The study for isolation and purification of R-phycoerythrin from a red alga

An effective procedure for the rapid extraction and purification of the biliprotein R-phycoerythrin from a red alga,Ceramium isogonum, was developed. The purified R-phycoerythrin ofC. isogonum consisted of three components with mol wt 180,000 (6β subunits), 70,000 (6α subunits), and 30,400 (γ subunit), respectively. The phycoerythrin is suitable for use as a natural food coloring and can also be used as a fluorescent label.     

Thermal stability and energy of deactivation of free and immobilized amyloglucosidase in the saccharification of liquefied cassava starch

Amyloglucosidase from Novo (Copenhagen, Denmark) was immobilized in controlled pore silica particles with the silane-glutaraldehyde covalent method. Thermal stability of the free and immobilized enzyme (IE) was determined with 30% (w/v) α-amylase liquefied cassava starch, pH 4.5, temperatures from 35 to 75°C. Free amyloglucosidase maintained its activity practically constant for 240 min and temperatures up to 50°C. The IE has shown higher stability retaining its activity for the same period up to 60°C. Half-life for free enzyme was 20.6, 6.44, 2.07, 0.69, and 0.24 h for 55, 60, 65, 70, and 75°C, respectively, whereas the IE at the same temperatures had half-lives of 116.4, 30.88, 8.52, 2.44, and 0.73 h. The energy of thermal deactivation was thus 50.6 and 57.6 kcal/mol, respectively for the free and IE, confirming stabilization by immobilization.     

Broad spectrum and mode of action of an antibiotic produced byScytonema sp. TISTR 8208 in a seaweed-type bioreactor

A photobioreactor was constructed using anchored polyurethane foam strips (1 x 1 x 40 cm) fixed onto a stainless-steel ring to prevent flotation, as a biomass support material (BSM). This type of reactor was named a seaweed-type bioreactor. A filamentous cyanobacterium, Scytonema sp. TISTR 8208, which produces a novel cyclic dodecapeptide antibiotic, was immobilized in seaweed-type photobioreactor and cultivated with air containing 5% CO₂ sparged at a gas flow rate of 250 mL/min under illumination at a light intensity of 200 μmol photon m^-2s^-1. The antibiotic produced in the seaweed-type photobioreactor was purified by HPLC and examined regarding its spectrum and mode of action. The antibiotic effectively inhibited the growth of Gram-positive bacteria, pathogenic yeasts, and filamentous fungi, but it had only a weak effect on Gram-negative bacteria. Scanning electron micrograph analysis showed that the most characteristic change was swelling of the cells after exposure to the antibiotic. The antibiotic seems to alter the conformation of the microbial cell membrane, thereby changing its permeability, leading to osmotic shock.     

Production of ethanol from starch by co-immobilizedZymomonas mobilis-glucoamylase in a fluidized-bed reactor

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilizedZymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. TheZ. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of κ-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35κC and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.     

Biosorption of nickel in complex aqueous waste streams by cyanobacteria

A study was undertaken to determined if a suitable biosorbent could be found for removal of nickel at low concentrations (< 20 parts per million [ppm]) from a chemically complex wastewater effluent generated by electroplating operations. Algae and cyanobacteria were chosen as candidate biosorbent materials because they are easy to grow and they have the ability to withstand processing into biosorbent materials. Several species were screened for nickel-biosorption capacity initially, and three species of cyanobacteria were selected for further study based on their performance in the scoping tests. When compared to live controls, autoclaving improved the binding capacities of all three species, but usually biosorption data from experiments with live cells were more consistent. None of the three species was able to bind nickel efficiently in actual effluent samples. Further experimentation indicated that sodium ions, which were present in high concentrations in the effluent, were interfering with the ability of the cells to bind nickel. Adsorption isotherm plots for biosorption of nickel by two species ofAnabaena in NiCl₂-deionized water solutions were prepared. Managed by Martin Marietta Energy Systems, Inc., for the US Department of Energy under contract No. DEAC05-84OR21400.     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Tributyl phosphate degradation by immobilized cells of aCitrobacter sp.

Tributyl phosphate (TBP), a plasticizer and solvent, is used in nuclear fuel reprocessing, generating TBP wastes laden with residual uranium. ACitrobacter sp. accumulated heavy metals via a phosphohydrolase(s) that precipitated metals with inorganic phosphate liberated from an organic phosphate “donor” molecule (TBP). Mutant analysis suggested that TBP hydrolysis was not attributable to a previously documented acid phosphatase (monoesterase). Purified monoesterase had little activity against phospho di- and triesters, had no requirement for Mg²⁺ or Mn²⁺, and was EDTA-resistant. Conversely, TBP cleavage by immobilized cells was enhanced by Mg²⁺, and ininhibited by Mn²⁺ and EDTA. A separate phosphotri/diesterase was implicated.     

