Anaerobic detoxification fermentation by Rhodospirillum rubrum for rice straw as feed with moderate pretreatment

A novel and effective process was put forward for converting rice straw into feed by combining diluted acid hydrolysis and ammonization with Rhodospirillum rubrum fermentation. After pretreatment with dilute sulfuric or phosphoric acid (1%, w/w) at 100°C, materials were subjected to fermentation under several gases (N₂, CO₂, and air) and different light intensities in a 2-L fermentor. The key indexes of feed for fermented materials were estimated and several toxic substances were investigated during the fermentation. Following sulfuric acid treatment, the true protein of rice straw increased from 29 to 143?g?kg^?1 and the crude fiber decreased from 359 to 136?g?kg^?1 after fermentation at 0.3?L?min^?1?L^?1 of N₂ flow and a light intensity of 3400 lux; and following phosphoric acid treatment, the true protein increased by 286% and the crude fiber decreased by 52% after fermentation at 0.4?L?min^?1?L^?1 of N₂ flow and a light intensity of 3000 lux. Other key contents were also improved for use as feed, and some toxic substances (i.e., furfural, hydroxymethylfurfural, acetic acid, phenol, cresol) produced by the pretreatments could be removed at low levels during the fermentations.     

A shaking bioreactor equipped with twin ceramic membranes for acetic acid production using Acetobacter pasteurianus

A shaking bioreactor system with twin internal ceramic membranes was developed for effective perfusion culture and applied to the continuous production of acetic acid using Acetobacter pasteurianus. The system makes it possible to carry out the back-washing of the membrane without stopping the continuous operation because one membrane can be washed by medium feed flow while another membrane provides filtration of the broth by the simple switching of the medium and the broth flow direction. The medium flow through the membrane could successfully wash the surface of the membrane thereby effectively maintaining the filtration ability. By using the system, continuous operation of more than 800 h was achieved and the maximum acetic acid productivity reached 13.4 g l^–1 h^–1 using air enriched with 40% O₂.     

Butanol production from sweet sorghum bagasse with high solids content: Part I—comparison of liquid hot water pretreatment with dilute sulfuric acid

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H₂SO₄ (2 g L^?1) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250–300 g L^?1. This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L^?1 sugars, 7.60 g L^?1 acetic acid, 0.33 g L^?1 furfural, and 0.07 g L^?1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L^?1 additional sugars, 2.40 g L^?1 acetic acid, zero g L^?1 furfural, and zero g L^?1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone‐butanol‐ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L^?1 ABE of which butanol was the main product. Use of 2 g L^?1 H₂SO₄ as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6–93.8% (w/w) sugars from 250 to 300 g L^?1 SSB, respectively. LHW or dilute H₂SO₄ were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960–966, 2018     

Competing two enzymatic reactions realizing one‐step preparation of cell‐enclosing duplex microcapsules

The usefulness of cell‐enclosing microcapsules in biomedical and biopharmaceutical fields is widely recognized. In this study, we developed a method enabling the preparation of microcapsules with a liquid core in one step using two enzymatic reactions, both of which consume H₂O₂ competitively. The microcapsule membrane prepared in this study is composed of the hydrogel obtained from an alginate derivative possessing phenolic hydroxyl moieties (Alg‐Ph). The cell‐enclosing microcapsules with a hollow core were obtained by extruding an aqueous solution of Alg‐Ph containing horseradish peroxidase (HRP), catalase, and cells into a co‐flowing stream of liquid paraffin containing H₂O₂. Formation of the microcapsule membrane progressed from the surface of the droplets through HRP‐catalyzed cross‐linking of Ph moieties by consuming H₂O₂ supplied from the ambient liquid paraffin. A hollow core structure was induced by catalase‐catalyzed decomposition of H₂O₂ resulting in the center region being at an insufficient level of H₂O₂. The viability of HeLa cells was 93.1% immediately after encapsulation in the microcapsules with about 250 µm diameter obtained from an aqueous solution of 2.5% (w/v) Alg‐Ph, 100 units mL^?1 HRP, 9.1 × 10⁴ units mL^?1 catalase. The enclosed cells grew much faster than those in the microparticles with a solid core. In addition, the thickness of microcapsule membrane could be controlled by changing the concentrations of HRP and catalase in the range of 13–48 µm. The proposed method could be versatile for preparing the microcapsules from the other polymer derivatives of carboxymetylcellulose and gelatin. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1528–1534, 2013     

Biosorption of Malathion by immobilized cells of Bacillus sp. S14

Abstract

Biosorption is potentially an attractive technology for the treatment of wastewater by removing pesticide molecules from dilute solutions. This study investigated the feasibility of an isolated Bacillus sp. S₁₄ immobilized in calcium alginate that was used as a biosorbent for Malathion removal from aqueous solutions in batch mode. The highest value of Malathion uptake by isolated Bacillus sp. S14 (1.33g L^?1, dry basis) immobilized in 3% calcium alginate was 64.4% at 25°C and pH7.0 when the initial Malathion concentration was 50 mg L^?1. Equilibrium was attained at 8h. The sorption data conformed well to the Fruendlich isotherm model.     

