Wheat Seed Colonized with Atoxigenic Aspergillus flavus: Characterization and Production of a Biopesticide for Aflatoxin Control

Biocontrol of aflatoxin contamination using atoxigenic Aspergillus flavus to competitively exclude aflatoxin-producing strains has previously been reported, and is currently in the third year of commercial-scale tests (treating 50-200 ha per annum). Wheat seed colonized with atoxigenic A. flavus has been used in the commercial trials. Requirements for production of this colonized wheat seed are described and the spore yield of wheat is compared to other substrates. The study suggests that the most cost-effective inoculum production would require colonization of wheat (106 conidia kg -1 of wheat seed) at 25% (w/w) moisture for 18 h at 31 C. To prevent fungal growth and associated wheat aggregation in storage, seed had to be dried below 15% (w/w) moisture, although a moisture content of 35% (w/w) did not reduce viability in sealed containers stored at 18-25 C over an 8-month period. The dry biopesticide had multi-year stability without refrigeration and withstood temperatures of 70 C for 20 min. Sporulation of the product occurred within 3 days at 31 C and 100% relative humidity with yields averaging 4.9 X 109 conidia g -1 by day 7.     

Optimization of erythritol and glycerol accumulation in conidia of Beauveria bassiana by solid-state fermentation, using response surface methodology

Entomopathogenic fungi are widely produced for use as mycoinsecticides. Therefore, improvement of the shelf life of fungal propagules under good and adverse conditions should be a pre-requisite of their production. In order to improve conidial physiology as well as mycoinsecticide efficiency, culture conditions may be varied. The Doehlert design was used to generate response surfaces with an estimation of the parameters of the quadratic model allowing the study of three different factors at a different number of levels. This experimental design was applied to optimize water activity (a _w), pH, and fermentation time for Beauveria bassiana conidial production and accumulation of polyols in solid-state fermentation. Thus, it was possible to identify the region in the experimental range in which the optimum values of these parameters were simultaneously achieved. Maximal conidia production was achieved at pH 5–6 and a _w=0.999. Under these conditions, polyol accumulation was 3 mg erythritol/g conidia and 29.6 mg glycerol/g conidia. However, maximal polyol accumulation was achieved at pH 4.5 and a _w 0.950; erythritol production increased 33-fold and glycerol production 4.5-fold. Under these conditions conidia production was 1,000 times lower. The possibilities of increasing the quality of the biocontrol agent without neglecting yield are discussed.     

Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates

On agar media the maximum conidia production of Exserohilum monoceras occurred on V-8 juice agar (VA) or centrifuged V-8 juice agar, whereas the optimal radial mycelial growth occurred on Czapek-Dox agar. The optimal temperatures for radial mycelial growth and conidia production were 28 and 27°C respectively. Light prohibited E. monoceras conidia production. The best sporulation occurred under continuous dark conditions. Echinochloa leaf decoction significantly increased conidia production on potato dextrose agar (PDA) and VA, and significantly increased germ tube length on PDA, lima bean agar and VA, but did not affect conidia germination. No conidia were produced in liquid media. Of 22 agricultural-based products evaluated as solid substrates, the most abundant sporulation (1.8 × 10⁶ conidia g^-1 of dry weight) occurred on corn leaves. The conidia production of E. monoceras on corn leaves was affected by incubation period, moisture content and substrate quantity. There were no differences in germination rate, germ tube length and virulence of conidia produced on agar media or corn leaves.     

The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-batch cultivation system

The present investigation deals with role of Ca⁺⁺ ions in increasing the yield of citric acid in a repeated-batch cultivation system (working volume 9-1) and its kinetic basis. Five different hyper-producing strains of Aspergillus niger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/1, 6 days after mycelial inoculation. The addition of CaCl₂ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl₂ at a level of 2.0 M, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/1 was obtained when the sampling vs. substrate feeding was maintained at 4-1 (44.50% working volume). The incubation period was reduced from 6 to only 2 days. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by the selected Aspergillus niger NG^GCB101 (LSD = 0.456a, HS). Case studies are highly economical because of higher yield of product, lower energy consumption and the use of raw substrate without any additional supplementation.     

