Sporulation of Penicillium roqueforti in solid substrate fermentation

Summary A study of the influence of temperature, aeration rate, and substrate water content on sporulation of Penicillium roqueforti on buckwheat seeds in a fixed bed reactor is described. Use of an experimental procedure based on a 2³ factorial design allowed optimum to be determined as 23.5° C for temperature, 0.48 g/g dry matter for substrate water content and 4.42 VVH for aeration rate.     

Effect of substrate moisture content on fungal production of citric acid in a solid state fermentation system

Summary Under solid state fermentation conditions, the production of citric acid byAspergillus niger varied considerably with the initial moisture content of apple pomace. The stimulating effect of methanol on fungal production of citric acid decreased markedly as the substrate moisture decreased.     

Responses of soil respiration to elevated CO2, air warming, and changing soil water availability in a model old-field grassland

Responses of soil respiration to atmospheric and climatic change will have profound impacts on ecosystem and global carbon (C) cycling in the future. This study was conducted to examine effects on soil respiration of the concurrent driving factors of elevated atmospheric CO₂ concentration, air warming, and changing precipitation in a constructed old‐field grassland in eastern Tennessee, USA. Model ecosystems of seven old‐field species were established in open‐top chambers and treated with factorial combinations of ambient or elevated (+300 ppm) CO₂ concentration, ambient or elevated (+3 °C) air temperature, and high or low soil moisture content. During the 19‐month experimental period from June 2003 to December 2004, higher CO₂ concentration and soil water availability significantly increased mean soil respiration by 35.8% and 15.7%, respectively. The effects of air warming on soil respiration varied seasonally from small reductions to significant increases to no response, and there was no significant main effect. In the wet side of elevated CO₂ chambers, air warming consistently caused increases in soil respiration, whereas in the other three combinations of CO₂ and water treatments, warming tended to decrease soil respiration over the growing season but increase it over the winter. There were no interactive effects on soil respiration among any two or three treatment factors irrespective of time period. Treatment‐induced changes in soil temperature and moisture together explained 49%, 44%, and 56% of the seasonal variations of soil respiration responses to elevated CO₂, air warming, and changing precipitation, respectively. Additional indirect effects of seasonal dynamics and responses of plant growth on C substrate supply were indicated. Given the importance of indirect effects of the forcing factors and plant community dynamics on soil temperature, moisture, and C substrate, soil respiration response to climatic warming should not be represented in models as a simple temperature response function, and a more mechanistic representation including vegetation dynamics and substrate supply is needed.     

Effect of operating conditions on solid substrate fermentation

In this work the effects of environmental parameters on the performance of solid substrate fermentation (SSF) for protein production are studied. These parameters are (i) air flow rate, (ii) inlet air relative humidity, (iii) inlet air temperature, and (iv) the heat transfer coefficient between the outer wall of the fermentor and the air in the incubator. The air flow is supplied to effect cooling of the fermented mass by evaporation of water. A dynamic model is developed, which permits estimation of biomass content, total dry matter, moisture content, and temperature of the fermented matter. The model includes the effects of temperature and moisture content on both the maximum specific growth rate and the maximum attainable biomass content. The results of the simulation are compared with actual experimental data and show good agreement with them. The most important conclusions are that (i) the evaporative cooling of the biomass is very effective for temperature control and (ii) the air flow rate and the heat transfer coefficient have strong effects but they affect the biomass morphology and are not controllable easily. Also, a simple technique for the determination of the optimum temperature and moisture content profile for cell protein production is applied. The simulated biomass production increases considerably employing the optimum temperature and moisture content profiles. The ultimate goal is to implement the determined effects of the environmental parameters on the SSF biomass production and the temperature and moisture variation profiles to effectively control the SSF and optimize the biomass production. (c) 1993 John Wiley & Sons, Inc.     

Detection of bacterial chitinase activity in transformed plant tumour cells using a specific exochitinase substrate

Methods for the detection of bacterial chitinase activity were compared. The soluble substrate p-nitrophenyl-ß-D-N,N diacetyl chitobiose (NDC) was more sensitive in detecting purified chitinase of Serratia marcescens than assays measuring degradation of a solid chitin substrate by either radiochemical or colorimetric means. A chimaeric gene containing a S. marcescens chitinase gene under control of a Cauliflower Mosaic Virus 35S promoter and nopaline synthase terminator sequences was constructed and transferred to tobacco tumour cells using Agrobacterium tumefaciens as a vector. The rate of hydrolysis of the NDC substrate was three fold greater with cell extracts of both pooled and individual tumours carrying the chimaeric chitinase gene than in control tumours. It was calculated from the enzyme activity data that the foreign bacterial chitinase contributed 0.1% of the total soluble protein in transformed plant cells. This level of expression of this gene was not detectable using the less sensitive assays employing solid chitin substrate. These results indicate that NDC is a preferable substrate for assaying bacterial chitinase in transformed plant cells.     

