Article

Application of Scale-Up Criterion of Constant Oxygen Mass Transfer Coefficient (k(L)a) for Production of Itaconic Acid in a 50 L Pilot-Scale Fermentor by Fungal Cells of Aspergillus terreus

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Scale-up criterion of constant oxygen mass transfer coefficient(kLa) was applied for the production of itaconic acid(IA) in a 50L pilot-scale fermentor by the fungal cells of Aspergillus terreus. Various operating conditions were examined to collect as many kLa data as possible by adjusting stirring speed and aeration rate in both 5L and 50L fermentor systems. In the fermentations performed with the 5L fermentor, the highest IA production was obtained under the operating conditions of 200rpm and 1.5vvm. Accordingly, we intended to find out parallel agitation and aeration rates in the 50L fermentor system, under which the kLa value measured was almost identical to that (0.02 sec(-1)) of the 5L system. The conditions of 180rpm and 0.5vvm in the 50L system turned out to be optimal for providing almost the same volumetric amount of dissolved oxygen (DO) into the fermentor, without causing shear damages to the producing cells due to excessive agitation. Practically identical fermentation physiologies were observed in both fermentations performed under those respective operating conditions, as demonstrated by nearly the same values of volumetric(Qp) and specific(qp) IA production rates, IA production yield(Yp/s) and specific growth rate(μ). Especially, the negligible difference of the specific growth rate(μ) between the two cultures (i.e., 0.029 hr(-1) vs. 0.031 hr(-1)) was notable, considering the fact that μ normally has a significant influence on qp in the biosynthesis of secondary metabolite such as itaconic acid.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... There are several scale-up strategies; one efficient strategy is applying the scale-up criterion as the volumetric power input (P/VL), mixing time, impeller tip speed or constant volumetric mass transfer coefficient (k L a) in different size fermenters [21][22][23]. In the case of aerobic fermentation, maintaining constant oxygen transfer or k L a is a good choice [16]. ...
... k L a is a standard parameter for the characterization of mass transport by correlating the mass transfer rate with the concentration change [24]; this is the most important parameter for the design and operation of mixing/sparging [25]. As supplying adequate oxygen (gas-liquid mass transport) is a significant factor in aerobic cultures, maintaining a similar k L a has been frequently employed as the basis in the scaling-up process; besides that, the scale-up basis with the k L a criterion is commonly used in around of 30% of the fermentation industry [23,26,27]. The aim of this work was to demonstrate an efficient use of the k L a criterion in the scale-up process through three different sizes of bioreactor systems for the production of PRP by Hib. ...
... The linear profile of k L a obtained using the 1.5 L bioreactor was similar to the one obtained by Averkina et al. [34] under a stirrer range of 50-300 rpm. Therefore, there is a direct influence of agitation on gas; it is dispersed, caused by the impeller with a varying efficiency for breaking bubbles and produces the increase in the gas liquid interface area and residence time into the medium culture [26,33,35], while the k L a profile obtained in 15 L and 80 L was similar to that reported by Shin et al. [23] under evaluation of a different stirrer and aeration rate. ...
Article
Full-text available
Polyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b (Hib) is an active immunizing molecule used in the production of the vaccine against H. influenzae, and industrial production could contribute to satisfying a world demand especially in developing countries. In this sense, the aim of this study was to establish a scale-up process using the constant oxygen mass transfer coefficient (kLa) such as the criterion for production of PRP in three different sizes of bioreactor systems. Three different kLa values (24, 52 and 80 h−1) were evaluated in which the biological influence in a 1.5 L bioreactor and 52 h−1 was selected to scale-up the production process until a 75 L pilot-scale bioreactor was achieved. Finally, the fed-batch phase was started under a dissolved oxygen concentration (pO2) at 30% of the saturation in the 75 L bioreactor to avoid oxygen limitation; the performance of production presented high efficiency (9.0 g/L DCW-dry cell weight and 1.4 g/L PRP) in comparison with previous scale-up studies. The yields, productivity and kinetic behavior were similar in the three-size bioreactor systems in the batch mode indicating that kLa is possible to use for PRP production at large scales. This process operated under two stages and successfully produced DCW and PRP in the pilot scale and could be beneficial for future bioprocess operations that may lead to higher production and less operative cost.
... In various reports, it was found that the efficiency of the process-in terms of the technologically most important IA yield and productivitycould be affected by the conditions provided in the fermenter unit. Actually, the variables that have been confirmed to take strong influence on bioreactor performance during IA production cover the quality of the medium and type of substrate, temperature, pH, aeration and stirring (Mondala 2015;Hevekerl et al. 2014;Karaffa et al. 2015;Kolláth et al. 2019;Molnár et al. 2018;Shin et al. 2013). ...