β-Cyclodextrin production by simultaneous fermentation and cyclization

Production of β-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava starch, liquefied with α-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added yeast,S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38αC, pH 6.0, DE from 2 to 25, and 3.3 mL of CGTase/L. The yield of β-CD has decreased with an increase in DE, and maximum reaction yields were found for DE equal to 3.54, reaching 5.6, 14.7, and 11.5 mM β-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to 23.79), β-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times for SFC (from 11.5 to 3.73 mM), showing that this process is less sensitive to variations in the DE     

Glutamate production from CO2 by Marine CyanobacteriumSynechococcus sp.

A photobioreactor was constructed in the form of a Perspex column 900 mm tall with an internal diameter of 70 mm. The reactor volume was 1.8 L and the light source consisted of a metal-halide lamp to reproduce sunlight. Light was distributed through the culture using a new type of optical fiber that diffuses light out through its surface, perpendicular to the fiber axis. A cluster of 661 light-diffusing optical fibers (LDOFs) pass from the light source through the reactor column (60-cm culture depth) and are connected to a mirror at the top of the reactor. This biosolar reactor has been used for the production of glutamate from CO₂ by the marine cyanobacterium Synechococcus sp. NKBG040607. We present here details of the construction of the biosolar reactor and characterization of its properties. The effect of light intensity on glutamate production was measured. Carbon dioxide-to-glutamate conversion ratios were determined at different cell densities: the maximum conversion ratio (28%) was achieved at a cell density of 3x10⁸ cells/mL. A comparison of glutamate production using the LDOF biosolar reactor described here with production by batch culture using free or immobilized cells showed that use of an optical-fiber biosolar reactor increased glutamate-production efficiency 6.75-fold. We conclude that as a result of its high surface-to-volume ratio (692/m) increased photoproduction of useful compounds may be achieved. Such a system is generally applicable to all aspects of photobiotechnology.     

Selective polarity- and adsorption-guided extraction/purification ofAnnona sp. Polar acetogenins and biological assay against agricultural pests

Annonaceae acetogenins (AG) comprise a family of natural chemical modifications of long-chain fatty acids (C₃₅_₃₇) bearing one to several hydroxyls (less often oxo), middle-chain tetrahydrofuran rings, and a γ-lactonized, α/β-unsaturated carboxyl group. Acetogenins’ strong biological activity as larvicides, pesticides, and antitumorals is dependent on these structural variations. The hydroxylation degree is particularly important for these etfects. Seeds, albeit rich in fats (mostly triacylglycerols, [TAG]), are a nonpredatory source of these drugs as compared to other botanical parts such as roots and stems. Conventional lipid extractions lead to quantitative lipid recovery and then the unfavorable natural ratio of TAG:AG in the range >90:<0.1 These extracts thus require, for instance, partitions and extensive sílica gel column chromatographic steps, in order to enrich or purify the AG fraction(s). Great operational difficulties result from the similar polarity and mol. wt. range of TAG and AG when carrying out these purification steps. An alternative fast two-step procedure to obtain polar acetogenin (pAG)-enriched preparations was developed. The extraction procedure forAnnona spp. seeds pAG was carried out with acetonitrile (Eβ = 0.65; log P = - 0.33) as a polar organo-solvent, followed by the adsorption of the solvent-free extract on activated charcoal, then washed with hexane and/or chloroform (Eβ = 0.0 and 0.40: log P = 3.5 and 2.0) for most of the contaminating TAG removal, and then with acetone (Eβ = 0.56; log P = - 0.23) to the desorption of an enrichedpAG fraction. An alternative procedure for pAG extraction was supercritical fluid extraction (SFE) at moderate thermopressurization conditions (65-82βC; 120-130 atm) using CO2, with 10% acetonitrile as the polarity modifier. The pAG fractions’ bioactivity was evaluated with the brine-shrimp test (BST), and for feed deterrance, growth inhibition, and lethality against the high-impact agricultural pestsAnticarsia gemmatalis andPseudaletia sequax caterpillars feeding on soya or grass leaves sprayed with a 10% alcohol-stabilized emulsion of pAG.     

