Influence of ultrasound amplitude and duty cycle on fungal morphology and broth rheology of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Effects of ultrasound amplitude and duty cycle on cultures of Aspergillus terreus are reported in a 25 l slurry bubble column sonobioreactor. Fermentations were carried out batchwise. A 2^k-factorial design with added central points was used. Sonication at any cycle and amplitude level did not affect biomass growth rate and yield relative to nonsonicated control, but did affect growth morphology. Ultrasound disrupted fungal pellets and caused the biomass to grow mainly as dispersed hyphae. Production of lovastatin was reduced by medium- and high-cycle sonication. Sonication affected broth rheology. In view of these results, sonication can be used to modify growth morphology and broth rheology without affecting growth rate and yield of filamentous fungi.     

Morphology and physiology of an alpha-amylase producing strain of Aspergillus oryzae during batch cultivations

The microscopic morphology, that is, total hyphal length and total number of tips, has been characterized during batch cultivations of Aspergillus oryzae. The specific growth rate estimated by measuring the total hyphal length (mu(h)) corresponds well with the specific growth rate estimated from dry weight measurements during cultures grown as free hyphal elements. The average tip extension rate can be described with a saturation type kinetics with respect to the average total hyphal length, and the branching frequency is closely related to the total hyphal length. For the applied strain of A. oryzae, pellet formation occurs by coagulation of spores. The agglomeration process is pH dependent and pellets are formed at pH values higher than 5, whereas low pH (<3.5) results in growth as freely dispersed hyphal elements. The maximum specific growth rate has a broad pH optimum between 3 and 7, whereas the alpha-amylase production has a sharper maximum at about pH 6. During batch cultivation with pellets the growth is described well by the cube-root law when pellet fragmentation can be neglected. The kinetic parameter k in the cube-root law is derived from the growth kinetics with no mass transfer limitation, k = mu(h)/3. Based on an oxygen balance, the active growth layer in the pellet is estimated to be 200 to 325 mum and, consequently, up to 50% of the biomass is limited by oxygen for large pellets. Ethanol production (up to 1 g L(-1)) was observed during batch cultivations with pellets, suggesting that ethanol is produced in the oxygen limited part of the biomass. A constitutive, low alpha-amylase production was observed at high glucose concentration. The specific alpha-amylase production was significantly higher for filamentous growth than for pellets and oxygen appears to be necessary for production of alpha-amylase. (c) 1996 John Wiley & Sons, Inc.     

Comparative analysis of mixing distribution in aerobic stirred bioreactor for simulated yeasts and fungus broths

The study on mixing distribution for an aerobic stirred bioreactor and simulated (solutions of carboxymethylcellulose sodium salt), yeasts (S. cerevisiae) and fungus (P. chrysogenum pellets and free mycelia) broths indicated the significant variation of mixing time on the bioreactor height. The experiments suggested the possibility to reach a uniform mixing in whole bulk of the real broths for a certain value of rotation speed or biomass concentration domain. For S. cerevisiae broths the optimum rotation speed increased to 500 rpm with the biomass accumulation from 40 to 150 g/l d.w. Irrespective of their morphology, for fungus cultures the existence of optimum rotation speed (500 rpm) has been recorded only for biomass concentration below 24 g/l d.w. The influence of aeration rate depends on the apparent viscosity/biomass concentration and on the impellers and sparger positions. By increasing the apparent viscosity for simulated broths, or biomass amount for real broths, the shape of the curves describing the mixing time variation is significantly changed for all the considered positions. The intensification of the aeration induced the increase of mixing time, which reached a maximum value, decreasing then, due to the flooding phenomena. This variation became more pronounced at higher viscosities for simulated broths, at higher yeasts concentration, and at lower pellets or filamentous fungus concentration, respectively. By means of the experimental data and using MATLAB software, some mathematical correlations for mixing time have been proposed for each broth and considered position inside the bioreactor. These equations offer a good agreement with the experiment, the maximum deviation being ±7.3% for S. cerevisiae broths.     

Identifying the conditions for development of beneficial mycelium morphology for chitosan-producing Absidia spp. in submersed cultures

Summary The first step when producing chitosan from fungi grown submersed, i.e. the cultivation technique of some chitosan-producing Abisidia spp/, was studied. Various strains have their own specific requirements for development of morphology beneficial for a high growth rate. Regarding the species studied (A. coerulea, CBS 100.38; A. fusca, CBS 102.35; A. glauca, Dep. Microbial Ecology, Lund; A. repens; CBS 102.32), these demands are primarily those concerning agitation. Increasing the number of spores (from 1.4·10⁷ to 5.8·10⁷ per liter) of A. coerulea increased the initial growth rate, which on the other hand (due to agglomeration of pellets) levelled off at a comparatively earlier stage. Too low stirrer speed generally caused the formation of a pulp with a strong decline in growth rate. Elevated stirrer speeds caused the formation of a more compact morphology, often in the form of small pellets. The use of an open paddle impeller with a large diameter (with satisfactory pumping and mixing characteristics) ran at a sufficiently high stirrer speed to avoid the formation of a viscous pulp allowed most of the species to grow satisfactorily. Continuous cultivations were hard to establish due to lack of new growth spots and accreted growth.     

