Excessive excretion of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1.

At 25 degrees C, the optimal temperature for growth of Rhizobium trifolii TA-1, extracellular and capsular polysaccharide (EPS and CPS) were the main carbohydrate products synthesized in mannitol-rich medium (10 g of mannitol and 1 g of glutamic acid per liter). In the same medium at 33 degrees C, EPS and CPS production was inhibited, and up to 3.9 g of cyclic beta-(1,2)-glucan was produced during an incubation period of 20 days with a total biomass of 0.55 g of protein. In a medium containing 50 g of mannitol and 10 g of glutamic acid per liter, high cell densities (3.95 g of protein) were obtained at 25 degrees C. This biomass excreted 10.9 g of cyclic beta-(1,2)-glucan within 10 days. Concomitantly, 4.8 g of EPS were synthesized, while CPS production was strongly suppressed. The excreted cyclic beta-(1,2)-glucans were neutral and had degrees of polymerization ranging from 17 to 25, with a degree of polymerization of 19 as the major glucan cycle.     

Excessive excretion of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1

At 25 degrees C, the optimal temperature for growth of Rhizobium trifolii TA-1, extracellular and capsular polysaccharide (EPS and CPS) were the main carbohydrate products synthesized in mannitol-rich medium (10 g of mannitol and 1 g of glutamic acid per liter). In the same medium at 33 degrees C, EPS and CPS production was inhibited, and up to 3.9 g of cyclic beta-(1,2)-glucan was produced during an incubation period of 20 days with a total biomass of 0.55 g of protein. In a medium containing 50 g of mannitol and 10 g of glutamic acid per liter, high cell densities (3.95 g of protein) were obtained at 25 degrees C. This biomass excreted 10.9 g of cyclic beta-(1,2)-glucan within 10 days. Concomitantly, 4.8 g of EPS were synthesized, while CPS production was strongly suppressed. The excreted cyclic beta-(1,2)-glucans were neutral and had degrees of polymerization ranging from 17 to 25, with a degree of polymerization of 19 as the major glucan cycle.     

Fungal growth on C1 compounds: quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture.

A study was made of some salient parameters that influence growth of the methanol-utilizing fungus Trichoderma lignorum growing in batch culture on a minimal medium containing methanol as the sole source of carbon. Maximum cell yield was recorded at the expense of 1.58 g of methanol per liter. Inhibition was observed with methanol concentrations in excess of 4.7 g/liter. The optimum temperature for fungal growth was 23 degrees C. Growth of the fungus was directly proportional to an inorganic nitrogen concentration up to 0.2 g of NH4NO3 per liter. No inhibition of growth occurred at any concentration of NH4NO3 up to 11 g/liter. The pH of the growth medium decreased from 7.0 to 3.5 during growth of the fungus on methanol, which may have been due, in part, to the accumulation of trace amounts of organic acids in the growth medium. An analysis of the commercial potential of the fungus, as a source of edible protein, indicated that the strain of methanol-utilizing T. lignorum used was uneconomical in terms of the yield and the specific growth rate.     

Fungal growth on C1 compounds: quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture.

A study was made of some salient parameters that influence growth of the methanol-utilizing fungus Trichoderma lignorum growing in batch culture on a minimal medium containing methanol as the sole source of carbon. Maximum cell yield was recorded at the expense of 1.58 g of methanol per liter. Inhibition was observed with methanol concentrations in excess of 4.7 g/liter. The optimum temperature for fungal growth was 23 degrees C. Growth of the fungus was directly proportional to an inorganic nitrogen concentration up to 0.2 g of NH4NO3 per liter. No inhibition of growth occurred at any concentration of NH4NO3 up to 11 g/liter. The pH of the growth medium decreased from 7.0 to 3.5 during growth of the fungus on methanol, which may have been due, in part, to the accumulation of trace amounts of organic acids in the growth medium. An analysis of the commercial potential of the fungus, as a source of edible protein, indicated that the strain of methanol-utilizing T. lignorum used was uneconomical in terms of the yield and the specific growth rate.     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Production, characterization and application of keratinase from Streptomyces gulbargensis

A Streptomyces gulbargensis newly isolated, thermotolerant feather-degrading bacterial strain was investigated for its ability to produce keratinase enzyme. Maximum keratinolytic activity was observed at 45 degrees C and pH 9.0 at 120 h of incubation. Activity was completely stable (100%) between 30 and 45 degrees C and pH 7.0-9.0, respectively. Addition of starch to the growth medium affects the activity by means of increase in keratinase secretion. After seven days of cultivation, 10-fold increase (14.3 U ml(-1)) in keratinase activity was observed in the presence of 3g starch (per liter) of the medium. The enzyme was monomeric and had a molecular mass of 46 kDa. The enzyme activity was significantly inhibited by CaCl(2) and partly inhibited by EDTA, whereas, Na(2)SO(3) enhance the enzyme activity by 2.9 times more. In addition, native chicken feather was completely degraded at 96 h of incubation. The results obtained showed that newly isolated strain S. gulbargensis could be a useful in biotechnology in terms of valorization of keratin-containing wastes or in the leather industry.     

