Characterization of pellet morphology during submerged growth of Streptomyces tendae by image analysis

Many filamentous bacteria and fungi tend to form pellets, or mixtures of dispersed mycelium and pellets in liquid fermentation broths. In some cases, a specific kind of morphology is required for optimum product yield. When quantitative analysis and characterization of the pellet morphology are needed, an image processing system can be used. It allows a fast and reproducible analysis of the frequency distribution of pellet size, mean pellet size, contents of pellets, or their shape. The use of such a system allows for an on-line analysis. For a demonstration of the method, results of two fermentations of Streptomyces tendae are shown.     

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%.     

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.     

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron [German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 h Aspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.     

Adding talc microparticles to Aspergillus terreus ATCC 20542 preculture decreases fungal pellet size and improves lovastatin production

Changing fungal morphology with the use of morphological engineering techniques leads to improving the production of metabolites by filamentous fungi in the submerged culture. Adding mineral microparticles is one such simple method to change fungal pellet size. Here, it was studied for a lovastatin producer, Aspergillus terreus ATCC 20542. The experiments were conducted in shake flasks and 10 μm talc microparticles were added to the preculture. Intrapellet oxygen concentration profiles were determined by an oxygen microprobe. Talc microparticles caused a decrease of A. terreus pellets diameter from about 2000 to 900 μm, dependent on their concentration in the preculture. Smaller pellets produced more lovastatin, whose titre exceeded then 120 mg L^?1, utilising more lactose. The decrease in pellet size resulted in changes of oxygen concentration profiles in the pellets. The estimated critical pellet diameter, at which the non‐oxygenated zone was observed in the centre of the pellets, was 1700 μm. Smaller pellets were fully penetrated by oxygen. To conclude, facilitated diffusion of oxygen into the pellets of smaller diameter and their less dense structure made lactose utilisation by A. terreus more efficient, which ultimately increased lovastatin production in the runs with talc microparticles added, compared to the control runs.     

Hyphal vocuolation and fragmentation inpenicillium chrysogenum

A link between vacuolation and fragmentation of Penicillium chrysogenum mycelia in stirred tank submerged fermentations is reported. Quantitative information on vocuolation and morphology was obtained by image analysis. In fed-batch fermentations the coincidence of the events of rapid vacuolation and the fall of the mean total and main hyphal lengths suggests that hyphal fragmentation is not necessarily due to "shear" alone. The physiological state of the hyphae, characterized by the proportions of vaccuoles, was found to have a significant influence on the breakage of mycelial hyphae, It was found that the fragmentation was greater when the hyphae became heavily vacuolated following nutrient limitation in the culture, i.e., during the switch from the rapid growth to the production phase. (c) 1994 John Wiley & Sons, Inc.     

A current approach to the control of filamentous fungal growth in media: microparticle enhanced cultivation technique

Various strategies have been carried out to date in order to overcome the problem of the adverse effects of bulk fungal growth in bioreactors. Nevertheless, previous conventional methods such as modifying the cultivation temperature or pH resulted in limited biomass production and consequently lower yields. In recent years microparticle enhanced cultivation (MPEC) techniques are one of the most remarkable and novel methods employed for submerged fungal production to overcome bulk microbial growth. In addition to low cost advantages, MPEC also provides benefits such as not interfering with fungal metabolism, enhancing final product concentration and improving homogeneity in the fermentation broth. In this review, a comparison of conventional and novel methods to control fungal morphology has been discussed. Additionally, the application of microparticles in fungal fermentations, their benefits to the process in terms of fungal morphology, biomass accumulation, substrate consumption, and product formation also effect mechanisms of microparticle function are discussed in detail.     

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.     

Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3.4-22.5 g kg(-1) biomass) and the power-law model adequately described the rheological properties. In the cultivations there were pellets, clumps, and freely dispersed hyphal elements. The different morphological fractions were quantified using image analysis. The apparent viscosity of the fermentation broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations.     

Utilization of orange peel,a food industrial waste,in the production of exo-polygalacturonase by pellet forming <Emphasis Type="Italic">Aspergillus sojae</Emphasis>

The production of exo-polygalacturonase (exo-PG) from orange peel (OP), a food industrial waste, using Aspergillus sojae was studied in submerged culture. A simple, low-cost, industrially significant medium formulation, composed of only OP and (NH₄)₂SO₄ (AS) was developed. At an inoculum size of 2.8 × 10³ spores/mL, growth was in the form of pellets, which provided better mixing of the culture broth and higher exo-PG activity. These pellets were successfully used as an inoculum for bioreactors and 173.0 U/mL exo-PG was produced. Fed-batch cultivation further enhanced the exo-PG activity to 244.0 U/mL in 127.5 h. The final morphology in the form of pellets is significant to industrial fermentation easing the subsequent downstream processing. Furthermore, the low pH trend obtained during this fermentation serves an advantage to fungal fermentations prone to contamination problems. As a result, an economical exo-PG production process was defined utilizing a food industrial by-product and producing high amount of enzyme.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

Submerged citric acid fermentation: rheological properties ofAspergillus niger broth in a stirred tank reactor

Summary A large number of submerged citric acid fermentations in a beet molasses substrate was studied. The development of Aspergillus niger from conidia to pellets was followed. Rheological characteristics of the fermentation broth including the pellets were determined. The results obtained confirm the fact that the non-Newtonian pseudoplastic behaviour of the fermentation broth was due to the presence of mycelial pellets. The most significant changes in rheological properties occurred during the period of maximal citric acid production and increase in biomass. Offprint requests to: M. Berovi     

Theoretical Characterization of Slurry Fermentation

This is an extensive study of the three classical fermentation systems (emerged, submerged and solid‐state fermentations), thus uncovering, a system that is an intrinsically concomitant overlap of submerged and solid‐state fermentations – slurry fermentation. It is a convenient method of fermentation with hardly any theoretical characterization. This paper details the specifics of this system and its potential fields of application.     

