<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Spore Production Under Solid-State Fermentation of Lignocellulosic Residues

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47?×?10¹⁰ spores g^?1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82?×?10¹⁰ spores g^?1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH₂PO₄, 1 g MgSO₄·7H₂O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105?×?10¹⁰ spores g^?1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10?×?10¹¹ spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.     

鸟巢蕨孢子繁殖技术研究

研究了鸟巢蕨孢子无菌播种和常规播种两种繁殖方法。结果表明,鸟巢蕨无菌播种中孢子萌发率最高可达66.7%,原叶体在固体培养基上培养不能诱导出孢子体,而需经过振荡培养后才能诱导出孢子体;常规播种法更容易诱导出孢子体,每盆播0.02 g孢子时,每克孢子可产生孢子体4 000株以上,比无菌播种操作简便,成本低。因此,孢子常规播种法更适合于鸟巢蕨规模化生产。     

Spore productivity in Cladosporium

The high incidence ofCladosporia in the airspora indicates a prolific production of spores. Six species ofCladosporium were sampled over a period of 9 weeks, using dry and wet (mist-laden) air, and over a period of 4 weeks using humid air. Many more spores were released in wet air than in dry air: numbers released in humid air were generally intermediate between those of wet and dry samples. None of the cultures was exhausted of spores at the end of the sampling periods although samples generally decreased in size from the fifth or sixth week onwards. Removal of spores would seem to be conducive to further sporulation provided the substrate is not exhausted. Maximum productivities recorded for the six species (all in mist-laden air) ranged from 730 to 26 100 spores per mg dry weight of mycelium. Differences in the levels of spore production in culture by the six species do not correlate with their individual frequencies in the airspora, indicating that the latter are more dependent on the distribution and substrate relationships of each species.     

Production of ochratoxin A by Aspergillus ochraceus NRRL-3174 before and after exposures to 60Co irradiation.

Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A.     

根霉12号固体发酵产生纤溶酶工艺条件的研究*

研究了根霉12号固体发酵产生纤溶酶的工艺条件。采用单因素试验、均匀设计方法对固体发酵培养基的碳源、氮源、碳氮比、初始pH、加水量、无机盐加量进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果表明,实验范围内根霉12固体发酵产纤溶酶的适宜培养基组成为:麸皮∶豆粕=1∶2,初始pH5.0,加水量0.75ml/g物料, MnSO4H2O和 (NH4)2SO4加量分别为0.25%和 1.42%(对物料)。适宜培养条件为接种量107个孢子/g物料,培养时间72h。优化条件下的纤溶酶产量平均达791.81u/g物料。     

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O₂, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.     

金矿床区蜡质芽孢杆菌孢子数与金矿化关系

土壤中蜡质芽孢杆菌（Bacilluscereus）孢子数目是寻找隐伏金矿床的一种较理想的探矿法。利用此法对我国2个金矿床区的土壤样品进行了该菌的分离鉴定。从55份样品中分离到阳性菌对9株,从739林中挑取11株进行了生理生化特性鉴定,结果证明,该分离株均为典型的蜡质芽孢杆菌。分析矿区每克土壤中蜡质芽孢杆菌数量最高可达19000余个,与背景矿区比较要高达几百倍至几千倍。同时对55份土壤也进行了金含量的分析;结果发现,该菌孢子异常与金异常有一空间上的分离。依据这些研究,作者认为蜡质芽孢杆菌孢子数是指示金矿床区金含量多少的一项重要指标。     

Metarhizium anisopliae conidia production in packed-bed bioreactor using rice as substrate in successive cultivations