... Aeration and mixing intensity are crucial factors for aerobic bioprocess optimization (Garcia-Ochoa and Gomez 2009). As the production of IA is obligatory aerobic, it requires the maintenance of sufficient oxygen supply in the whole working volume of the bioreactor, influenced by the oxygen mass transfer conditions (Shin et al. 2013). For instance, Molnár et al. (2018) proved recently that the dissolved oxygen concentration as high as 30% of saturation level notably determined the fermentation profile with A. terreus, particularly in terms of IA rate and yield. ...
... As in the aerobic bioreactor unit, the factor that takes both aeration and stirring conditions into account is the so-called K L a (oxygen mass transfer coefficient) (Garcia-Ochoa and Gomez 2009), the next study in our research sequence ought to deal with the examination of K L a in assisting IA production. For instance, Shin et al. (2013) documented the increment of K L a in response to more vigorous mixing (from 100 to 300 rpm) and larger air loading (0.5-1.5 L/L min) for pilot-scale IA fermenter with A. terreus. Nevertheless, it is to stress that simultaneous setting of aeration and mixing for favorable K L a mustn't threaten the growth of A. terreus since the appearance of extreme shearing forces may damage its filamentous network and deteriorate biosynthesis of IA consequently. ...
Article
Full-text available
The effects of the bioreactor conditions, in particular the mode and intensity of aeration and mixing were studied on itaconic acid (IA) fermentation efficiency by Aspergillus terreus strain from glucose substrate. IA was produced in batch system by systematically varying the oxygen content of the aeration gas (from 21 to 31.5 vol% O2) and the stirring rate (from 150 to 600 rpm). The data were analyzed kinetically to characterize the behavior of the process, and besides, the performances were evaluated comparatively with the literature. It turned out that the operation of the bioreactor with either the higher inlet O2 concentration (31.5 vol% O2) or faster stirring (600 rpm) could enhance biological IA generation the most, resulting in yield and volumetric productivity of 0.31 g IA/g glucose and 0.32 g IA/g glucose and 3.15 g IA/L day and 4.26 g IA/L day, respectively. Overall, the significance of fermentation settings was shown in this work regarding IA production catalyzed by A. terreus and notable advances could be realized by adjusting the aeration and stirring towards an optimal combination. Graphic abstract Open image in new window
... A plot of ln (1 2 C i /C*) against time can be used to estimate the k L a, while the final concentration can be used to estimate the maximum oxygen concentration and to set the probe at 100%. This technique is classical (Atkinson & Mavituna, 1992;Shin et al., 2013), but still important because (1) it accommodates specificities of the fermentation conditions, from bioreactor geometry and stirring rate to temperature and pressure, to culture media composition; and (2) because if k L a can be replicated in scaling-up, then the OTR can possibly be replicated too, leading to the desired, similar physiological conditions in both scales. ...
... These methods involve the determination of impeller speed and aeration rate, which rely on empirical correlations, and keeping one or more parameters constant (Badino et al., 2001;García-Ochoa & Gó mez, 2009;Mahdinia et al., 2019;Marques et al., 2010;Vasconcelos et al., 2000). The gas surface velocity, specific gas flow rate, or gas flow number are the criteria adopted to estimate the aeration rate (Shin et al., 2013;Shukla et al., 2001). ...
... The volumetric oxygen transfer coefficient (k L a) reflects the bioreactor's ability to transfer oxygen to microbial cultures. Oxygen transport is an essential parameter to be considered in the scale-up of aerobic processes, since it is needed in microbial metabolism (Shin et al., 2013) especially in processes that demand large amounts of oxygen, such as the production of antibiotics , itaconic acid (Magalhaes et al., 2019), citric acid (Mores et al., 2021), and yeast and bacterial fermentation for different biomolecules' production (Junker, 2004). Besides, oxygen can also act as a final electron receptor in the reoxidation of electron-carrying molecules in bioprocesses (García-Ochoa & Gó mez, 2009;Liu, 2017). ...
Chapter
The definition of bioprocesses’ parameters generally occurs at bench scale. However, the reproduction of these processes’ conditions at pilot and/or industrial scale is certainly a challenge. These conditions are for example aeration, which involves critical aspects such as dissolved oxygen and oxygen uptake rate, and agitation, which promotes shear stress and needs high energy consumption. It is important not to lose process efficiency and productivity, which impact on bioeconomy of biotechnological processes. So, some well-known scale-up strategies can be employed, combined or individually, to maintain bioprocesses’ performance. These strategies consider geometric similarity aspects of bioreactors, agitation and aeration conditions, and some rheological aspects of the fluid that must be considered and maintained at the new scale. Some operational conditions have a great influence on cell growth and on the biosynthesis of different biomolecules and should be reproduced at higher scales. Accordingly, one or more operating factors can be maintained constant during scale-up and some procedures can be employed, for example, to determine the power consumption of large-scale bioreactors or determine aeration conditions in an aerobic culture. Stirred tank reactors will be employed as models for scale-up. However, the scale-up of other bioreactors models such as bubble column reactors and solid-state bioreactors will also be described.
