The half-saturation coefficient for dissolved oxygen: A dynamic method for its determination and its effect on dual species competition

A simple approach was developed to determine the half-saturation coefficient for dissolved oxygen (K(DO)) for three bacteria by maintaining a constant oxygen concentration in continuous culture, and employing a dynamic method to obtain the specific growth rate (mu) for each species. Measurement of mu at selected dissolved oxygen concentrations (DO) resulted in a typical Monod curve for a plot of mu vs. DO. Values for K(DO) and mu(max) were obtained from the Lineweaver-Burk reciprocal plot. The bacteria studied included representative strains of three microorganisms isolated in pure culture from poorly settling activated sludge: two filamentous microorganisms, Sphaerotilus natans and a second Sphaerotilus sp., and an unidentified floc-forming microorganism. The K(DO) values obtained for Sphaerotilus sp., S. natans, and the floc former were 0.014, 0.033, and 0.073 mg/L, respectively. Dual species competition experiments were conducted in continuous culture under low and high DO conditions. Successful growth competition by these microorganisms under DO-limiting conditions was consistent with experimentally determined K(DO) values. The finding of lower K(DO) values for the two Sphaerotilus species, compared to the floc former, confirmed the hypothesis that these filamentous microorganisms can outgrow floc-forming microorganisms in activated sludge when DO in the aeration basin is low.     

Morphological characterization of filamentous microorganisms in submerged cultures by on-line digital image analysis and pattern recognition

The morphology of filamentous microorganisms in submerged culture is of great interest. On the one hand, morphology influences rheology and mass transfer in the fermentation broth. On the other hand, morphology could be a visible expression of physiology and metabolism of the microorganisms. An algorithm for the morphological characterization and the estimation of biomass of filamentous microorganisms by means of digital image analysis has been developed. After measurement of eight features the objects in the broth are classified into different morphological classes, i.e., pellet aggregates, rough pellets, smooth pellets, mycelial flocks, and medium components. The classification is based on the measured object parameters and a knowledge base, which was generated in a preceding training phase. The method was tested on Streptomyces tendae Tü 901/8c. A typical batch fermentation in a defined medium is presented. It could be shown that both morphology and physiology have been changed in the course of the fermentation, especially during the transition from trophophase to idiophase. In order to supervise the fermentation processes continuously, an on-line image analysis system has been developed. Sampling, dilution, and image acquisition of the culture were performed under the control of a personal computer. (c) 1997 John Wiley & Sons, Inc.     

A mathematical model for the growth of mycelial pellet populations

In liquid culture, filamentous organisms often grow in the form of pellets. Growth result in an increase in radius, whereas shear forces result in release of hyphal fragments which act as centers for further pellet growth and development. A previously published model for pellet growth of filamentous microorganisms has been examined and is found to be unstable for certain parameter values. This instability has been identified as being due to inaccuracies in estimating the numbers of fragments which seed the pellet population. A revised model has been formulated, based on similar premises, but adopting a finite element approach. This considers the population of pellets to be distributed in a range of size classes. Growth results in movement to classes of increasing pellet size, while fragments enter the smallest size class, from which they grow to form further pellets. The revised model is stable and predicts changes in the distribution of pellet sizes within a population growing in liquid batch culture. It considers pellet growth and death, with fragmentation providing new centers of growth within the pellet population, and predicts the effects of shear forces on pellet growth and size distribution. Predictions of pellet size distributions are tested using previously published data on the growth of fungal pellets and further predictions are generated which are suitable for experimental testing using cultures of filamentous fungi or actinomycetes. (c) 1995 John Wiley & Sons, Inc.     

华根霉固态与液态培养产胞外蛋白的比较蛋白质组学分析

【目的】考察固态和液态两种培养方式对丝状真菌华根霉(Rhizopus chinensis)CCTCC M201021产胞外蛋白的影响,以加深对微生物固、液态培养特异性产酶的认识。【方法】利用成分相同的培养基对华根霉分别进行平板固态培养和液态培养,提取华根霉所产胞外蛋白,采用二维凝胶电泳(2-DE)结合质谱分析,分离鉴定差异蛋白。【结果】固态培养下华根霉所产胞外蛋白的蛋白酶活性是液态培养的9.2倍。胞外蛋白质组数据分析表明,华根霉固态和液态培养所产胞外蛋白存在显著差异,其中约70%是固态和液态培养下各自产生的特有蛋白。质谱鉴定进一步表明,固、液态培养对华根霉产胞外蛋白的种类和表达量都有显著影响,其中水解酶类所占比例较大,主要是与蛋白质降解相关的蛋白。【结论】固态和液态不同培养方式影响了华根霉产胞外蛋白的组成,有些基因只在特定培养方式下表达。固态培养下华根霉产胞外蛋白酶种类相对更多以及大部分蛋白酶的上调表达,可能是固态培养下胞外蛋白酶活性很高的原因。研究结果提示需要注意固液态不同培养方式下丝状真菌胞外酶的筛选和生产可能存在的不一致性。     