This paper presents a study on scale-up and cost reduction of the production of spores of Metarhizium anisopliae IBCB 425, entomopathogenic fungus used in sugarcane crops. Rice was mixed with sugarcane bagasse (9:1 w/w) for substrate composition, assuring adequate physical structure for cultivation in packed-beds. Spores yield from only rice in bench-scale packed-bed bioreactor was 56 % of the one obtained from the mixture 9:1 w/w. In comparison to plastic packages used in bioindustries, equivalent spores yields per gram of substrate have been achieved in bench and pilot-scale bioreactors built by cylindrical jacketed modules, that provide better control of operational and environmental variables, attested by little variability among replicates. Although non-negligible temperature rise (5 °C above the ideal) occurred within the pilot-scale bioreactor, spores production was not harmed in comparison to bench-scale. By reusing rice up to three successive cultivations, a 2.5-fold increase of spores yields was achieved in comparison to single use. Temperatures and CO₂ profiles corroborates the fungus adapted differently to substrate at each usage. Such results are valuable for industrial producers of commercial formulations of the fungus spores, allowing process modernization by using packed-bed bioreactors and production costs and rice demand reduction by recycling the substrate.     

Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235

The effect of solid substrates, inoculum and incubation time were studied using response surface methodology (RSM) for the production of polygalacturonase enzyme and spores in solid-state fermentation using Aspergillus sojae ATCC 20235. Two-stage optimization procedure was applied using D-optimal and face-centered central composite design (CCD). Crushed maize was chosen as the solid substrate, for maximum polygalacturonase enzyme activity based on D-optimal design. Inoculum and incubation time were determined to have significant effect on enzyme activity and total spore (p<0.01) based on the results of CCD. A second order polynomial regression model was fitted and was found adequate for individual responses. All two models provided an adequate R(2) of 0.9963 (polygalacturonase) and 0.9806 (spores) (p<0.001). The individual optimum values of inoculum and incubation time for maximum production of the two responses were 2 x 10(7) total spores and 5-6 days. The predicted enzyme activity (30.55 U/g solid) and spore count (2.23 x 10(7)spore/ml) were very close to the actual values obtained experimentally (29.093 U/g solid and 2.31 x 10(7)spore/ml, respectively). The overall optimum region considering the two responses together, overlayed with the individual optima. Solid-state fermentation provided 48% more polygalacturonase activity compared to submerged fermentation under individually optimized conditions.     

Spore production and mycorrhizal development in various tropical crop hosts infected with Glomus clarum

Plant growth, mycorrhizal development and vesicular arbuscular spore production were examined in five tropical crop host species inoculated with Glomus clarum and grown in a glasshouse. In one of the two experiments, sequential harvests of maize, sorghum and chickpea were made in order to study spore production in relation to plant growth and mycorrhizal development. Spore numbers in each of these hosts increased at a fairly constant rate until maximum plant dry weight, when spore production ceased. Sorghum and maize produced considerably more spores than chickpea, with spore numbers being closely correlated with mycorrhizal root length. In the second experiment, Glomus clarum was cultured on each of maize, millet, sorghum, groundnut and chickpea for three consecutive generations before cross-inoculation of the spores from each host onto all five hosts. Sporulation with respect to host size was generally greatest when the inoculum used to infect a host had been produced on that host. The growth-promoting effects of the fungus were not influenced by the source of the inoculum. More spores were produced on the cereals than the legumes. Differences in spore numbers amongst hosts and plant generations were apparently influenced mainly by infected root length and by the growth period.     

Production of Soy Sauce Koji Mold Spore Inoculum in Plastic Bags

An innovation is described for producing soy sauce koji mold spore inoculum by using inexpensive autoclavable plastic bags and reuseable plastic enclosures to make culture vessels. After growth, the spore mass could be dried and packaged in the same bag after removing the enclosure. Broken rice was used as the substrate for mold cultivation. Viable spore counts of 10⁹ spores per g were obtained under optimal conditions. After drying at 50°C for 6 h, the moisture content of the spore mass decreased from 35.22 to 6.32% with no significant effect on spore viability. The dry spores could be stored in the refrigerator or at room temperature for at least 3 months.     

Optimized preparation and determination of pea seedling diamine oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio.

Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state.     