Small-scale experiments aimed at optimization of large-scale production of the microalga <Emphasis Type="Italic">Rhodomonas salina</Emphasis>

The cryptophyte Rhodomonas is an important feed item for live feed organisms in aquaculture and although large-scale cultivation of Rhodomonas in photobioreactors (PBRs) is feasible, the production needs to be optimized through further studies of specific factors. Through small-scale experiments, several factors relevant for an on-going large-scale production of Rhodomonas were studied and the results presented here provide a useful insight on factors that can help future large-scale production. The content of polyunsaturated fatty acids (PUFAs) and the temporal sedimentation was compared in five strains of Rhodomonas. Strain K-1487 (R. salina) was chosen as the most suitable for cultivation in PBRs due to a good biochemical content of PUFAs and low cell sedimentation. The f/2 growth medium used for cultivation was modified by excluding CoCl₂ which did not affect either growth rate or cell content of the PUFAs, DHA, EPA, and ARA. Furthermore, the growth medium was modified by adding the nitrogen source as ammonium (NH₄⁺), nitrate (NO₃^?), urea, or combinations of these, with NH₄⁺ yielding a significantly higher growth rate of 1.30?±?0.07 day^?1. The seawater used for cultivation was exposed to three types of treatments which gave no significant difference in the growth rate: (1) filtration (0.2 μm)?+?autoclaving, (2) filtration (0.2 μm)?+?UV-radiation, and (3) filtration (0.2 μm). Finally, the results for growth rates of inocula at initial densities ranging from 2000 to 200,000 cells mL^?1 showed that growth rate decreased with increasing density but a final density of 10⁶ cells mL^?1 was obtained fastest with the highest initial density. With the present findings, several barriers for effective cultivation of Rhodomonas are solved and future large-scale production has become a great step closer.     

Vitamin analysis with use of a yeast electrode

A vitamin B₁ (thiamin)-sensitive electrode has been devised by combining an oxygen electrode with a yeast-containing membrane. The assembly was used for assaying thiamin at concentrations down to 10^?11 gl^?1. The analytical procedure developed should allow the measurement of 10–20 samples per hour. The performance of the yeast electrode was improved when alginate membranes reinforced with a nylon network were used. An apparatus for preparing such membranes is described together with a magnetic membrane holder facilitating handling of membranes in combination with electrodes.     

Reduction and separation of nitrate and nitrite by liquid membrane-encapsulated enzymes

Purified enzymes encapsulated in liquid surfactant membranes have been shown to retain their catalytic activity. In general, previous work on encapsulation has been confined to single enzymes. The system has now been extended to encapsulate a bacterial cell-free homogenate. Liquid membrane-encapsulated bacterial cell-free homogenate reduces effectively NO₃^? to NO₂^? and other nitrogen compounds of lower oxidation state. This technique of removing nitrates and nitrites may have application in waste-water treatment. Also, it has been shown that encapsulated cell-free homogenate does not leak and there is no absorption of the substrate onto the liquid surfactant membrane surfaces. The reduction in the reaction rates is discussed in terms of solubility of the substrate and the rate of permeation of the substrates through the liquid surfactant membrane.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Regulation of Potato Immune Responses by Laminarin

Laminarin blocks potato immune responses by inhibiting the reaction of oversensitivity, formation of phytoalexins, wound repair, and the activity of proteinase inhibitors. It was found that laminarin exhibits antielicitor activity. Addition of salicylic acid to laminarin enhances its immunosuppressing effect, which becomes systemic.     