... When the impeller tip speed was 94.2 cm s −1 at a fixed aeration rate of 0.5 vvm, IA production was 3.6 and 1.6 times higher than those in the impeller speed of 62.8 and 125.7 cm s −1 , respectively. Shin et al. [81] applied the scale-up criterion of constant oxygen mass transfer coefficient (k L a) for production of IA from glucose in 5 L and 50 L scale fermenters by A. terreus cells. The highest IA production (0.71 g g −1 glucose) was obtained under operating conditions of 200 rpm and 1.5 vvm in a 5 L fermenter, and 180 rpm and 0.5 vvm in a 50 L fermenter. ...
Article
Recently, itaconic acid (IA), an unsaturated C5-dicarboxylic acid, has attracted much attention as a biobased building block chemical. It is produced industrially (>80 g L⁻¹) from glucose by fermentation with Aspergillus terreus. The titer is low compared with citric acid production (>200 g L⁻¹). This review summarizes the latest progress on enhancing the yield and productivity of IA production. IA biosynthesis involves the decarboxylation of the TCA cycle intermediate cis-aconitate through the action of cis-aconitate decarboxylase (CAD) enzyme encoded by the CadA gene in A. terreus. A number of recombinant microorganisms have been developed in an effort to overproduce it. IA is used as a monomer for production of superabsorbent polymer, resins, plastics, paints, and synthetic fibers. Its applications as a platform chemical are highlighted. It has a strong potential to replace petroleum-based methylacrylic acid in industry which will create a huge market for IA.
... This may be caused in a changed flow profile, energy input, or oxygen supply in the 15-L scale compared to the 1.5-L scale. All mentioned parameters influence the itaconic acid production with A. terreus (Park et al. 1993;Gyamerah 1995b;Riscaldati et al. 2000;Shin et al. 2013;Yahiro et al. 1997). The yield of itaconic acid decreased to 0.56 (w/w) in the 15-L scale as a result of a higher CDW. ...
Article
Full-text available
Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.
... According to Jin et al. (2004), the size of the bubbles in the medium depends primarily on such factors as the properties of the liquid phase and the gas velocity. Shin et al. (2013) used a scale-up criterion of constant oxygen transfer for the scale-up of itaconic acid production, and found that appropriate kLa values for the process were 0.03 s -1 to 0.05 s -1 . Their results are comparable to the kLa value found in the present study. ...
Article
Full-text available
The goal of this work was to scale up the simultaneous saccharification and fermentation (SSF) of lactic acid using microwave-alkali-pretreated empty fruit bunches (EFB) from a scale of 16 L to a scale of 150 L. To facilitate the scaling-up process of lactic acid production by Rhizopus oryzae NRRL 395, a scaling-up criterion of constant kLa value was applied. Operating conditions, such as aeration rate and superficial velocity, were varied and evaluated on both scales (16-L and 150-L). The highest lactic acid yield of 6.8 g/L was obtained under an operating condition of 1 vvm (0.061 s⁻¹). Parallel aeration rates were determined for the 150-L fermenter system to obtain the same kLa value as the 16-L fermenter. An operational condition of 0.5 vvm dissolved oxygen supply in the 150-L fermenter was optimal to support an identical value of kLa and production rate of lactic acid for both scales.
... Scaling-up is necessary for the microbial nematicide production process to be cost-effective for industrial development. Multiple strategies for scaling-up with constant oxygen transfer coefficient [28,29], mixing model [30], constant impeller tip speed [13,24], and minimization of impurity levels [31] have been developed. Of these scaleup criteria, constant impeller tip speed was used as a criterion in our study. ...
Article
Full-text available
Oxalic acid has potent nematicidal activity against the root-knot nematode Meloidogyne incognita. In this study, fermentation parameters for oxalic acid production in submerged culture of Aspergillus niger F22 at 23, 25, and 30 °C were optimized in 5-L jar fermenters. The viscosity of the culture broth increased with increasing temperature. There was a negative correlation between oxalic acid production and the apparent viscosity; high volumetric productivity of oxalic acid was obtained at low apparent viscosity (less than 1000 cP), with a productivity of more than 100 mg/L h. When the apparent viscosity was over 2500 cP, the volumetric productivity decreased below 50 mg/L h. In addition, the volumetric mass transfer coefficient, KLa, positively correlated with volumetric productivity. When the KLa value increased from 0.0 to 0.017 /s, the volumetric productivity proportionally increased up to 176 mg/L h. When the temperature decreased, KLa increased due to the decrease in viscosity, leading to increased volumetric productivity. The highest productivity of 7453.3 mg/L was obtained at the lowest temperature, i.e., 23 °C. The nematicidal activity of culture filtrate was proportional to the content of oxalic acid. Based on a constant impeller tip speed, oxalic acid production was successfully scaled up to a 500-L pilot vessel, producing a final concentration comparable to that in the 5-L jar.