高山被孢霉发酵生产花生四烯酸的宏观形态

采用图像处理技术对高山被孢霉(Mortierella alpina)发酵过程中的不同形态进行分析,并对其产花生四烯酸(ARA)能力进行了比较。研究发现:复合N源中蛋白胨与酵母粉比例是影响高山被孢霉宏观形态的重要因素,球形形态生长的菌体中ARA产量较分散,菌丝体中ARA产量高;在球形形态中,空心球的菌体生物量低,ARA比例低,蓬松球可以兼顾菌体高生物量、高油脂比例及高ARA比例。产ARA能力由生物量、油脂比例及油脂中ARA比例共同决定。结果表明:直径大约4 mm、成核区域面积大约为43.6%、紧密度为71.36的蓬松球形态,是高山被孢霉一种相对较佳的发酵形态,其菌体产ARA能力分别是空心球和分散丝状菌体产ARA能力的2.01和2.70倍。     

Summer water relations of the desert phreatophyte Prosopis glandulosa in the Sonoran Desert of southern California

Summary Two shoot populations of the rhizomatous, patchforming herb Solidago canadensis were studied throughout a developmental cycle in two abandoned pasture sites in southern Ontario. The shoot cohorts that emerged in spring dominated the two populations; subsequent recruitment was very low. Shoot mortality was highest in June and was concentrated in the smallest size classes. Both populations showed a pronounced bimodal size structure for most of the growth cycle. Relative growth rate of shoots declined as the growing season progressed and tended to be lowest in the smallest size classes. Inflorescence production depended on shoot size. The calculated relationship between log mean weight and log density of shoots was not constant during the growth cycle and the calculated maximum biomass values do not transgress the ultimate thinning line suggested from previously published data.Address for proofs and present address: Department of Geography, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX. England     

Effect of medium composition on 1-octen-3-ol formation in submerged cultures of Pleurotus pulmonarius

The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O₂ to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium. Correspondence to: D. Levanon     

不同施肥方法对马来沉香和土沉香苗期根系生长的影响

以珍贵树种马来沉香、土沉香1年生播种苗木为材料进行试验,研究指数施肥、平均施肥对2种沉香苗期根系生长动态及对N的响应特征。结果表明,经氮素处理的马来沉香、土沉香的根系生物量、根系长度、根系表面积、根系平均直径、根体积等指标均显著高于对照处理(P<0.05)。经指数施肥处理的苗木根系生长及各形态指标均高于平均施肥处理。同一时期在相同施肥处理方式下,马来沉香苗根系生长及根系形态指标值均高于土沉香。洛伦兹模型对不同施肥方法处理下马来沉香、土沉香苗木生长指标与根系生物量进行拟合,具有较高的R²(0.95-0.99)和较低的RSMD(0.538-2.352);抛物面模型对不同施肥方法处理下马来沉香、土沉香苗木生长指标与比根长进行拟合,具有较高的R²(0.92-0.99)和较低的RSMD(3.218-6.692)。     

The morphological and physiological evolution of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mycelium in the submerged culture and its relation to the formation of secondary metabolites

The influence of the morphology and differentiation of Aspergillus terreus hyphae on the formation of mevinolinic acid (lovastatin) and (+)-geodin was tested. Lovastatin titre was the highest (above 60 mg l⁻¹) in the system with smaller pellets (diameter below 1.5 mm) and high biomass concentration (above 10 g l⁻¹ in the idiophase). These biomass features were induced by the higher initial number of spores in the preculture (above 2 × 10¹⁰ l⁻¹). At the initial number of spores below 2 × 10⁹ l⁻¹ (+)-geodin biosynthesis was the most efficient but it was rather connected with the elevated C/N ratio than with the pellet size. In order to quantify the hyphal differentiation in fungal pellets a special approach was used. The sectioning of the stained pellets together with the image analysis and calculation procedures were applied. The analysis of hyphal differentiation indicated that lovastatin formation was correlated with the fraction of the active, growing hyphae.     

