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.     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

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.     

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。     

Cultivation of E. coli in single- and ten-stage tower-loop reactors

E. Coli was cultivated in batch and continuous operations in the presence of an antifoam agent in stirred-tank and in single- and ten-stage airlift tower reactors with an outer loop. The maximum specific growth rate, mu(m), the substrate yield coefficient, Y(x/s), the respiratory quotient, RQ, substrate conversion, U(s), the volumetric mass transfer coefficient, K(L)a, the specific interfacial area, a, and the specific power input, P/V(L), were measured and compared. If a medium is used with a concentration of complex substrates (extracts) 2.5 times higher than that of glucose, a spectrum of C sources is available and cell regulation influences reactor performance. Both mu(m) and Y(X/S), which were evaluated in batch reactors, cannot be used for continuous reactors or, when measured in stirred-tank reactors, cannot be employed for tower-loop reactors: mu(m) is higher in the stirred-tank batch than in the tower-loop batch reactor, mu(m) and Y(x/s) are higher in the continuous reactor than in the batch single-stage tower-loop reactor. The performance of the single-stage is better than that of the ten-stage reactor due to the inefficient trays employed. A reduction of the medium recirculation rate reduces OTR, U(s), Pr, and Y(X/S) and causes cell sedimentation and flocculation. The volumetric mass transfer coefficient is reduced with increasing cultivation time; the Sauter bubble diameter, d(s), remains constant and does not depend on operational conditions. An increase in the medium recirculation rate reduces k(L)a. The specific power input, P/V(L), for the single-stage tower loop is much lower with the same k(L)a value than for a stirred tank. The relationship k(L)a vs. P/V(L) evaluated for model media in stirred tanks, can also be used for cultivations in these reactors.     

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.     

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.     

Verbesserung alter biotechnologischer Verfahren

By the example of the Penicillin G production, it is shown, how the production process can be improved by using pellet suspension instead of highly viscous filamentous mycel. In order to optimize the pellet geometry, dissolved oxygen concentration profiles were measured, and the overall mass transfer resistance was calculated in the pellets; histological investigations were carried out with pellets also. All measurements indicated that the optimal pellet diameter is 400 μm. When using this pellet, the specific power input was reduced from 4–5 kW/m³ to 0.8 kW/m³. The losses during the recovery of Penicillin G can be reduced below 1% by using reactive extraction. The thermodynamics, kinetics and reaction technique of reactive extraction of Penicillin G are also taken into account.     

Use of the chemiluminescent probe lucigenin to monitor the production of the superoxide anion radical in a recombinant Aspergillus niger (B1-D)

Direct detection of intracellular superoxide anion radical (O(2)(.-)) production is of critical importance for investigating the responses of filamentous fungi to oxidative stress in bioprocesses. The purpose of this study is to establish a reliable method to monitor the O(2)(.-) production within pellets of Aspergillus niger. Addition of pure oxygen and the redox cycling agent paraquat to fungal pellet suspensions resulted in a considerable increase in lucigenin-derived chemiluminescence (LDCL). In the presence of exogenous superoxide dismutase (SOD), the LDCL of a disrupted cell solution was inhibited. In contrast, with addition of diethyldithiocarbamate and sodium azide, respectively, the inhibitors of Cu, Zn-SOD and Mn-SOD, an increased LDCL was observed. Further, as a probe, lucigenin can be absorbed and accumulated in fungal pellet within a few minutes. Various pretreatments of the bioreactor sample for the measurement of LDCL, were also investigated in the present study, and the use of intact pellets was adopted here rather than disrupting cells because the latter treatment led to difficulties in LDCL measurement. These results show that lucigenin may be used as a convenient chemiluminescent probe to monitor intracellular production of O(2)(.-) in filamentous fungi, and thus to follow changes in the level of this stressor within fungi Copyright 2001 John Wiley & Sons, Inc.     

Nikkomycin production in pellets of Streptomyces tendae.

In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

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.     

Nikkomycin production in pellets of Streptomyces tendae

S.E. VECHT-LIFSHITZ, Y. SASSON AND S. BRAUN. 1992. In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw

ABSTRACT: BACKGROUND: Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost. RESULTS: The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm. CONCLUSION: This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.     

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.     

The influence of morphology on geldanamycin production in submerged fermentations of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The diverse morphology of the filamentous organism Streptomyces hygroscopicus var. geldanus was characterised by image analysis under various environmental conditions. In the presence of surfactant compounds, a significant decrease in the mean pellet diameter was observed. Cell aggregation was also influenced by spore inoculum level, with high concentrations reducing pellet size. In addition, the dispersion of pellets was found to increase with the inclusion of glass beads to submerged shake-flask cultures. In all cases, production of the secondary metabolite geldanamycin was determined to be dependent on the morphological profile of the organism, with a concomitant increase of 88% in geldanamycin yield observed as the mean pellet diameter was reduced by 70%. Thus, to maximise the yield of geldanamycin, it is necessary to limit pellet formation in Streptomyces hygroscopicus var. geldanus to an appropriate size.     

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

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.     

A novel multi-stage preculture strategy of Rhizopus oryzae ME-F12 for fumaric acid production in a stirred-tank reactor

This study has developed a novel multi-stage preculture strategy to control the morphology of filamentous Rhizopus oryzae in a 3.3-l stirred-tank reactor (STR), consequently enhancing the fumaric acid production. Using multi-stage precultures as inoculum, the morphology of R. oryzae was maintained as large clumps, small fluffy pellets, and entangled filaments. The highest fumaric acid production of 42.5 g/l with a yield of 55.6% was obtained in association with the small fluffy pellets which were formed in the third preculture at pH 3.0. The results indicated that the use of the multi-stage precultures as inoculum in the STR is a promising approach for commercial production of fumaric acid.     

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%.