... The configuration of each fermenter was as follows: 7-L fermenter; 2 impellers with 6 blades, ring typed sparger of 12 holes, bottom-driven, 160 mm of tank diameter, and 50-L fermenter; 3 impellers with 8 blades, ring typed sparger of 12 holes, top-driven, 310 mm of tank diameter. For determining volumetric oxygen mass transfer coefficient (k L a) in the 7-L and 50-L fermenter systems, a gas analyzer (Autolab-LK930A from Lokas, South Korea) was connected 58 . Fed-batch cultivation was conducted based on the initial feeding medium that was 7. times more concentrated than the SLP medium and phosphate was not added for the regulation of cell growth. ...
Article
Full-text available
Abstract Muconic acid (MA) is a valuable compound for adipic acid production, which is a precursor for the synthesis of various polymers such as plastics, coatings, and nylons. Although MA biosynthesis has been previously reported in several bacteria, the engineered strains were not satisfactory owing to low MA titers. Here, we generated an engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshikimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. To accumulate key intermediates in the MA biosynthetic pathway, aroE (shikimate dehydrogenase gene), pcaG/H (PCA dioxygenase alpha/beta subunit genes) and catB (chloromuconate cycloisomerase gene) were disrupted. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroE-pcaG/H-catB triple knock-out Corynebacterium strain. This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. These results show highest levels of MA production demonstrated in Corynebacterium, suggesting that the rational cell factory design of MA biosynthesis could be an alternative way to complement petrochemical-based chemical processes.
... Karaffa et al. (2015) provided evidence that the overproduction of itaconic acid requires a medium in which the Mn(II) concentration is in the same range as in citric acid fermentations. Interestingly, however, there are also reports on itaconic acid fermentations with high yields which do not attempt to remove metal ions (Shin et al. 2013. This issue therefore needs further investigation. ...
Article
Full-text available
Citric acid production by Aspergillus niger and itaconic acid production by Aspergillus terreus are two major examples of technical scale fungal fermentations based on metabolic overflow of primary metabolism. Both organic acids are formed by the same metabolic pathway, but whereas citric acid is the end product in A. niger, A. terreus performs two additional enzymatic steps leading to itaconic acid. Despite of this high similarity, the optimization of the production process and the mechanism and regulation of overflow of these two acids has mostly been investigated independently, thereby ignoring respective knowledge from the other. In this review, we will highlight where the similarities and the real differences of these two processes occur, which involves various aspects of medium composition, metabolic regulation and compartmentation, transcriptional regulation, and gene evolution. These comparative data may facilitate further investigations of citric acid and itaconic acid accumulation and may contribute to improvements in their industrial production. Electronic supplementary material The online version of this article (10.1007/s00253-018-09607-9) contains supplementary material, which is available to authorized users.
... Certainly, the properties of the actual starting material will influence the achievable itaconic acid formation efficiency [9][10][11], and besides that, process control via the maintenance of adequate environmental conditions will play a key role. As a matter of fact, ensuring suitable aeration, broth composition, mixing, temperature and pH are crucial criteria for the improved formation of itaconic acid by A. terreus [12][13][14][15]. ...
Article
Full-text available
Biological itaconic acid production can by catalyzed by Aspergillus terreus (a filamentous fungi) where the fermentation medium pH is of prominent importance. Therefore, in this work, we investigated what benefits the different pH regulation options might offer in enhancing the process. The batch itaconic acid fermentation data underwent a kinetic analysis and the pH control alternatives were ranked subsequently. It would appear that the pH-shift strategy (initial adjustment of pH to 3 and its maintenance at 2.5 after 48 h) resulted in the most attractive fermentation pattern and could hence be recommended to achieve itaconic acid production with an improved performance using A. terreus from carbohydrate, such as glucose. Under this condition, the itaconic acid titer potential, the maximal itaconic acid (titer) production rate, the length of lag-phase and itaconic acid yield were 87.32 g/L, 0.22 g/L/h, 56.04 h and 0.35 g/g glucose, respectively.
... It is also known that the microbial physiology of filamentous fungal cells is significantly influenced by the DO concentration in suspended cultures, and it has been suggested that the critical DO concentration for fungal cells in culture should be greater than around 20% of the saturation DO value [45,46]. In this study, no effort was made to maintain the DO above 20% in order to investigate the effect of DO on enzyme production by the fungus T. reesei MUM 97.53. ...