Spent brewery grains as substrate for the production of cellulases byTrichoderma reesei QM9414

Summary The cellulolytic fungusTrichoderma reesei QM9414 can be cultivated on spent brewery grains for the production of cellulases. The levels of the cellulase components endoglucanase and exoglucanase synthesized, and the complexes filter paper cellulase and grain-hydrolyzing cellulase synthesized by the organism on spent grains were as high as 287, 182, 187, and 449 units per g available cellulose, respectively. Scaling up the spent grains fermentation system by up to 40-fold (200g dry substrate/tray) demonstrated that cellulase production was comparable to laboratory scale (5g dry substrate/flask) yields. Cultivation of the fungus was feasible on spent grains without pretreatment or further adjustment, although the enzyme yield was somewhat lower than that on dried grains moistened with water orTrichoderma medium. This suggested the possible reutilization of spent grains, with minimal pretreatment, in the cultivation ofT.reesei QM9414 for cellulase synthesis and for future incorporation into animal feed.     

Production cost of a real microalgae production plant and strategies to reduce it

The cost analysis of a real facility for the production of high value microalgae biomass is presented. The facility is based on ten 3 m³ tubular photobioreactors operated in continuous mode for 2 years, data of Scenedesmus almeriensis productivity but also of nutrients and power consumption from this facility being used. The yield of the facility was close to maximum expected for the location of Almería, the annual production capacity being 3.8 t/year (90 t/ha·year) and the photosynthetic efficiency being 3.6%. The production cost was 69 €/kg. Economic analysis shows that labor and depreciation are the major factors contributing to this cost. Simplification of the technology and scale-up to a production capacity of 200 t/year allows to reduce the production cost up to 12.6 €/kg. Moreover, to reduce the microalgae production cost to approaches the energy or commodities markets it is necessary to reduce the photobioreactor cost (by simplifying its design or materials used), use waste water and flue gases, and reduce the power consumption and labor required for the production step. It can be concluded that although it has been reported that production of biofuels from microalgae is relatively close to being economically feasible, data here reported demonstrated that to achieve it by using the current production technologies, it is necessary to substantially reduce their costs and to operate them near their optimum values.     

Production of conidia of Beauveria bassiana in solid-state culture: current status and future perspectives

Beauveria bassiana is an important entomopathogenic fungus widely commercialized in the world. Recent progress and achievements on conidia production have focused on a yield goal of 10⁹ to 10¹⁰ conidia per gram of dry substrate. Due to cost-competitive perspectives, these yields should be associated with better production rates or productivities. This study presents a review of relevant studies of B. bassiana conidia production on solid-state cultures and the parameters that should be taken into account to maintain constant quality in the product to be commercialized. Conditions for maximizing production and infectivity of B. bassiana conidia are also analysed.     

Conidia production ofBeauveria sp. by solid-state fermentation for biocontrol ofIlex paraguariensis caterpillars

Conidia production of Beauveria sp. strain LAG by solid-state fermentation (SSF) using blends of agro-industrial residues (residual potatoes and sugar-cane bagasse) was optimized with respect to cultivation conditions and the composition of substrate mixture in Erlenmeyer flasks and column-type bioreactor. With a blend of 60 % residual potatoes and 40 % sugar-cane bagasse the optimum conditions achieved were: incubation temperature 26 degrees C, initial substrate pH 6, inoculum concentration 10(7) conidia per g substrate; optimal initial moisture of the substrate was 70 % for Erlenmeyer flasks, in column-type bioreactor (with forced aeration) the optimal initial moisture of the substrate was 65 % with airflow of 60 mL/min. The highest production (1.07 x 10(10) conidia per g dry substrate) was achieved after a 10-d fermentation. The conidia were used in laboratory assays against Thelosia camina and Hylesia sp., caterpillars that are serious pests of mate plants. The mortality of T. camina was >90 % 10 d after spraying caterpillars with 1 mL conidia suspension at a concentration 10(5)-10(8)/mL. For Hylesia sp., the mortality was 70 %, 7 d after immersion in the conidia suspension containing 108 conidia per mL. Therefore, the Beauveria sp. LAG can be considered to be an important biocontrol instrument in the prospect of the Integrated Pest Management for mate plants.     