High-temperature production of protein-enriched feed from cassava by fungi.

A simple, nonaseptic, low-cast process for the conversion of cassava, a starchy tropical root crop, into microbial protein for use as animal feed was sought. Screening tests culminated in the isolation of a thermotolerant, amylase-producing mold, designated I-21, which was identified as Aspergillus fumigatus. The optimum pH for protein synthesis was 3-5, but the optimum temperature was less than the desired temperature (larger than or equal to 45 C) required for a nonaseptic fermentation. A. fumigatus I-21 and its asporogenous mutant I-21A grew equally well in a medium prepared from whole cassava roots with a mean protein doubling time at 45 C and pH 3.5 of 3.5 h. In batch culture, approximately 4% carbohydrate, supplied as whole cassava, could be feremented in 20 h, giving a final yield of 24 g of dry product, containing 36.9% crude protein, per liter. The conversion of carbohydrate used to crude protein was 22.1%. When determined as amino acids, the protein content of the product, which contained cassava bark and other unfermented residues, was 27.1%. With urea as the nitrogen source, no pH control was necessary. Preliminary data indicated that medium prepared from whole cassava roots was inhibitory to the mold unless the cassava pulp was heated to 70 C immediately after being ground. Heating to 70 C was required to gelatinize the starch and permit its complete utilization.     

Key physiology of anaerobic ammonium oxidation.

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1. 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.     

Decolorization and detoxification of extraction-stage effluent from chlorine bleaching of kraft pulp by Rhizopus oryzae

Rhizopus oryzae, a zygomycete, was found to decolorize, dechlorinate, and detoxify bleach plant effluent at lower cosubstrate concentrations than the basidiomycetes previously investigated. With glucose at 1 g/liter, this fungus removed 92 to 95% of the color, 50% of the chemical oxygen demand, 72% of the adsorbable organic halide, and 37% of the extractable organic halide in 24 h at temperatures of 25 to 45 degrees C and a pH of 3 to 5. Even without added cosubstrate the fungus removed up to 78% of the color. Monomeric chlorinated aromatic compounds were removed almost completely, and toxicity to zebra fish was eliminated. The fungal mycelium could be immobilized in polyurethane foam and used repeatedly to treat batches of effluent. The residue after treatment was not further improved by exposure to fresh R. oryzae mycelium.     

Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on alpha-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His(6)) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-alpha-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley beta-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and beta-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing beta-glucan as the sole carbon source and could directly ferment 45 g of beta-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli.

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus

Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus. Experiments were performed to evaluate the potential role of A. oligospora in destroying third-stage larvae of Cooperia spp. on agar plates and in cattle faeces. The fungus had a growth rate optimum at 23 degrees C and pH 6. Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C. Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp. was captured within three days of the experiment. The trapping efficiency in faeces was shown to depend upon the inoculation level. At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed. The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.     

豆薯种籽中一种抗真菌蛋白PaAFP的分离纯化和部分特性研究

各类植物由于缺少自身免疫系统的支持,因而必须依赖于其它机制来抵御外来微生物的入侵．其中的一种重要机制就是通过合成体内各类能抑制微生物生长的蛋白质来完成的［１］．已报道从植物中分离出多种不同的抗真菌蛋白．广为研究的是几丁质酶和β－１,３－葡聚糖酶,认为它们在植物对真菌病的抗性中起重要作用［２,３］;核糖体失活蛋白（ＲＩＰｓ）和一类富含半胱氨酸的碱性多肽Ｔｈｉｏｎｉｎｓ也显示有非专一的抗真菌活性［４,５］．但仍有一些蛋白质,体外表现强烈的抗真菌活性,却不属于以上范围［６,７］．本文报道了豆薯种籽中一…     

Production of 2-Ketogluconic Acid by Serratia marcescens

Production of 2-ketogluconic acid from glucose by fermentation with Serratia marcescens NRRL B-486 was studied in 20-liter stainless-steel fermentors. Conditions for 2-ketogluconic acid production included the following: glucose-salt medium, aeration rate of 0.75 volumes per volume per minute, agitation rate of 400 rev/min, temperature of 30 C, CaCO₃ to neutralize the acid formed, and a 5% (v/v) inoculum. Foaming was controlled with an antifoam agent added at intervals during the fermentation. When 120 g per liter of glucose were supplied, 95 to 100% yields of 2-ketogluconic acid were obtained in 16 hr. Larger amounts of glucose could be used in the fermentation provided that the carbohydrate was fed continuously. Continuous feeding of glucose to a total amount of 180 g per liter gave 95 to 100% yields of 2-ketogluconic acid in 24 hr; feeding glucose to a total amount of 240 g per liter gave 85 to 90% yields in 32 to 40 hr.     