Article
Full-text available
Enzymatic hydrolysis accounts for 20% of the total cost in the conversion process of lignocellulosic biomass into bioethanol. Therefore, production of biomass-degrading enzymes by using lignocellulosic residue as a fermentation substrate may be an alternative to decrease the production costs. In this study, corncob (CC) has been pretreated by liquid hot water (LHW) at 200 °C for 30 min and used as inducer source for production of biomass-degrading enzymes by Trichoderma reesei MUM 97.53. The pretreatment was used to increase the cellulose content and the accessibility to lignocellulosic material. Although the filamentous fungus secreted a broad range of cellulolytic and hemicellulolytic enzymes when grown on untreated CC, higher enzyme productions were obtained when cultured on LHW-pretreated CC in a 2-L stirred tank bioreactor (STB). Besides, the effects of aeration (2 and 4 vvm) and agitation (150 and 250 rpm) rates on enzyme production were studied by submerged fermentation in a batch STB and correlated with the volumetric oxygen transfer coefficient (kLa). Maximal cellulase, xylanase, and β-xylosidase productions were found at 150 rpm and 4 vvm, while the highest β-glucosidase levels were obtained at 150 rpm and 2 vvm, that corresponded to kLa values of 32.50 h−1 and 16.41 h−1, respectively. At higher agitation, a lower enzymatic production was observed probably due to the high shear stress in the fungal hyphae.
... By subsequent transportation outside the cell, IA can be recovered from the broth by technologies such as membrane filtration and electrodialysis [12][13][14][15]. Although A. terreus has been proven as the most promising candidate for IA production, the actual bioprocess performance is highly-dependent on the environmental settings, in terms of substrate concentration, pH, temperature, media composition with respect to N-sources as well as the presence/absence of certain minerals, dissolved oxygen levels, stirring rate, etc. [16][17][18][19]. On that matter, to track how efficiently the biosynthesis proceeds under given bioreactor operating conditions, the most essential information can be acquired from the fermentation kinetics with special interest on substrate consumption, IA (as product) formation and biomass growth. ...
Article
Full-text available
In this work, itaconic acid (IA) was produced biotechnologically by Aspergillus terreus fungal strain from glucose. The performance of the batch fermentation was kinetically assessed and the maximal IA production potential, maximal production rate and lag-phase time were determined as 28.1 g/L, 3.83 g/L day and 1.52 days, respectively. In addition, the bioprocess was evaluated based on the most frequently used parameters, in particular IA titer (26.3 g/L), yield (0.22 g/g substrate) and productivity (0.1 g/L h), which were comparable to the already published literature. Furthermore, an on-line monitoring system was installed to the fermenter in order to measure the CO2 content of the bioreactor of-gas. Actually, it was indicated by the results that the CO2 production could have a linear-like relationship with the quantity of fungal biomass. Hence, the data collected in such a way may have the potential to establish an alternative methodology for the monitoring of biomass growth in the course of the biological transformation taking place.
... By controlling the pH at 3.4 after the itaconate initiating phase, product titers up to 160 g L −1 could be achieved [2]. Further productivity could be increased by media optimization and pHshift experiment to 1.15 g L −1 h −1 [16] and the highest reported yield with 0.72 g ITA g −1 GLC was reached by optimizing oxygen transfer [17]. Following submerged fermentation with A. terreus, the itaconic acid is typically purified by repeated crystallization in industrial settings [18]. ...
Article
Full-text available
Background: Ustilago cynodontis ranks among the relatively unknown itaconate production organisms. In comparison to the well-known and established organisms like Aspergillus terreus and Ustilago maydis, genetic engineering and first optimizations for itaconate production were only recently developed for U. cynodontis, enabling metabolic and morphological engineering of this acid-tolerant organism for efficient itaconate production. These engineered strains were so far mostly characterized in small scale shaken cultures. Results: In pH-controlled fed-batch experiments an optimum pH of 3.6 could be determined for itaconate production in the morphology-engineered U. cynodontis Δfuz7. With U. cynodontis ∆fuz7r ∆cyp3r PetefmttA Pria1ria1, optimized for itaconate production through the deletion of an itaconate oxidase and overexpression of rate-limiting production steps, titers up to 82.9 ± 0.8 g L-1 were reached in a high-density pulsed fed-batch fermentation at this pH. The use of a constant glucose feed controlled by in-line glucose analysis increased the yield in the production phase to 0.61 gITA gGLC-1, which is 84% of the maximum theoretical pathway yield. Productivity could be improved to a maximum of 1.44 g L-1 h-1 and cell recycling was achieved by repeated-batch application. Conclusions: Here, we characterize engineered U. cynodontis strains in controlled bioreactors and optimize the fermentation process for itaconate production. The results obtained are discussed in a biotechnological context and show the great potential of U. cynodontis as an itaconate producing host.
... Similar to the findings with citric acid fermentation [6], lowering the concentration of inorganic phosphate alleviated the inhibitory effect of Mn 2+ on itaconic acid production [58]. Interestingly, however, there are also reports on itaconic acid fermentations with high yields which do not attempt to remove metal ions [59,60]. This issue therefore needs further investigation. ...