Mass Production of Conidia of Penicillium frequentans,a Biocontrol Agent Against Brown Rot of Stone Fruits

Conidial production of Penicillium frequentans , a biocontrol agent of the fungal pathogen Monilinia laxa , was tested in liquid and solid-state fermentation. Conidial production of P. frequentans in solid-state fermentation was higher than in liquid-state fermentation. Solidstate fermentation was made in specially designed plastic bags (VALMIC ® ) containing peat:vermiculite (1:1 w/w). Addition of nutrients to the peat:vermiculite increased conidial production of P. frequentans , especially when lentil meal was added. The number of conidia obtained in this solid-state fermentation was maintained in the range of 10 8 -10 9 conidia g -1 from 5 to 120 days after inoculation. Germinability of these conidia was > 90% until 90 days of incubation and declined at 120 days. Optimal initial moisture content in the substrate was 30-40% (v/w). At lower moisture contents, significant reductions in conidial production and germinability were observed, particularly at 10% (v/w). Conidial production was similar when the substrate was inoculated with 10 5 , 10 6 or 10 7 conidia g -1 dry substrate. Fresh conidia produced by solid-state fermentation reduced the incidence of brown rot on plums by 75%.     

A computer model for the growth and differentiation of a fungal colony on solid substrate

Mold growth and differentiation are closely related to the formation of secondary products. In solid-substrate fermentations this interrelationship is often more completely realized than in submerged cultures. Solid substrate reactions are used commercially in a limited manner in the western world, but are relatively common in Asia. Basic studies in solid-substrate fermentation should yield results applicable to all types of commercial mold fermentations for the production of a secondary product. This paper presents a relatively simple model for the growth of a mold colony on a solid surface with a defined medium utilizing glucose. Unlike submerged cultures the model must account for both cellular differentiation and the spatial heterogeneity in the system. Model parameters were estimated independently using literature values. The results of the simulation studies suggest that mass transfer limitations are at least partially responsible for the proliferation of differentiated structures on solid substrates as compared to liquid cultures. Since the concentration profile depends on the depth of the substratum, conditions that enhance conidia production can be achieved by controlling the depth of the solid medium.     

Batch cultures of supplemented whey permeate using Lactobacillus helveticus: unstructured model for biomass formation, substrate consumption and lactic acid production

The Luedeking and Piret expression can not account for the cessation of production observed at the end of batch; so an empiric term has been previously added to this equation which accounted in a global way for possible substrate limitations. In the model developed in this work, a carbon substrate limitation appeared explicitly in the production expression. Assuming a sigmoidal variation with time of specific growth rate previously validated, the new production model matched well the entire experimental production kinetics. It has been successfully tested for a wide range of nitrogen supplementations, i.e. from an almost total coupling between growth and production for largely supplemented media, to a high decoupling in case of few available nitrogen. Since all the parameters of this model have an obvious biologic meaning, it may be an unvaluable tool for the comprehension of the phenomenon. The model accounted also well for the variation of the specific production rate versus specific growth rate, avoiding the noise due to the direct differentiation of experimental data.     

酵母发酵蔗渣半纤维素水解物生产木糖酶

采用二次正交旋转组合设计研究了蔗渣半纤维素水解过程中硫酸浓度与液 固比对木糖收率的影响。回归分析表明 ,这两个因素与木糖的收率之间存在显著的回归关系。通过回归方程优化水解条件 ,当硫酸浓度 2 .4g L ,液 固 =6 .2 ,在蒸汽压力 2 .5× 10 4Pa的条件下水解 2 .5h ,10 0g蔗渣可水解生成木糖约 2 4g。大孔树脂吸附层析处理蔗渣半纤维素水解物 ,能有效地减少其中的酵母生长抑制物含量 ,显著改善水解物的发酵性能。用大孔树脂在pH 2条件下处理过的蔗渣半纤维素水解物作基质 ,含木糖 2 0 0g L ,产木糖醇酵母菌株CandidatropicalisAS2 .1776发酵 110h耗完基质中的木糖 ,生成木糖醇 12 7g L ,产物转化率 0 .6 4(木糖醇g 木糖g) ,产物生成速率 1.15g L·h .     

