification procedure was conduced by precipitation of the crude extract with 80% saturation ammonium sulfate (F0/80) followed by affinity chromatography on guar-gum column. The lectin of P. capillacea was purified 14.5 fold and had a recovery of 27.4% of the original total specific activity present in the crude extract. The absence of carbohydrate suggested that the lectin is not a glycoprotein. The molecular mass of P. capillacea lectin, determined by gel filtration, was 5.8 kDa. SDS-PAGE in the presence of ß-mercaptoethanol gave one band, indicating that the native lectin is a monomeric protein. The activation energy of denaturation process (D G') was calculated to be 106.87 kJ . mol-1 at 70 ºC.

Purification and characterization of a phytoalexin elicitor from spores of the saprobe Mucor ramosissimus

Plants accumulate antimicrobial compounds (phytoalexins) in response to a wide variety of microorganisms. Mucor ramosissimus Samutsevitsch is a saprobe capable of inducing phytoalexin production in soybean cotyledons and in the leaves of tropical Rubiaceae on whose surface it has been found. In the present study, the elicitor from M. ramosissimus was partially purified and the activity compared to that of a glucan elicitor isolated from Phytophthora sojae. Optimal isolation of the elicitor (based on fungal growth, yield of spores and elicitor activity) was achieved by autoclaving spores obtained from nine day-old cultures of the fungus. The elicitor was precipitated with ethanol and purified by chromatography on an anion exchange column, which retained the elicitor, and a Concanavalin A-affinity matrix, to which the elicitor did not bind. The purification resulted in a considerable increase (six-fold) in the specific activity of the elicitor. Neutral sugar composition, analyzed by HPLC, revealed the predominance of mannose, followed by glucose and galactose, whereas colorimetric quantification showed the presence of uronic acids. GC-MS analysis of the elicitor revealed the predominance of glucuronic acid and mannose. These results suggest that fragments of mucoran-type polysaccharides are the phytoalexin elicitors present in the spores of the saprobe M. ramosissimus. Our results also indicate for the first time that soybean cotyledon tissues can recognize fragments of glucuronic-acid heteropolymers as phytoalexin elicitors.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Purification of human albumin by the combination of the method of Cohn with liquid chromatography

Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG). The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F)-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998) Brazilian Journal of Medical and Biological Research, 31: 1375-1381). The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60ºC for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 ± 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were <=5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.

Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography

Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.