Article
Full-text available
Organic acid accumulation is probably the best-known example of primary metabolic overflow. Both bacteria and fungi are capable of producing various organic acids in large amounts under certain conditions, but in terms of productivity-and consequently, of commercial importance-fungal platforms are unparalleled. For high product yield, chemical composition of the growth medium is crucial in providing the necessary conditions, of which the concentrations of four of the first-row transition metal elements, manganese (Mn2+), iron (Fe2+), copper (Cu2+) and zinc (Zn2+) stand out. In this paper we critically review the biological roles of these ions, the possible biochemical and physiological consequences of their influence on the accumulation of the most important mono-, di- and tricarboxylic as well as sugar acids by fungi, and the metal ion-related aspects of submerged organic acid fermentations, including the necessary instrumental analytics. Since producing conditions are associated with a cell physiology that differs strongly to what is observed under “standard” growth conditions, here we consider papers and patents only in which organic acid accumulation levels achieved at least 60% of the theoretical maximum yield, and the actual trace metal ion concentrations were verified.
... The aeration of the growing microorganism must be carried out equally in the entire work volume of the bioreactor and needs to be enough for its maintenance. Another benefit of a good system of aeration and agitation is to diminish the size of mycelial aggregates, making the access to cells easier to oxygen [28][29][30][31]. The online bioreactor used provides a good system of aeration and agitation. ...
Chapter
The global organic acid market is expected to reach USD 36.86 Billion in 2026. This study aimed to produce citric, itaconic, and gluconic acids by using a biotechnological process in a lightly stirred bioreactor with fungus from the genus Aspergillus spp. These fermentations processes provided good yields, 22.6%, 99.9%, and 24.8% (48 h) for citric, gluconic, and itaconic acids, respectively.
... The aeration of the growing microorganism must be carried out equally in the entire work volume of the bioreactor and needs to be enough for its maintenance. Another benefit of a good system of aeration and agitation is to diminish the size of mycelial aggregates, making the access to cells easier to oxygen [28][29][30][31]. The online bioreactor used provides a good system of aeration and agitation. ...
... A higher IA titre was achieved for the later conditions (48.5 g⋅L − 1 , compared with 30.5 g⋅L − 1 ), which was linked to the lower extent of damage to mycelia at a fixed agitation of 300 rpm, explaining why most of the studies (cited in Table 3) using A. terreus have used agitation varying from 300 to 400 rpm. Shin et al. [84], in 2013, scaled-up IA production from 1.5 to 50 L, keeping the kLa at 0.02 s − 1 and reported the highest yield until thus far in an STR (0.72 g IA /g glucose ). This value is remarkably high and near the maximum theoretical yield, productivities, and titres for different A. terreus strains (productivities: 0.32-0.8 ...
Article
Full-text available
The discovery of itaconic acid as a product of citric acid pyrolytic distillation in 1837 opened the possibility of using it as a polymer building block. Itaconic acid, featuring two carboxylic acids and an unsaturated group, can potentially be used as a building block in several chemical syntheses, with a particular emphasis on polymer manufacture. The elucidation of biochemical pathways originating from itaconic acid, first in Aspergillus terreus and, recently, in several species of the Ustilago genus, has intensified and diversified research focused on microbe-based itaconic acid production, including at an industrial scale. These efforts include the engineering of naturally producing species/strains along with the exploration of other species that do not naturally produce itaconic acid but may offer potential benefits. The use of renewable wastes or sugar-enriched residues as substrates to produce itaconic acid, from a circular bioeconomy perspective, is another important aspect of the advancements in microbial itaconic acid production. This review provides an overview of the achievements as well as the challenges concerning the engineering of the producing strains/species, substrate selection, optimisation of bioreactor operation, and downstream itaconic acid purification methods.
Article
Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98,5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its biosynthesis.
Article
The 11α-hydroxylation of canrenone can be catalyzed by Aspergillus ochraceus in bioreactors, where the geometry of the impeller greatly influences the biotransformation. In this study, the effects of the blade number and impeller diameter of a Rushton turbine on the 11α-hydroxylation of canrenone were considered. The results of fermentation experiments showed that 3.40% and 11.43% increases in the conversion ratio were achieved by increasing the blade number and impeller diameter, respectively, when mixed with a 50 mm four-blade impeller. However, with an impeller diameter of 60 mm, the conversion ratio with a six-blade impeller was 14.42% lower than that with a four-blade impeller. Data from cold model experiments with a large-diameter six-blade impeller equipped indicated that the serious leakage of inclusions and a 22.08% enzyme activity retention led to a low conversion ratio. Numerical simulations suggested that there was good gas distribution and high fluid flow velocity when the fluid was stirred by large-diameter impellers, resulting in a high dissolved oxygen content and good bulk circulation, which positively affected hyphal growth and metabolism. However, a large-diameter six-blade impeller created overly high shear compared to a large-diameter four-blade impeller, thereby decreasing the conversion ratio. The average shear rates of the former and latter cases were 43.25 s-1 and 35.31 s-1, respectively. It was concluded that appropriate shear should be provided in the 11α-hydroxylation of canrenone. Overall, this study provides basic data for the scaled-up production of 11α-hydroxycanrenone.