Permeability to ions of bovine retinal disk membrane vesicles in the bleached state

The permeability of the bleached disk membrane of retinal rod outer segments to univalent and divalent ions is studied by light scattering. The membranes are isolated from frozen dark-adapted bovine retinae, swollen into spherical vesicles in a hypotonic medium and bleached in dilute suspension and their size is determined by elastic and quasi-elastic light scatterings. Various electrolytes are then added to the suspending medium in order to examine their osmotic activity relative to the vesicles deformation characteristics. By following the deformation behavior of the membrane vesicles by elastic light scattering in terms of the oblate ellipsoidal shell model, the osmotic activity of a given electrolyte is qualitatively deduced and thereby the permeability of the membrane to the electrolyte is ranked in reference to a chosen standard, i.e., sucrose. By this method, we show that the permeabilities to Na⁺, K⁺, Mg²⁺ and Ca²⁺ are all alike, and those to halides (F^?, Cl^?, Br^?, I^?), nitrate and phosphates (HPO₄^2?/H₂PO₄^?) are similar. Acetate, however, is about 3-times more permeative, while sulfate is less permeative than the other anions by about the same factor. The viability of our method is checked with use of an ionophore, lasolocid (X-537A), by establishing partial recovery from the osmotic deformation through the suppression of the cation osmotic effect. Ion-induced aggregation and pH-dependent size and shape changes are both found to be insignificant.     

Optical resolution and mechanism using enantioselective cellulose,sodium alginate and hydroxypropyl‐β‐cyclodextrin membranes

Chiral solid membranes of cellulose, sodium alginate, and hydroxypropyl‐β‐cyclodextrin were prepared for chiral dialysis separations. After optimizing the membrane material concentrations, the membrane preparation conditions and the feed concentrations, enantiomeric excesses of 89.1%, 42.6%, and 59.1% were obtained for mandelic acid on the cellulose membrane, p ‐hydroxy phenylglycine on the sodium alginate membrane, and p ‐hydroxy phenylglycine on the hydroxypropyl‐β‐cyclodextrin membrane, respectively. To study the optical resolution mechanism, chiral discrimination by membrane adsorption, solid phase extraction, membrane chromatography, high‐pressure liquid chromatography ultrafiltration were performed. All of the experimental results showed that the first adsorbed enantiomer was not the enantiomer that first permeated the membrane. The crystal structures of mandelic acid and p ‐hydroxy phenylglycine are the racematic compounds. We suggest that the chiral separation mechanism of the solid membrane is “adsorption – association – diffusion,” which is able to explain the optical resolution of the enantioselective membrane. This is also the first report in which solid membranes of sodium alginate and hydroxypropyl‐β‐cyclodextrin were used in the chiral separation of p ‐hydroxy phenylglycine.     

Exogenous Gangliosides GD1b and GD1b-Lactone, Stably Associated to Rat Brain P2 Subcellular Fraction, Modulate Differently the Process of Protein Phosphorylation

GD1b and GD1b-lactone (GD1b-L) gangliosides bind to the same extent to a P₂ crude membrane preparation from rat brain. After 30 min of incubation with 10^?4, 10⁵, and 10^?6 Absolutions of ganglioside, 1,800, 450, and 100 pmol of ganglioside/mg of protein, respectively, were found to be stably associated to the P₂ fraction. This association modifies the phosphorylation process of the P₂ membrane proteins in a dose-dependent manner, the maximal effect being reached at a ganglioside association of 1.85 nmol/mg of protein and in large part at 450 pmol/mg of protein. The effects of GD1b and GD1b-L on the phosphorylation of five proteins, showing apparent molecular masses of 17, 20, 36, 41, and 44 kDa, were different after 0.5 min of phosphorylation reaction as well as after 15 min. After 0.5 min of reaction, in the presence of stably associated GD1b, the phosphorylation of the 36-, 41-, and 44-kDa proteins was increased with reference to the control, whereas the phosphorylation of the 17- and 20-kDa proteins was decreased. GD1b-L exerted qualitatively similar effects only on the 44-, 41-, and 36-kDa proteins and to a strongly reduced degree. After 15 min of reaction, only the phosphorylation of the 36-kDa protein was stimulated by GD1b; GD1b-L exerted a similar effect, but to a low degree.     

Isolation and properties of the outer membrane of plant mitochondria.

The outer membrane of turnip (Brassica rapa L.) mitochondria was isolated by incubating the mitochondria with a dilute digitonin solution and differential centrifuging. The outer membrane fraction was not contaminated by inner membrane enzymes and lacked an NADPH-cytochrome c reductase. However it possessed very active NADH-cytochrome c, dichloroindophenol and ferricyanide reductases which were insensitive to antimycin A, Amytal and low (less than 10 μm) concentrations of Dicumarol. p-Chloromercuribenzoate (ClHgBzO^?) and high concentrations (greater than 10 μm) of Dicumarol inhibited the reductases, ClHgBzO^? almost completely. Preincubation of the outer membrane with NADH protected it from ClHgBzO^? inhibition. An acid phosphatase and an NADPH-ferricyanide reductase were also detected, but the latter was only loosely bound to the membrane. The NADH dehydrogenase of the outer membrane was insensitive to ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetate (1 mm) and was not stimulated by CaCl₂ (0.5 mm), thus differing from the external NADH oxidase of the inner membrane (Coleman, J. O. D., and Palmer, J. M. (1971) FEBS Lett., 17, 203–208). Respiratory-linked oxidation of exogenous NADH by intact mitochondria showed a similar pattern of inhibition by ClHgBzO^? as did the outer membrane, but was inhibited strongly by low concentrations of Dicumarol (5 μm inhibited by 70%).     