Process analysis of fluidized bed granulation

This study assesses the fluidized bed granulation process for the optimization of a model formulation using in-line near-infrared (NIR) spectroscopy for moisture determination. The granulation process was analyzed using an automated granulator and optimization of the verapamil hydrochloride formulation was performed using a mixture design. The NIR setup with a fixed wavelength detector was applied for moisture measurement. Information from other process measurements, temperature difference between process inlet air and granules (T_diff), and water content of process air (AH), was also analyzed. The application of in-line NIR provided information related to the amount of water throughout the whole granulation process. This information combined with trend charts of T_diff and AH enabled the analysis of the different process phases. By this means, we can obtain in-line documentation from all the steps of the processing. The choice of the excipient affected the nature of the solid-water interactions; this resulted in varying process times. NIR moisture measurement combined with temperature and humidity measurements provides a tool for the control of water during fluid bed granulation.     

Some characteristics of the cellulase of Aspergillus candidus

Summary The effects of pH, temperature and substrate concentration on the cellulase (B-1,4-endoglucanase) activity ofA. candidus were studied. Maximum enzyme activities were obtained when the concentration of the substrate (CMC) was 6 mg per ml, at pH 4, and temperature 50 C. The enzyme retained 85% of its original activity under optimal conditions of pH and temperature after 36 hours of incubation. The Km constant of the reaction was calculated as 2.3 mg of CMC per ml and the energy of activation of the enzyme as 7.92 k cal per mole.     

Computer-linked HPLC system for feedback control of fermentation substrates

Summary A data acquisition/control microcomputer system was interfaced to a commercial HPLC data transmission module. Control of substrate (ethanol) levels for four 7.5 L fermenters containing 100 g/L wet weight of the yeastCandida norvegensis was accomplished by employing intermittent, automated HPLC monitoring and a BASIC-encoded proportional integral policy for controlling substrate feed rates. Ethanol levels were maintained at 0.25, 0.50, 0.75 and 1.00% w/v.     

Development of a fermentation procedure to produce a tempe-related food using common beans as substrate

Summary A basic procedure was developed to produce a tempe-like product using the mouldRhizopus oligosporus and black common beans (Phaseolus vulgaris) as substrate. The initial pH of the substrate was 5.8, and fermentation was conducted at 37°C with a relative humidity of 70% for 72 hrs. Levels of soluble solids and soluble protein increased dramatically as a result of fermentation. Some changes were as well observed in fatty acid contents of fermented samples. It was concluded that the common bean used was an acceptable substrate for preparing this product.     

Evaporative temperature and moisture control in a rocking reactor for solid substrate fermentation

Summary In the solid substrate fermentation of cooked yellow corn grits with Rhizopus oligosporus in a rocking drum fermenter, temperature was controlled by blowing air through the substrate, forcing water evaporation. The rate of evaporation was controlled by the relative humidity of the air, according to the rate of heat generation during fermentation. Moisture content was maintained constant by spraying cold water on the substrate regulated by the water balance equation of the system. Both controls were operated by computer programs. The rocking motion in the reactor allowed even distribution of air and water in the substrate without disturbing the growing mycelia.     

Dynamics of growth in batch and continuous cultures of Saccharomyces cerevisiae during shifts from aerobiosis to anaerobiosis and reverse

Summary Saccharomyces cerevisiae H 1022 was cultivated in batch and continuous culture on a glucose substrate. The yeast was subjected to a sudden change from aerobic to anaerobic growth conditions by switching the inlet-gas stream from air to dinitrogen. The dynamics of growth and product formation during the periods of adaptation were studied. A structured growth model based on Monod-Blackman-kinetics was applied to simulate these shift-experiments. The immediate switch of the yeast to maximum anaerobic growth and ethanol production predicted by this model was confirmed very well in the chemostat-experiments. However, a slow adaptation was evident for the switch from anaerobiosis back to aerobiosis.Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig-Stöckheim, Federal Republic of Germany     

Enzymatic hydrolysis of penicillin V to 6-aminopenicillanic acid byFusarium oxysporum

Summary A strain ofFusarium oxysporum was identified as having an intracellular penicillin V acylase activity (penicillin V amidohydrolase EC 3.5.1.11). Activity was induced by phenoxyacetic acid and had a good tolerance for high substrate and product concentrations. Washed cells could be used repeatedly for the complete hydrolysis of 5% penicillin V solutions. The enzyme was partially purified and concentrated from disrupted cells by fractional precipitation with water miscible solvents.     