Article
Present study used Aspergillus terreus strain C1 isolated from mangrove soil for itaconic acid (IA) production from potato starch waste. Fermentation parameters were optimized by classical one factor approach and statistical experimental designs, such as Plackett‐Burman and response surface designs. Anionic deionization of potato waste was found to be a very effective, economic and easy way of improving IA production. The increase in IA production by deionization was found to correlate with removal of phosphate. In our knowledge, this is the first report on application of deionization of potato waste to enhance IA production. Other parameters like inoculum development conditions, pH, presence of peptone and certain salts in the medium also significantly affected IA production. IA production by strain C1 increased 143‐fold during optimization when compared with the starting condition. The optimized IA level (35.75 g/L) was very close to the maximum production predicted by RSM (38.88 g/L). Bench scale production of IA was optimized in 3‐L stirred tank reactor by varying parameters like agitation and aeration rate. The maximum IA production of 29.69 g/L was obtained under the agitation speed of 200 rpm and aeration rate of 0.25 vvm. To the best of our knowledge, it is the first report on IA production from potato starch waste at bioreactor level. This article is protected by copyright. All rights reserved.
Article
Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4Mb and 32 variants, 0.6Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration.
Article
Full-text available
Itaconic acid is one of the basic chemicals for the polymer industry, which can be produced on the basis of renewable raw materials. Since the middle of the twentieth century, itaconic acid has been produced industrially using the filamentous fungus Aspergillus terreus. But the demand for the organic acid is low due to the high production costs compared to alternative petrochemical manufactured raw materials. The high production costs are based on a low final titer, low productivities, and the usage of pure sugars, purified molasses, or starch hydrolysates, since the fungus reacts very sensitively to impurities in a culture medium. This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of itaconic acid. The technological achievements in the biotechnological production of itaconic acid are presented. Particular attention is paid to current achievements in terms of suitable alternative substrates and their applicability in fermentation processes. Also, the pathway of itaconic acid and especially the influences on the fermentation process, which must be known in order to achieve a high final titer of itaconic acid, a yield close to the theoretical yield, and high productivity.
Article
Fucoxanthin is one of the most vital pigments during photosynthesis and is extracted from golden-brown micro-algae such as Tisochrysis lutea. The present study investigates the constant volumetric mass transfer coefficient (kLa), for the first time as the scale-up strategy to change the scale from 500 mL to 2-L cultivation flasks, and 5-L bubble column photobioreactor for fucoxanthin production in T. lutea. The cell density and fucoxanthin content were improved because of through fine aeration, nutrients, and light availability by successful laboratory scale up. Fucoxanthin productivity obtained 21.20, 22.99, and 24.96 mg L–1day–1 for 500 mL, 2-L bottle, and 5-L bubble column photobioreactor, respectively. In addition, the biomass productivity enhanced from 267.5 to 275 and 284 mg L–1day–1 in three mentioned scales, respectively. Eventually, the scale up process for the production of fucoxanthin was succeeded from 500 mL bottle to 5-L photobioreactor using constant (kLa) under laboratory conditions.
Article
Full-text available
An appropriately available correlation through a series of formulas derivation and reasonable simplifications was established using some hypotheses. Then a wide range of culture conditions such as gas flow rate, stirrer speed and volume of the liquid in the vessel were correlated with volumetric oxygen mass transfer coefficient and the correlation is determined as: k La=KQxNyVL-y/3.15. The correlation factors K, x, y for the 30 l bioreactors calculated by sodium sulfite oxidation method were 0.004, 0.525 and 1.685 respectively. The correlation proposed here could guide the scale-up of fermentation to obtain an optimized initial volume of fermentation broth from lab-scale to a new higher scale and could inspect intuitively to the relationship between kLa and the other three parameters.
Article
Full-text available
Lovastatin, a secondary metabolite, was produced by fermentation process usingAspergillus terreus in an internal loop airlift reactor. It is a highly aerobic fermentation process. Biomass concentration and cell morphology were evaluated and observed to contribute significantly to the high viscosity and pseudoplastic non-Newtonian behavior of the broth. Typical morphological changes over 10 days in the fermentation broth were studied. The viscosity increased from the start of the fermentation with an increasing cell mass content, reached to a maximum of 60 N/m2·s at 160 h and then declined after the branching of the hyphae with the formation of arthrospores. Rheological parameters like consistency index and fluidity index were evaluated. The consistency index was observed to increase from 9.8 to 66.85 N/m2, while fluidity index decreased from 0.69 to 0.48 s−1 during 10 days of lovastatin production. A correlation between growth and consistency index of the broth has been evaluated.