Pearl millet, a source of alpha amylase production by Bacillus licheniformis

The present study is concerned with the selection of economically available agricultural starchy substrate for the production of alpha amylase by Bacillus licheniformis. Different agricultural starchy substrates such as soluble starch, hordium, pearl millet, rice, corn, gram and wheat starch were tested for the production of alpha amylase by parental and its mutant derivatives. The production of alpha amylase was 10-folds better by the mutant strain B. licheniformis GCUCM-30 than the parental strain when pearl millet starch at 1.5% level and nutrient broth concentrations at the level of 0.25% was supplemented to the fermentation medium.     

Optimized Preparation and Determination of Pea Seedling Diamine Oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Bioindicator production with <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>’ thermal-resistant spores cultivated by solid-state fermentation

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.     

Improved Medium for Sporulation of Clostridium perfringens

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.     

Surface Charge Properties of and Cu(II) Adsorption by Spores of the Marine Bacillus sp. Strain SG-1

Spores of marine Bacillus sp. strain SG-1 are capable of oxidizing Mn(II) and Co(II), which results in the precipitation of Mn(III, IV) and Co(III) oxides and hydroxides on the spore surface. The spores also bind other heavy metals; however, little is known about the mechanism and capacity of this metal binding. In this study the characteristics of the spore surface and Cu(II) adsorption to this surface were investigated. The specific surface area of wet SG-1 spores was 74.7 m² per g of dry weight as measured by the methylene blue adsorption method. This surface area is 11-fold greater than the surface area of dried spores, as determined with an N₂ adsorption surface area analyzer or as calculated from the spore dimensions, suggesting that the spore surface is porous. The surface exchange capacity as measured by the proton exchange method was found to be 30.6 μmol m⁻², which is equal to a surface site density of 18.3 sites nm⁻². The SG-1 spore surface charge characteristics were obtained from acid-base titration data. The surface charge density varied with pH, and the zero point of charge was pH 4.5. The titration curves suggest that the spore surface is dominated by negatively charged sites that are largely carboxylate groups but also phosphate groups. Copper adsorption by SG-1 spores was rapid and complete within minutes. The spores exhibited a high affinity for Cu(II). The amounts of copper adsorbed increased from negligible at pH 3 to maximum levels at pH >6. Their great surface area, site density, and affinity give SG-1 spores a high capability for binding metals on their surfaces, as demonstrated by our experiments with Cu(II).     

Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% tryptone, at 35 degrees C, and at inoculum concentration 2x107-2x108 spores 5 g-1 substrate; for A. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% yeast extract, at 35 degrees C, and at an inoculum concentration of 2x107-2x108 spores 5 g-1 substrate. Under these conditions, A. terreus produced 68.9 IU ml-1 of xylanase, and A. niger, 74.5 IU ml-1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.     

Verticillium lecanii spore production in solid-state and liquid-state fermentations

Verticillium lecanii has been recognized as an entomopathogen with high potential in biological control of pests. Two types of cultivation methods, the solid-state fermentation (SSF) and the liquid-state fermentation (LSF), were examined for V. lecanii. In SSF, the substrate types including rice, rice bran, rice husk, and the mixtures of these components were tested. The results showed that both cooked rice with appropriate water addition and rice bran gave significantly higher spore production of 1.5 2 10⁹ spores/g substrate and 1.4 2 10⁹ spores/g substrate, respectively. In LSF, SMAY liquid medium was used as a base, and the effects of environmental conditions on the spore production of V. lecanii were investigated. From the time course study, on the 9th day the spore yield reached 1.2 2 10⁹ spores/ml of broth at 24v°C, 150 rpm for this strain. A series of medium volumes in the shaker-flask have been tested for the requirement of aeration. The largest surface aeration test, one tenth of the medium volume in the shaker-flask for cultivation, gave the highest spore count. The optimal pH value was tested and the initial pH 5 in the SMAY medium produced a high spore density. Finally, V. lecanii spores from SSF and LSF were different in size, shape, and size distribution; while mean spore length from SSF was 6.1 7m, and mean spore length from LSF was 5.0 7m.