Medium Selection and Effect of Higher Oxygen Concentration Pulses on <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var<Emphasis Type="Italic">. lepidiotum</Emphasis> Conidial Production and Quality

Rice and oat flours were analyzed as media for the production of conidia by M. anisopliae var. lepidiotum. The presence of peptone increased conidia yield regardless of the substrate used; however, the highest yield was achieved on oat flour media. The effect of oxygen on conidia production using oat-peptone medium was also studied at two levels: Normal atmosphere (21% O₂) and Oxygen-rich pulses (26% O₂). Maximum conidia production (4.25 × 10⁷ conidia cm⁻²) was achieved using 26% O₂ pulses after 156 h of culture, which was higher than 100% relative to conidial levels under normal atmosphere. Conidia yield per gram of biomass was 2.6 times higher with 26% O₂ (1.12 × 10⁷ conidia mg⁻¹). Conidia quality parameters, such as germination and hydrophobicity, did not show significant differences (P < 0.05) between those treatments. Bioassays parameters, using Tenebrio molitor adults, were analyzed for conidia obtained in both atmospheres and data were fitted to an exponential model. The specific mortality rates were 2.22 and 1.26 days⁻¹, whereas lethal times for 50% mortality were 3.90 and 4.31 days, for 26% O₂ pulses and 21% O₂ atmosphere, respectively. These results are relevant for production processes since an oxygen increase allowed superior levels of conidia by M. anisopliae without altering quality parameters and virulence toward Tenebrio molitor adults.     

Optimising inoculum yield and shelf life of Plectosphaerella cucumerina,a potential bioherbicide for Cirsium arvense

Plectosphaerella cucumerina was identified as a potential bioherbicide for controlling Cirsium arvense in Canada and New Zealand. The current study evaluated production conditions using two isolates (one from each country) to determine whether the yield and shelf life of inoculum are suitable for mass production. Mycelial growth and sporulation in culture both increased from 15°C to 25°C and declined at higher temperatures with no mycelial growth at 37°C. The Canadian isolate produced fewer conidia than a New Zealand isolate. Potato dextrose-based liquid media with moderate to high concentrations of carbohydrates (25%, 50%, and 100%) maximised conidia production and these base media produced conidia with the highest germination rate (>80%) both at harvest and after 4 weeks stored at 4°C in 2.5% glycerol, 40% milk glycerol or after air drying. However, after 10-week storage, the conidia failed to germinate. Sporulation occurred during growth on all solid substrates tested (rice, rolled barley, and triticale), but conidial germination was highest on rice and barley, both before and after air drying. By contrast to conidia, 90% of mycelia-infested barley grains were viable after 3 years of storage at room temperature, although viability was lost by this time on the other substrates. This study has shown that the nutritional base is an important determinant of sporulation and shelf life for P. cucumerina. Although the yield of conidia in liquid medium was adequate to justify further development of P. cucumerina as a bioherbicide, improvement in its shelf life, or alternate formulation types that extend the shelf life, must be made for commercial efficiency.     

Conidia production by Isaria fumosorosea on solid substrates and its pathogenicity towards Bemisia tabaci

Large amounts of conidia must be recovered to use Isaria fumosorosea as a biological control agent. Aerial conidia are mass-produced in solid substrate culture. Conidia obtained from growth on different substrates must be similar in quantity and quality (viability, purity and virulence). In the present work, several solid substrates were evaluated: rice, sorghum, sugarcane, corncob, plantain and wheat (derived from the production of Trichogramma sp.) to find alternatives that are economical and available in different regions. The highest conidia production was obtained on rice, plantain and corncob substrates, which yielded 5.33 × 10⁹ conidia/g, 5.24 × 10⁹ conidia/g and 4.8 × 10⁹ conidia/g substrate, respectively. At concentrations of 1 × 10⁷ conidia/mL, the mortality rates obtained with conidia developed on rice, plantain and corncob substrates were 98%, 97% and 88%, respectively. The lethal concentration 50 (LC₅₀₎ values of conidia obtained from rice and plantain were 7.2 × 10⁴ conidia/mL and 5.1 × 10⁴ conidia/mL, respectively.     