Submerged culture screening of two strains ofStreptomyces sp. with high keratinolytic activity

Keratinases can be used for the production of potentially important hydrolyzed proteins and chemicals. This study investigated the keratinolytic activity ofStreptomyces sp on keratinaceous materials like wool. High levels of proteolytic and keratinolytic activity were obtained after 96 h of culture when two Streptomyces sp strains were grown on basal medium containing mineral salts and 3% (w/v) of defatted wool as a source of energy, carbon, and nitrogen. The cell-free culture filtrates exhibited rapid proteolytic digestion of keratin powder. Currently, the authors are testing whether the enzymatic activity obtained is in fact keratinolytic, and not only an alkaline protease activity.     

Enhanced conversion of starch to cyclodextrins in ethanolic solutions by bacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase

Improved formation of cyclodextrins (CDs) from starch in ethanolic solutions byBacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase was studied. The β- and γ-CD yields increased and α-CD yield gradually decreased as the ethanol concentration was raised. The ethanol concentration required for maximal CD yield depended essentially on starch concentration. The ethanol's effect was pronounced at high starch concentrations. For example, with 30% (w/v) starch, the CD yield was 2.4-fold (146.5 g/L) in the presence of 15% (v/v) ethanol. The effect of dimethylsulfoxide on the formation of CDs was similar to that of ethanol. The disintegration of β- and γ-CDs were narrowly interdependent on the formation of a α-CD and malto-sugars. The amount of reducing sugars decreased from a dextrose equivalent value of roughly 7.5 to 4.5 in the presence of ethanol at starch concentrations 1-30% (w/v). The effect of ethanol on starchy materials from various sources was similar. It was concluded that ethanol retards the decomposition of β-CD by a general mechanism involving a decreased activity of water.     

A novel fermentation pathway in anEscherichia coli mutant producing succinic acid,acetic acid,and ethanol

Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E.coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

An integrated bioconversion process for production of l-lactic acid from starchy potato feedstocks

The potential market for lactic acid as the feedstock for biodegradable polymers, oxygenated chemicals, and specialty chemicals is significant. L-lactic acid is often the desired enantiomer for such applications. However, stereospecific lactobacilli do not metabolize starch efficiently. In this work, Argonne researchers have developed a process to convert starchy feedstocks into L-lactic acid. The processing steps include starch recovery, continuous liquefaction, and simultaneous saccharification and fermentation. Over 100 g/L of lactic acid was produced in less than 48 h. The optical purity of the product was greater than 95%. This process has potential economical advantages over the conventional process.     

Urease purification from the seeds ofCajanus Cajan and its application in a biosensor construction

Urease has been purified from the seeds of Cajanus Cajan. The purification process involves three solvent extraction steps followed by DEAE-cellulose column chromatography. The specific activity of the purified enzyme is found to be 1920 U/mg with the recovery of 8%. The application of the purified enzyme in a biosensor construction is discussed.     

Purification and characterization of a xylanase from the thermophilic ascomyceteThielavia terrestris 255b

Thielavia terrestris 255B, a thermophilic ascomycete, produced two major forms of xylanase with pIs of 4.6 (xylanase I) and 6.1 (xylanase II). The latter enzyme could be purified to > 99% homogeneity using anion-exchange chromatography and gel filtration. Xylanase II had a mol wt of 25.7 kDa (SDS-PAGE) and a pH and a temperature optimum of 3.6–4.0 and 60–65°C, respectively. The ratio of the enzyme’s activity against xylan and carboxymethylcellulose was 500–1000 to 1, indicating a possible application of this enzyme in biobleaching processes. The amino acid sequence of this protein is being determined, and initial data suggest that the enzyme belongs to a group of low-mol wt xylanases that have been isolated from both bacteria and fungi.     

Microbial oxidation of mixtures of methylmercaptan and hydrogen sulfide

Refinery spent-sulfidic caustic, containing only inorganic sulfides, has previously been shown to be amenable to biotreatment withThiobacillus denitrificans strain F with complete oxidation of sulfides to sulfate. However, many spent caustics contain mercaptans that cannot be metabolized by this strict autotroph. An aerobic enrichment culture was developed from mixedThiobacilli and activated sludge that was capable of simultaneous oxidation of inorganic sulfide and mercaptans using hydrogen sulfide (H₂S) and methylmercaptan (MeSH) gas feeds used to simulate the inorganic and organic sulfur of a spent-sulfidic caustic. The enrichment culture was also capable of biotreatment of an actual mercaptancontaining, spent-sulfidic caustic but at lower rates than predicted by operation on MeSH and H₂S fed to the culture in the gas phase, indicating that the caustic contained other inhibitory components.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.