Modeling the exponential growth of filamentous fungi during batch cultivation

In certain conditions, filamentous fungi are observed to grow exponentially during batch submerged growth. It is shown for three cases, with simple mechanistic models, that an exponential growth assumption is reasonable. The basis of these models is the identification of a growth unit, and a mechanism for its doubling with a constant generation time. The importance of the variation of morphological properties within populations is demonstrated by the comparison of computer simulations of simplified models using average values and either experimental data or computer simulations of detailed stochastic models. Copyright 1998 John Wiley & Sons, Inc.     

Mixed culture biooxidation of phenol. I. Determination of kinetic parameters

A mixed culture derived from soil and activated sludge organisms was used to degrade phenol which was inhibitory to microorganisms at higher concentrations. The purpose of the experiments was to determine the kinetic parameters governing growth of the organisms by measuring growth rates in batch culture. To maintain a constant inoculum for the experiments inoculum was taken from a continuously operating continuous culture. Two populations were studied corresponding to two separate residence times in the continuous culture apparatus. One contained predominantly filamentous organisms, the other nonfilamentous. Five kinetic models were applied to the data and the best kinetic parameters for each model were determined by nonlinear least squares techniques. The models were then evaluated for best relative fit to the data. No significant differences were found between the models on the basis of fit and so a choice was made on the grounds of simplicity. A model proposed by Haldane was chosen as the best. No function however gave a satisfactory fit at the highest growth rates obtained. This experimental maximum in the plot of growth rate against substrate concentration was very sharp.     

SParticle,an algorithm for the analysis of filamentous microorganisms in submerged cultures

Streptomycetes are filamentous bacteria that produce a plethora of bioactive natural products and industrial enzymes. Their mycelial lifestyle typically results in high heterogeneity in bioreactors, with morphologies ranging from fragments and open mycelial mats to dense pellets. There is a strong correlation between morphology and production in submerged cultures, with small and open mycelia favouring enzyme production, while most antibiotics are produced mainly in pellets. Here we describe SParticle, a Streptomyces Particle analysis method that combines whole slide imaging with automated image analysis to characterize the morphology of submerged grown Streptomyces cultures. SParticle allows the analysis of over a thousand particles per hour, offering a high throughput method for the imaging and statistical analysis of mycelial morphologies. The software is available as a plugin for the open source software ImageJ and allows users to create custom filters for other microbes. Therefore, SParticle is a widely applicable tool for the analysis of filamentous microorganisms in submerged cultures.     

培养条件对里氏木霉306菌体形态和t-PA生物合成的影响

里氏木霉（Trichoderrna reesei）306是能够生物合成组织型纤溶酶原激活剂（t-PA）的基因工程菌株。在对其液态深层培养时,发现随培养夸件和培养时间的变化,其菌体能以松散和菌丝球的两种形态存在。菌体形态和t-PA的产生密切相关。培养基中无机盐和表面活性剂的种类和添加量以及接种量和pH等培养条件是影响里氏木霉306菌体形态和t-PA合成的主要因素。在液态深层培养过程中,菌体以松散的菌丝体形态生长,形成纸浆状发酵液,利于t-PA的合成。     

Growth mechanisms and growth kinetics of filamentous microorganisms

Filamentous microorganisms are of major biotechnological importance, being responsible for production of the majority of secondary metabolites, particularly antibiotics. Two main groups are involved, filamentous fungi and filamentous actinomycetes, particularly the streptomycetes. In terms of cellular growth mechanisms, these groups differ greatly. Eukaryotic fungi possess subcellular organelles and cytoskeletal structures directing growth while prokaryotic streptomycetes have no such cellular organization. Despite these fundamental differences, both groups exhibit similar morphologies, growth patterns, growth forms, and hyphal and mycelial growth kinetics on solid media and in liquid culture both grow as dispersed mycelia and pellets. The article therefore discusses the relationship between cellular growth mechanisms and vegetative growth in both filamentous fungi and actinomycetes, the conceptual and theoretical models applicable to both groups, and the significance of such models in industrial fermentation processes.     

Acid phosphatase distribution and localization in the fungus Humicola lutea

Acid phosphatase activities in a culture liquid and mycelial extract were studied in submerged cultures of the filamentous fungus Humicola lutea 120-5 in casein-containingmedia with and without inorganic phosphate (Pi). The Pi-repressible influence on the phosphatase formation was demonstrated. Significant changes in the distribution of acid phosphatase between the mycelial extract and culture liquid were observed at the transition of the strain from exponential to stationary phase. Some differences in the cytochemical localization of phosphatase in dependence of Pi in the media and the role of the enzyme in the release of available phosphorus from the phosphoprotein casein for fungal growth were discussed.     