Laboratory scale bioreactor for solid state processes

Engineering aspects of solid state processes, design of fermentors, control systems for maintaining parameters are important problems for the research in this area. Laboratory scale reactors (2 kg dry-matter capacity) were constructed to allow control of the temperature and moisture level of the substrate without agitation. Forced aeration is accomplished by means of thermostated air injected at the bottom of the reactor. By bubbling in a waterbath and after heating, the thermostated air allows the regulation of temperature and moisture content of the medium during the cultivation. These reactors have been used for the study of protein enrichment of leached sugar-beet pulp by solid state processes.     

Effects of temperature and moisture on embryonic diapause of the veiled chameleon (Chamaeleo calyptratus)

The development of lizard embryos is typically initiated at fertilization and continues until birth or hatching. In contrast, embryonic development of some chameleons is arrested at the gastrula stage, and embryos remain at this stage for months after the eggs are laid. Our research tested the hypothesis that increased temperature, moisture, or both, are associated with the resumption of development by diapausing embryos of Chamaeleo calyptratus, the veiled chameleon. After 40 days of incubation at 25 degrees C in a relatively dry substrate, eggs were subjected to: 1) no change in temperature or moisture, 2) no change in temperature but change from a dry to a wet substrate, 3) change to a warmer temperature but no change in substrate moisture, or 4) an increase in both temperature and substrate moisture. Overall, embryos initiated development after 50-60 days to 80 or more days of incubation. Neither substrate moisture nor water uptake by eggs was related to the interval when development resumed. In contrast, development was initiated about 10 days earlier for eggs in the high temperature treatment compared to eggs in the low temperature treatment. Our results suggest that neither water availability nor water uptake by eggs affect the length of diapause but that an increase in ambient temperature initiates development of diapausing embryos of C. calyptratus.     

Culture method to study fungal growth in solid fermentation

Summary A new culture method is described to study the growth of Aspergillus niger on cassava meal in the solid state. This method uses preparations of the cooked starchy substrate as a homogeneous granulated product containing spores, salts and water. An incubation device aerates the mass with humidified air at a controlled temperature. Homogeneous development of mycelia, without sporulation, occurred in the substrate mass. From physiological studies, optimal conditions for A. niger growth on cassava in the solid state were 50–55% moisture, 35°C, a nitrogen source comprising 60% ammonium and 40% urea (on a nitrogen basis) and 2×10⁷ spores/g of substrate. Growth kinetics were established and changes in pH, protein, carbohydrate and water content were determined during the incubation. Growth rate and yield were quite similar to those described in the literature for A. niger cultivated in liquid media under optimal conditions.     

The role of substrate feeding earthworms (Lumbricidae) for bioturbation in a beechwood soil

Summary Burrow formation (burrow length and faeces production) of the substrate feeding earthworm Aporrectodea caliginosa (Savigny) in relation to three temperatures (5, 10 and 15°C) and soil moisture contents (48, 60 and 73% water of dry wt) was determined. Soil moisture content affected the burrowing activity of A. caliginosa only below a distinct threshold (60% water of dry wt). At sufficient moisture an, increase in temperature by 5° C approximately doubled the amount of egesta produced. Another substrate feeding earthworm (Octolasion lacteum (Örley)) showed a similar pattern of burrowing activity.The data for the dependence of the faeces production of A. caliginosa on soil temperature and moisture content were combined with field data. The amount of faeces produced by the population of this earthworm species in a beechwood on limestone was calculated to be about 4.23 kg ha^-1 a^-1. The amount of egesta produced by all substrate feeding species is assumed to exceed 6 kg ha^-1 a^-1, which is equivalent to a soil layer of approximately 9 mm. This group of earthworms is therefore considered to be of greater importance for bioturbation and the formation of mull than previously assumed.     

A model substrate for solid-state fermentation

Summary A model substrate consisting of cassava starch embedded in kappa-carrageenan was used to mimic the growth ofRhizopus oligosporus on cassava tubers. Growth on the model substrate was similar to that during solid-state fermentation of the actual cassava. However, protein production and starch utilization were slower on the model substrate.     