Interrelation between penicillin productivity and growth rate

Summary Fermentations were carried out in an 801 tower-loop reactor with pellets of Penicillium chrysogenum. The development of the inner structure of the pellets with regard to various fermentation conditions was observed by means of histological preparations of the pellets. Under conditions of energy-source-limitation mycelial tip growth and lysis of other mycelial parts exist simultaneously. Thus the net growth rate (formation rate of cell mass) is higher than the gross growth rate (multiplication rate of cell mass). Under conditions of nitrogen limitation, gross growth rate and net growth rate are identical. A very strict correlation between gross growth rate and penicillin production rate was found as long as sufficient oxygen supply could be maintained and carbon catabolite repression was avoided. The energy source requirement of the biomass can be described with the sum of three terms that correspond to gross growth, lysis compensation growth and maintenance.Symbols a Constant 1/l h - b Constant - K Decay rate constant for product 1/h - K ₁ Substrate inhibition constant g/l - K _op Controls saturation constant for oxygen g/l - K _p Saturation constant for substrate g/l - m Maintenance coefficient 1/h - ms Apparent maintenance coefficient 1/h - O Dissolved oxygen concentration g/l - P Product concentration g/l - p Exponent of O - q Specific productivity 1/h - S Substrate concentration g/l - t Time h - t ₁ Beginning of production phase h - t ₂ Time of pellet dissolution h - V Liquid volume of fermentation broth l - X Dry cell mass concentration g/l - Y Yield of dry cell mass from energy substrate - g Specific gross growth rate of biomass 1/h - l Specific lysis rate of cell mass 1/h - n Specific net growth rate of cell mass 1/h - p Maximum specific rate of product formation 1/h     

Effect of agitation intensities on fungal morphology of submerged fermentation

Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 +/- 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 +/- 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.     

Dynamics of ginsenoside biosynthesis in suspension culture of <Emphasis Type="Italic">Panax quinquefolium</Emphasis>

Biosynthesis of six saponins (ginsenosides) in suspension culture of P. quinquefolium Z₅ was investigated. Ginsenoside content in biomass reached the highest level, nearly 30 mg g⁻¹ d.w., between 25 and 30 days of the culture. Saponins were synthesized simultaneously with cell growth but their synthesis rate was not proportional to the growth rate. During the phase of rapid biomass multiplication, after which biomass reached 90% of its maximum yield, only half examined ginsenosides was produced. The second half of the final saponins yield was produced during the slow growth phase, in which only 10% of biomass was grown. During the intensive growth phase the productivity of six saponins examined per biomass (dry weight) unit was 3.4 μg mg⁻¹ d.w. day⁻¹, however, this parameter calculated for slow growth phase reached nearly 30 μg mg⁻¹ d.w. day⁻¹. There were differences in increase of the contents of six saponins determined in biomass, and it was the highest for saponins Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) and Rg₁ (20(S)-protopanaxatriol-6,20-di-O-β-d-glucoside).     

Simulation of growth of a filamentous fungus in 3 dimensions

The tridimensional growth of a filamentous fungus was simulated, based on a model for the evolution of the microscopic morphology of Trichoderma reesei. When supplemented with a spatial representation of growth, the model correctly simulates the evolution from a single spore to a pellet. Diffusion of oxygen is included in the model. The simulated tridimensional structures have a fractal nature; and the fractal dimension, determined by a box-counting method, increases during growth. The fractal dimension only depends on the mass of the pellet and is not affected by model parameters such as tip extension rate and branching frequency. Realistic pictures are obtained and the radius of the pellet increases at a constant rate. The influence of model parameters (tip extension rate, branching frequency, minimum porosity) on dissolved oxygen concentration profiles, biomass concentration profiles, rate at which the pellet diameter increases, and the evolution of the fractal dimension was determined. The dissolved oxygen profiles were found to be very different from the profiles, obtained by assuming a homogenous biomass distribution within the pellet. Finally, the formation of pellets from spore aggregates is calculated and the size of the spore aggregate is found to only influence the time needed before the appearance of a pellet and not its morphology. (c) 1997 John Wiley & Sons, Inc.     

Changes in miombo woodland structure under different land tenure and use systems in central Zambia