Studies on the ɛ-Lysine Acylase

Since Achromobacter pestifer EA isolated from soils shows markedly high ?-Iysine acylase activity compared with those of the other microorganisms ever tested, cultural conditions for the production of this enzyme were investigated.

As a result, it was confirmed that simple medium containing 1% peptone, 5% glucose and some inorganic salts is most suitable for the enzyme production and that much more ?-Iysine acylase is produced by shaken culture or submerged culture in jar fermentor than by stationary culture. α-Amino acylase activity in this organism was also studied.     

A Universally Applicable and Rapid Method for Measuring the Growth of Streptomyces and Other Filamentous Microorganisms by Methylene Blue Adsorption-Desorption

Quantitative assessment of growth of filamentous microorganisms, such as streptomycetes, is generally restricted to determination of dry weight. Here, we describe a straightforward methylene blue-based sorption assay to monitor microbial growth quantitatively, simply, and rapidly. The assay is equally applicable to unicellular and filamentous bacterial and eukaryotic microorganisms.     

Microparticle-enhanced cultivation of filamentous microorganisms: increased chloroperoxidase formation by Caldariomyces fumago as an example

Microparticle-enhanced cultivation (MPEC) was applied as a novel method for improved biomass and product formation during cultivation of filamentous microorganisms. Exemplarily, chloroperoxidase (CPO) formation by Caldariomyces fumago was analyzed in the presence and absence of microparticles of different size. Particles of approximately 500 microm in diameter had no effect on growth morphology or productivity of CPO formation by C. fumago. In contrast particles of < or =42 microm in diameter led to the dispersion of the C. fumago mycelia up to the level of single hyphae. Under these conditions the maximum specific productivity of CPO formation was enhanced about fivefold and an accumulated CPO activity in the culture supernatant of more than 1,000 U mL(-1) was achieved after 10-12 days of cultivation. In addition, the novel cultivation method also showed a positive effect on growth characteristics of other filamentous microorganisms proven by the stimulation of single hyphae/cell formation.     

Enhancement of monascus pigment production by the culture of Monascus sp. J101 at low temperature

In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 degrees C. In a batch culture at 30 degrees C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 degrees C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 degrees C was 10 times greater than at 30 degrees C.     

Biofilm formation and chemostat dynamics: Pure and mixed culture considerations

Time-dependent biofilm formation effects on continuous fermenter operation are modelled here in general for a mixed culture of N different microorganisms growing on a single substrate. Dynamic computer solutions are detailed for two versions of the general model: a pure culture and a simple two-cell mixed culture. Pure culture model predictions compare favorably with two pure culture experiments in the literature where significant biofilm formation was noted. A mixed culture of one microbe (C(1)) having a higher growth rate than a second microbe (C(2)) is simulated for two hypothetical scenarios of microbe C(2) having different magnitudes of cell deposition rate. Biofilm effects on the estimation of kinetic and stoichiometric parameters in both model versions, plus the impact of biofilms on mixed culture dynamics, are discussed.     

Growth Inhibition of Various Organisms by a Violet Pigment,Nostocine A,Produced by Nostoc spongiaeforme

A freshwater filamentous cyanobacterium, Nostoc spongiaeforme TISTR 8169, produced and excreted a violet pigment, named nostocine A, in the culture medium. Nostocine A inhibited the growth of some typical strains of microorganisms, algae, cultured plants, and established animal cell lines.     

Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter

The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.     

光照混菌培养应用研究进展

光合细菌与其他微生物在光照条件下混合培养是近年来的研究热点。综述了光照混菌培养的特点和目前光照混菌培养在水体净化、生物制氢和高价值物质生产方面的应用,并对影响混合菌株生长代谢与繁殖的因素做了总结。分析表明菌株之间存在的相互协同共生作用能促进微生物的生长繁殖,使底物被充分利用,提高物质产率。光照混菌培养工艺简单、成本较低,在水体净化、生物制氢、高价值物质生产方面的应用具有相当好的效果。在影响因素中对混合培养影响最大的因素是菌株接种量、接种比和培养基pH。在总结光照混菌培养应用现存不足的基础上,对其发展前景作出展望。     

替考游动放线菌发酵生产替考拉宁的动力学模型

根据丝状菌的生长机理和替考拉宁的发酵动力学特征提出了一个简化的形态学结构模型,并用于描述替考拉宁的发酵过程．模型将菌丝分为三种类型：具有生长活性的菌丝(G)、具有代谢活性的菌丝(N)和失活的菌丝(D)．菌丝G可因失去生长活性而转化为菌丝N,菌丝N可因失去代谢活性而转化为菌丝D．菌丝G与菌丝生长有关,而菌丝N则与替考拉宁的合成有关．该模型能较好地描述替考拉宁发酵过程、