Air humidity as key determinant of morphogenesis and productivity of the rare temperate woodland fern Polystichum braunii

(1) Most ferns are restricted to moist and shady habitats, but it is not known whether soil moisture or atmospheric water status are decisive limiting factors, or if both are equally important. (2) Using the rare temperate woodland fern Polystichum braunii, we conducted a three‐factorial climate chamber experiment (soil moisture (SM) × air humidity (RH) × air temperature (T)) to test the hypotheses that: (i) atmospheric water status (RH) exerts a similarly large influence on the fern's biology as soil moisture, and (ii) both a reduction in RH and an increase in air temperature reduce vigour and growth. (3) Nine of 11 morphological, physiological and growth‐related traits were significantly influenced by an increase in RH from 65% to 95%, leading to higher leaf conductance, increased above‐ and belowground productivity, higher fertility, more epidermal trichomes and fewer leaf deformities under high air humidity. In contrast, soil moisture variation (from 66% to 70% in the moist to ca. 42% in the dry treatment) influenced only one trait (specific leaf area), and temperature variation (15 °C versus 19 °C during daytime) only three traits (leaf conductance, root/shoot ratio, specific leaf area); RH was the only factor affecting productivity. (4) This study is the first experimental proof for a soil moisture‐independent air humidity effect on the growth of terrestrial woodland ferns. P. braunii appears to be an air humidity hygrophyte that, whithin the range of realistic environmental conditions set in this study, suffers more from a reduction in RH than in soil moisture. A climate warming‐related increase in summer temperatures, however, seems not to directly threaten this endangered species.     

Hydrolysis of animall fats by lipase at temperatures below their melting points

Summary We have studied the hydrolysis of high melting animal fats by the lipase fromCandida rugosa at temperatures between 20°C and 37°C without the addition of surfactants or organic solvents. To establish the practical applications of this process we investigated the optimal conditions of the reaction at high substrate concentrations (50% fat w/v) to achieve 95% hydrolysis (or better) in 24 hours. Experiments were conducted in solid emulsions without constant stirring (500 ml total reaction volume). Under all conditions tested, edible pork lard was a better substrate than inedible beef tallow yielding up to 96% hydrolysis with as low as 0.3 g lipase/Kg fat or 98% hydrolysis with 0.5 g lipase/Kg fat. The optimum temperature for the hydrolysis of edible pork lard was around 30°C. Inedible beef tallow and pork lard did not exhibit a clear optimum temperature. Inedible lard gave results intermediate between those of edible lard and inedible beef tallow.     

Effects of post-application irrigation and substrate moisture on the efficacy of entomopathogenic nematodes against western flower thrips, Frankliniella occidentalis

The efficacy of entomomatogenic nematodes (Steinernema bicornutum Tallosi, Peters and Ehlers and/or Heterorhabditis indica LN2 Poinar, Karunakar and David) against the soil‐dwelling life stages of western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) was assessed under different moisture conditions in a commercial plant‐growing substrate in laboratory experiments. In the first experiment, both nematode species were tested at substrate moisture ranges of 67, 78, 88, or 95% relative moisture content, that were maintained before applying the nematodes at 100 or 400 infective juveniles (IJs) cm^?2. In the second experiment, 10, 25, 50, 100, or 120 ml irrigation water, resulting in relative moisture contents of 72, 81, 90, 99%, or more than the saturation level of the substrate, respectively, was applied to the substrate. Heterorhabditis indica LN2 was applied either in 3 ml water and followed by irrigation, or by suspending the infective juveniles in the water amounts indicated above to apply the nematodes in higher volume. Results indicated that at the higher application rate, initial moisture content did not significantly affect the efficacy of H. indica LN2. On the other hand, increasing moisture content resulted in an improved efficacy of H. indica LN2 and S. bicornutum at lower and higher application rates, respectively. Similar thrips control levels of 44 and 60% at the lower and higher application rate of H. indica LN2, respectively, were obtained at 88% relative moisture content. In the second experiment, higher and statistically similar thrips mortality of 40 and 50% at lower and higher application rates of H. indica LN2, respectively, were obtained when the infective juveniles were applied in a high volume suspension of 100 ml, or when followed by irrigation with 25 ml water, resulting in both cases in 81% relative moisture content. Generally, efficacies of the nematodes for thrips control can be improved by using an appropriate moisture content and/or post‐application irrigation. Thus, the high nematode application rates required for successful F. occidentalis control can be partly attributed to substrate moisture content and/or post‐application irrigation.