Aim Population pressure and communal land ownership are often perceived as serious threats to forest conservation in savanna woodlands of central and southern Africa. I aimed at testing the hypothesis that the rate of miombo woodland recovery after clearing and re‐growth structure are determined by land tenure and use. Location Miombo woodland under customary, leasehold, forest reserve and national park on ten permanent and temporary sites was studied in central Zambia. Two sites were in mature woodland and eight sites were in re‐growth miombo ranging in age from 1 to 30 years. Methods I enumerated and measured girth at breast height (1.3 m above ground) of trees/stems in sixty‐four 20 × 10 m plots in 1982, 1986 and 2000 at six sites and annually from 1990 to 2001 at four sites to determine stem density and status (live, dead or cut) and wood biomass. A total of 239 trees were cut, wood biomass measured and the data used to develop equations for estimating wood biomass on study plots. Distance between each study site and the nearest human settlement was estimated during each sampling period using aerial photographs, topographical maps and the global positioning system. Results Land tenure was responsible for significant differences in stem density, wood biomass and rate of biomass accumulation in re‐growth following clearing of mature miombo woodland. Although stem density was highest on customary land, wood biomass and accumulation rate were lowest. The highest biomass was on plots in forest reserves, with intermediate values for leasehold and national park. Fire was responsible for tree mortality at all the study sites and its impact was highest at a site in a national park. Sites close to human settlements had the highest density of cut stems but this activity did not significantly reduce wood biomass. Rate of woodland recovery was higher on sites cleared in the 1970s than on sites cleared in the 1990s, irrespective of age of re‐growth. The development of the first, second and third re‐growths following successive woodland clearing in 1972, 1981 and 1990, respectively, was not significantly different, except for stem density which was highest in the second re‐growth. Analysis of interactions between five land tenure and use factors (independent variables) and re‐growth structure revealed that 52% (P=0.0000) of the variation in stem density was because of re‐growth age and decade in which the woodland was cleared while distance to human settlements and age of re‐growth explained 42% (P=0.0000) of the variation in wood biomass. Individually, distance to human settlements explained 25% (P=0.0000) of the variation in wood biomass accumulation rate. Conclusion The results supported the hypothesis that rate of miombo woodland recovery and structure were influenced by land tenure and use. However, analysis of interactions between factors revealed that use related factors (i.e. decade in which woodland was cleared and distance to human settlements) and re‐growth related factors (age and type of re‐growth) were more important than land tenure per se in explaining variation in miombo recovery. The conclusion from these results is that regulation of land use is more important than change in land tenure to the proper management of miombo woodland.     

Batch and continuous transformation of progesterone by Rhizopus nigricans pellets in the presence of β-cyclodextrin

A pelleted morphological form of Rhizopus nigricans, which might be regarded as a naturally immobilized biomass, enabled development of a continuous process of progesterone 11α-hydroxylation in a laboratory-scale stirred tank bioreactor. β-Cyclodextrin was used to enhance steroid solubility in aqueous transformation media and consequently to increase the rate of steroid hydroxylation. Batch and continuous processes were performed with pellets of different average diameters, ranging from less than 1 mm to 7.5 mm. Mathematical model simulations of both operational modes confirmed the previously defined Michaelis–Menten kinetics for the biotransformation. A decrease in overall hydroxylation rate was noticed when pellets larger than 5 mm were used, which correlated with the lower area of outer surface of agglomerates where, presumably, most of the enzyme induction had taken place.     

Correlation between pellet morphology and glycopeptide antibiotic balhimycin production by Amycolatopsis balhimycina DSM 5908

Actinomycetes, a class of filamentous bacteria, are an important source of several industrially relevant secondary metabolites. Several environmental factors including the media composition affect both biomass growth and product formation. Likewise, several studies have shown that environmental factors cause changes in cellular morphology. However, the relationship between morphology and product formation is not well understood. In this study, we first characterized the effect of varying concentrations of phosphate and ammonia in defined media on pellet morphology for an actinomycete Amycolatopsis balhimycina DSM 5908, which produces balhimycin, a glycopeptide antibiotic. Our results show that higher balhimycin productivity is correlated with the following morphological features: (1) higher pellet fraction in the biomass, (2) small elongated pellets, and (3) shorter filaments in hyphal growth in the periphery of the pellets. The correlation between morphology and product formation was also observed in industrially relevant complex media. Although balhimycin production starts after 72 h with maximum production around 168 h, the morphological changes in pellets are observed as early as 24 h after commencing of the batch. Therefore, morphology may be used as an early predictor of the end-of-batch productivity. We argue that a similar strategy can be developed for other strains where morphological indicators may be used as a batch monitoring tool.     

Seasonal changes in growth rate, morphology and alginate content in Undaria pinnatifida at the northern limit in the Sea of Japan (Russia)

The growth, morphology, alginate yield and composition of Undaria pinnatifidawas studied from March to August in 2000 and 2001 at the northern limit of distribution of the species in Peter the Great Bay, the Sea of Japan (Russia). The changes in morphology, alginate yield and composition were caused by sporophyte growth and sporulation. The average rate of biomass change was 2–5% d⁻¹. The highest alginate content (51% d.wt) was obtained from blades, with lower values for sporophylls and midribs. An increase in alginate content was detected before sporulation. The conditions seem favourable for farming the alga in this region, with June the optimum month for harvesting. This revised version was published online in August 2006 with corrections to the Cover Date.