Regulation of potato immune responses by laminarin

Laminarin blocks potato immune responses by inhibiting the reaction of oversensitivity, formation of phytoalexins, wound repair, and the activity of proteinase inhibitors. It was found that laminarin exhibits antielicitor activity. Addition of salicylic acid to laminarin enhances its immunosuppressing effect, which becomes systemic.     

Purification and molecular characterization of exo-β-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2

The extracellular β-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific β-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40°C, respectively. The enzyme was significantly stimulated by Li⁺, Ni²⁺, and Ba²⁺. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the β-1,3-glucanases from other yeasts.     

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Brown algae are one of the largest groups of oceanic primary producers for CO₂ removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2^T. Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing d-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD⁺, and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.     

Effects of Free Radicals on the Fluidity of Myocardial Membranes

Free radicals, including superoxide anions (O₂^?_?), hydroxyl radical (HO'), and hypohalite radical (OCl'), as well as oxidants such as hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), have been indicated in the pathogenesis of myocardial ischemic and reperfusion injury. In this report, we compared the integrity of the myocardial membrane when exposed to these free radicals/oxidants. Isolated rat heart membrane preparations were exposed to chemically generated free radicals with or without their respective scavengers. Membrane fluidity was monitored by fluorescence polarization using the diphenylhexatriene probe, as well as by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethyl piperidine-n-oxyl as the spin labeling agent. HO', H₂O₂, and OCl' + HOCl increased the fluorescence polarization (FP) and microvis-cosity significantly by 1.7-fold, 1.8-fold, and 1.7-fold, respectively, as compared to an only 1.2– fold increase in FP by O₂^?_? O₂^?_? did not alter the fatty acid profiles of the membrane phospholipids. However, HO' and H₂O₂ reduced the arachidonic acid contents in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). These radicals also stimulated the lipid peroxidation by several-fold, while that by O₂^?_? was only insignificant. These results suggest that HO' and H₂O₂ decreased the membrane fluidity and induced lipid peroxidation by releasing the arachidonic acid from PC, PE. and PI.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

Cattle feed or bioenergy? Consequential life cycle assessment of biogas feedstock options on dairy farms

On‐farm anaerobic digestion (AD) of wastes and crops can potentially avoid greenhouse gas (GHG) emissions, but incurs extensive environmental effects via carbon and nitrogen cycles and substitution of multiple processes within and outside farm system boundaries. Farm models were combined with consequential life cycle assessment (CLCA) to assess plausible biogas and miscanthus heating pellet scenarios on dairy farms. On the large dairy farm, the introduction of slurry‐only AD led to reductions in global warming potential (GWP) and resource depletion burdens of 14% and 67%, respectively, but eutrophication and acidification burden increases of 9% and 10%, respectively, assuming open tank digestate storage. Marginal GWP burdens per Mg dry matter (DM) feedstock codigested with slurry ranged from –637 kg CO₂e for food waste to +509 kg CO₂e for maize. Codigestion of grass and maize led to increased imports of concentrate feed to the farm, negating the GWP benefits of grid electricity substitution. Attributing grass‐to‐arable land use change (LUC) to marginal wheat feed production led to net GWP burdens exceeding 900 kg CO₂e Mg^?1 maize DM codigested. Converting the medium‐sized dairy farm to a beef‐plus‐AD farm led to a minor reduction in GWP when grass‐to‐arable LUC was excluded, but a 38% GWP increase when such LUC was attributed to marginal maize and wheat feed required for intensive compensatory milk production. If marginal animal feed is derived from soybeans cultivated on recently converted cropland in South America, the net GWP burden increases to 4099 kg CO₂e Mg^?1 maize DM codigested – equivalent to 55 Mg CO₂e yr^?1 per hectare used for AD‐maize cultivation. We conclude that AD of slurry and food waste on dairy farms is an effective GHG mitigation option, but that the quantity of codigested crops should be strictly limited to avoid potentially large international carbon leakage via animal feed displacement.