Enhanced enzyme production with the pelleted form of <Emphasis Type="Italic">D. squalens</Emphasis> in laboratory bioreactors using added natural lignin inducer

White-rot fungi are extensively used in various submerged biotechnology processes to produce ligninolytic enzymes. Transfer of the process from the laboratory to the industrial level requires optimization of the cultivation conditions on the laboratory scale. An interesting area of optimization is pellet growth since this morphological form solves problems such as the decreased oxygen concentration, limited heat, and nutrient transport, which usually occur in dispersed mycelium cultures. Many submerged fermentations with basidiomycetes in pellet form were done with Phanerochaete, Trametes, and Bjerkandera species, among others. In our study, another promising basidiomycete, D. squalens, was used for ligninolytic enzyme production. With the addition of wood particles (sawdust) as a natural inducer and optimization of mixing and aeration conditions in laboratory stirred tank (STR) and bubble column (BCR) reactors on pellet growth and morphology, the secretion of laccase and the manganese-dependent peroxidase into the medium was substantially enhanced. The maximum mean pellet radius was achieved after 10 days in the BCR (5.1 mm) where pellets were fluffy and 5 days in the STR (3.5 mm) where they were round and smooth. The maximum Lac activity (1,882 U l⁻¹) was obtained after 12 days in the STR, while maximum MnP activity (449.8 U l⁻¹) occurred after 18 days in the BCR. The pellet size and morphology depended on the agitation and aeration conditions and consequently influenced a particular enzyme synthesis. The enzyme activities were high and comparable with the activities found for other investigations in reactors with basidiomycetes in the form of pellets.     

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of approximately 1200 microm initial diameter were reduced to a final stable size of approximately 900 microm when the agitation intensity was >/=600 rpm (u(t)>/=2.03 ms(-1)). A stable pellet diameter of approximately 2500 microm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.     

Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles

The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 μm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 μm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 μm (control) to 300 μm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 μm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 μm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production.     

两种方法形成菌丝球固定2-氯酚降解菌的对比

【目的】系统研究吸附法和同时培养法对所形成混合菌丝球的外观形态、内部结构及其去除2-氯酚效果的影响。【方法】采用吸附法和同时培养法将可降解2-氯酚的光合细菌PSB-1D固定在黄孢原毛平革菌(Phanerochaete chrysosporium)DH-1发酵而成的菌丝球上,形成混合菌丝球。以单一菌丝球为对照,利用光学显微镜、扫描电镜等仪器观察混合菌丝球的外观形态和内部结构,考察2种方法对混合菌丝球成球效果的影响;以无菌培养液为空白对照,考察游离光合细菌、单一菌丝球、2种方法形成混合菌丝球对2-氯酚的降解效能。【结果】在吸附法形成的混合菌丝球上,光合细菌主要集中在过渡区;而同时培养法将光合细菌牢固地包埋在菌丝球内核区,并大量簇状附着生长在菌丝交联的空隙处和每根菌丝上。在接种等量孢子和光合细菌的前提下,同时培养法较吸附法操作时间更短,成球数量更多,形成菌丝球干湿比更大,单位菌丝干重上固定的细菌数量更多。菌丝球降解体系和游离光合细菌对2-氯酚的降解均符合一级动力学特征。同时培养法形成的混合菌丝球降解效果最好,7 d内对初始浓度为50 mg/L的2-氯酚降解率可达89%以上,降解速率常数为0.3286 mg/(L·d),2-氯酚半衰期t1/2为2.8 d。【结论】首次报道黄孢原毛平革菌包埋固定化光合细菌形成混合菌丝球。该研究为生物质固定化材料的实际应用提供理论依据。     

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied—one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.     

Viability testing and characterization of germination of fungal spores by automatic image analysis

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. (c) 1993 John Wiley & Sons, Inc.     

Morphological and rheological properties of the three different species of basidiomycetes Phellinus in submerged cultures

AIMS: The objective of this work was to investigate the morphological and rheological properties in submerged culture of the three different basidiomycetes Phellinus (P. baumii, P. gilvus and P. linteus) that produce pharmacologically important exopolysaccharides (EPS). METHODS AND RESULTS: In flask cultures, pH proved to be a critical factor affecting mycelial growth, morphological change and EPS production. The macroscopic morphologies observed under different pHs in flask cultures were also comparable: i.e. starfish-like pellets with a lesser extent of free mycelium appeared in P. baumii, whereas smooth pellets with higher amounts of free mycelium were observed in P. gilvus and P. linteus. The pelleted fermentations were further characterized in a 5-l stirred-tank fermenter by image analysis with respect to mean diameter, core area and pellet circularity. Phellinus baumii showed the largest pellet size (5.2 mm in diameter), whereas P. linteus had extremely small and spherical pellets. The culture broth of P. baumii and P. gilvus yielded extremely high apparent viscosities, ranging from 5 to 7 Pa s. CONCLUSIONS: Three important species of Phellinus showed significantly different morphological and rheological properties. The morphological variation of the three Phellinus species was closely linked to EPS productivity and the apparent viscosity of the whole broth. SIGNIFICANCE AND IMPACT OF THE STUDY: The morphological change in the three species of Phellinus was a good indicator for identifying cell activity for EPS production. Our finding may be beneficial for further optimization of other fungal fermentation processes for large-scale production of EPS.