Isolation and characterization of Aspergillus sp. for the production of extracellular polysaccharides by response surface methodology

In this study, Aspergillus sp. was isolated for the production of extracellular polysaccharide. The process parameters were initially optimized by traditional methods. The cheap substrate, wheat bran was used for the production of extracellular polysaccharide in solid state fermentation. Supplementation of (1%, w/w) maltose, gelatin enhanced EPS production (5.36?mg/g). The salts such as, Cu²⁺ (4.9?mg/g), Ca²⁺ (3.5?mg/g), Zn²⁺ (2.9?mg/g), Mn²⁺ (3.4?mg/g) and Mg²⁺ (1.8?mg/g) stimulated EPS production. In two level full factorial experimental designs, the EPS yield varied from 3.18 to 11.65?mg/g wheat bran substrate with various combinations of the components supplemented with wheat bran substrate. Among these selected factors in central composite design, maltose significantly influenced on extracellular polysaccharide production.     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

A liquid medium for the production of Kabatiella zeae conidia.

A liquid medium, designated as Kabatiella zeae medium (KZM), containing 10.0 g of carboxymethylcellulose, 5.0 g of maltose, 1.5 g of peptone, 1.0 g of monobasic potassium phosphate, 1 L of distilled water, is described. Peptone was found to be the component most influential in stimulating sporulation. The optimum temperature for conidial production was 25 degrees C. In shake culture about 10(7) conidia/mL were produced in 5 days at room temperature (about 21 degrees C) when KZM was seeded with 4-day-old colony plugs. The optimum for conidial production was pH 5 with only a slight gradual reduction to pH 9. However, fungal development was abnormal at pH values of 4 and 5. Of the 29 isolates tested, only two did not sporulate in KZM or in any other medium. Because of the high yield of conidia in a short time, the ease of preparation and use, and its low cost, KZM is especially useful where large amounts of inoculum are needed.     

Microbial modification of sugars as building blocks for chemicals

Summary Investigations on the microbial modification of sucrose to the corresponding 3-keto-derivative were carried out with resting cells of Agrobacterium tumefaciens NCPPB 396. This highly specific oxidation to yield the 3-keto-derivative has been analysed kinetically with varying substrate and cell mass concentrations. The formation of the corresponding 3-keto-derivative depended strongly on the reaction time and the aeration rate, and was maximal at aeration rates up to 11.5 volume air/cultivation volume per minute with resting cells. The product formation increased with increasing substrate concentrations. However, the product yield was maximal at substrate concentrations below 20 g/l. Data pertaining to the production of active cell mass as well as for maximal 3-keto-derivative formation are presented in this paper. Also included are some applications for these derivatives.     

Use of a Novel Sporulation Monitor to Quantify the EVects of Formulation and Storage on Conidiation by Dried Mycelia of the Entomopathogenic Fungus Zoophthora radicans

A novel piece of equipment, the sporulation monitor, is described for the comparison of conidia production from mycelia receiving diVerent formulation and storage treatments. This equipment was used to compare the viability of Zoophthora radicans mycelial samples treated either with 10% maltose solution or with distilled water before drying and storage for 0-12 weeks at 4oC. Freshly dried maltose-treated mycelial mat samples produced significantly more conidia for a significantly longer time than distilled water-treated mat samples of the same age. Very few conidia were produced from mats in either treatment after storage for 4 weeks or longer. There were great diVerences in conidia production from mycelial mat samples produced in diVerent fermenter runs. These results are discussed in relation to the potential for the use of dried mycelia in biological control programmes.