Effect of SO2 and bisulfite on heterotrophic activity in an acid soil.

Glucose oxidation was inhibited in a forest soil (pH 4.01) previously exposed by 1.0 microliter of SO2 per liter, the extent of inhibition and the decline in pH being directly related to the length of exposure. The phase of rapid CO2 evolution in protein hydrolysate-amended soil previously treated with 5.0 microliter of SO2 per liter for 24 h or 1.0 microliter/liter for 48 h was delayed, but the degradation of the amino acid mixture then proceeded rapidly. Bacterial numbers in soil incubated for 48 h with 1.0 microliter of SO2 per liter were reduced, but the bacteria grew rapidly if glucose or an amino acid mixture was added after the exposure period. Low levels of bisulfite inhibited amino acid decomposition in soil at pH 3.89, but the effect was less pronounced in soil at pH 4.01. Comparable levels of sulfate were not toxic to carbon mineralization. Approximately 1.0 microgram of bisulfite S and about 20 microgram of sulfate S per g of soil appeared when the soil was treated with 1.0 microliter of SO2 per liter for 48 h. Bisulfite added to the soil disappeared readily. The possible ecological significance of the findings is discussed.     

Effect of SO2 and bisulfite on heterotrophic activity in an acid soil.

Glucose oxidation was inhibited in a forest soil (pH 4.01) previously exposed by 1.0 microliter of SO2 per liter, the extent of inhibition and the decline in pH being directly related to the length of exposure. The phase of rapid CO2 evolution in protein hydrolysate-amended soil previously treated with 5.0 microliter of SO2 per liter for 24 h or 1.0 microliter/liter for 48 h was delayed, but the degradation of the amino acid mixture then proceeded rapidly. Bacterial numbers in soil incubated for 48 h with 1.0 microliter of SO2 per liter were reduced, but the bacteria grew rapidly if glucose or an amino acid mixture was added after the exposure period. Low levels of bisulfite inhibited amino acid decomposition in soil at pH 3.89, but the effect was less pronounced in soil at pH 4.01. Comparable levels of sulfate were not toxic to carbon mineralization. Approximately 1.0 microgram of bisulfite S and about 20 microgram of sulfate S per g of soil appeared when the soil was treated with 1.0 microliter of SO2 per liter for 48 h. Bisulfite added to the soil disappeared readily. The possible ecological significance of the findings is discussed.     

Enzymatic hydrolysis of polysaccharide-rich particulate organic waste

Hydrolysis of organic particulates in rapid fermentative processes can be inhibited. The volatile fatty acids (VFA) released during fermentation reduce pH. Whether VFA or the drop in pH inhibits hydrolysis is unclear. The effects of pH and acetate on the enzymatic hydrolysis of a potato sample that contains both carbohydrate and protein were studied at fixed pH (5-9) in the presence/absence of 20 g/L of acetate. Experimental results showed that the effects of pH and acetate on the hydrolysis of carbohydrate differed from those on the hydrolysis of protein. Numerous kinetic models fitted the hydrolysis data obtained during the first 40 h of hydrolysis when inhibitory effects were insignificant. The Chen-Hashimoto model was used herein to fit the hydrolysis data obtained during 144 h of reaction. Also, the non-competitive inhibition model of three inhibitors (H(+), OH(-), total/undissociated/dissociated acetate) successfully described the inhibition of the hydrolysis of both carbohydrate and protein.     

The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modified malachite green method for determination of inorganic phosphate

The Malachite Green method for determination of inorganic phosphate (Pi) (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361-366) was modified to measure Pi in the range of 0.2-15 nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6 M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2 g of sodium molybdate, 0.3 g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in 1 liter of 0.7 M HCl, and the absorbance at 650 nm is then measured after a 35-40 min incubation at 25 degrees C. Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately using relatively low concentrations of native myosin and its subfragment-1. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8-0.9 mol per site in 0.1 M KCl, 10 mM MgCl2, pH 8.0 at 25 degrees C for fresh enzyme preparations.     

Bacterial conversion of glycerol to beta-hydroxypropionaldehyde

beta-hydroxypropionaldehyde (3-HPA) can be oxidized to acrylic acid, an industrially important chemical used in the manufacture of synthetic plastics and other polymers. Of 19 genera and 55 strains tested, 3 Klebsiella and 2 Enterobacter strains produced 3-HPA. The most efficient strain was Klebsiella pneumoniae NRRL B-4011. Under optimum conditions (28 degrees C; 40 g of semicarbazide hydrochloride per liter, 70 g of glycerol per liter; and pH 6.0), 3.1 g of B-4011 cells per liter accumulated 22 g of 3-HPA per liter at a specific rate of 0.83 g/g per h; however, 14.5 g of cells per liter accumulated 46 g of 3-HPA per liter at a specific rate of 0.41 g/g per h.