Article
Full-text available
Experimental data for the on-line estimation of cell concentration and growth rate are presented. For this purpose, we utilized the on-line calculation of the oxygen uptake rate (OUR), which was derived from a liquid phase dynamic mass balance for the oxygen during the active growth phase in cyclosporin A (CyA) fermentation. The cell yield coefficient, based on the oxygen (Yx/o) for both suspended and immobilized cells of Tolypocladium inflatum, was estimated as 1.9 g DCW/g O2 from a very good linear correlation between the cell mass produced and the total oxygen consumed. The calculated yield showed a good agreement with the value of Yx/o generated from the correlation between the cell growth rate and the oxygen uptake rate. In addition, further experimental data are given, which were also applied to determine the specific oxygen uptake rate of T. inflatum cells during the exponential phase of CyA fermentation. A theoretical basis for the analysis of these fermentation parameters is also provided.
Article
Full-text available
Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.
Article
Full-text available
More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields--dental, ophthalmic, and drug delivery--to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.
Article
Full-text available
Some of the Aspergilli are reputed for their versatile and efficient catabolism of soluble carbon sources and related metabolites as well as raw polymeric materials. Here, we present a detailed investigation of the genomic and evolutionary basis for this versatility, using seven Aspergillus and one Neosartorya genome sequences. We manually annotated about 155 genes per genome covering glycolysis, the pentose phosphate cycle, alternative routes of D-glucose metabolism, catabolism of D-galactose and pentoses, and the TCA cycle, as well as the utilization of acetate and ethanol, propionate metabolism, and gluconeogenesis.The annotation reveals that the Aspergilli have re-enforced several areas of their primary metabolism(notably glycolysis, TCA cycle, ethanol utilization, and pentose and polyol metabolism) by gene duplications,horizontal gene transfer or gene clustering. Results from the phylogenetic analysis of several enzymes encoded by duplicated genes also suggests that some gene products may have acquired new(physiological) functions, that render primary carbon metabolism of the Aspergilli more complex than previously thought.
Article
Full-text available
The kinetics of cell growth and Cyclosporin A (Cyc A) production by Tolypocladium inflatum were studied in shake flasks and bioreactors under controlled and uncontrolled pH conditions. In the case of the shake flask, the production time was extended to 226 h and the maximal antibiotic concentration was 76 mg/l. When scaling up the cultivation process to a bioreactor level, the production time was reduced to only 70 h with a significant increase in both the cell growth and the antibiotic production. The maximal dry cell weights in the case of the controlled pH and uncontrolled pH cultures in the bioreactor were 22.4 g/l and 14.2 g/l, respectively. The corresponding maximal dry cell weight values did not exceed 7.25 g/l with the shake flask cultures. The maximal values for Cyc A production were 144.72 and 131.4 mg/l for the controlled and uncontrolled pH cultures, respectively. It is also worth noting that a significant reduction was observed in both the dry cell mass and the antibiotic concentration after the Cyc A production phase, whereas the highest rate of antibiotic degradation was observed in the stirred tank bioreactor with an uncontrolled pH. Morphological characterization of the micromorphological cell growth (mycelial/pellet forms) was also performed during cultivation in the bioreactor.
Article
In order to investigate the effect of dissolved oxygen (DO) level on AVM B1a production by a high yielding mutant of Streptomyces avermitilis, five sets of bioreactor cultures were performed under variously controlled DO levels. Using an online computer control system, the agitation speed and aeration rate were automatically controlled in an adaptive manner, responding timely to the oxygen requirement of the producer microorganism. In the two cultures of DO limitation, the onset of AVM B1a biosynthesis was observed to casually coincide with the fermentation time when oxygen-limited conditions were overcome by the producing microorganism. In contrast, this phenomenon did not occur in the parallel fermentations with DO levels controlled at around 30% and 40% throughout the entire fermentation period, showing an almost growth-associated mode of AVM B1a production: AVM B1a biosynthesis under the environments of high DO levels started much earlier than the corresponding oxygen-limited cultures, leading to a significant enhancement of AVM B1a production during the exponential stage. Consequently, approximately 6-fold and 9-fold increases in the final AVM B1a production were obtained in 30% and 40% DO-controlled fermentations, respectively, especially when compared with the culture of severe DO limitation (the culture with 0% DO level during the exponential phase). The production yield (Yp/x), volumetric production rate (Qp), and specific production rate (q̄p) of the 40% DO-controlled culture were observed to be 14%, 15%, and 15% higher, respectively, than those of the parallel cultures that were performed under an excessive agitation speed (350 rpm) and aeration rate (1 vvm) to maintain sufficiently high DO levels throughout the entire fermentation period. These results suggest that high shear damage of the high-yielding strain due to an excessive agitation speed is the primary reason for the reduction of the AVM B1a biosynthetic capability of the producer. As for the cell growth, exponential growth patterns during the initial 3 days were observed in the fermentations of sufficient DO levels, whereas almost linear patterns of cell growth were observed in the other two cultures of DO limitation during the identical period, resulting in apparently lower amounts of DCW. These results led us to conclude that maintenance of optimum DO levels, but not too high to cause potential shear damage on the producer, was crucial not only for the cell growth, but also for the enhanced production of AVM B1a by the filamentous mycelial cells of Streptomyces avermitilis.
Article
This welcome new edition discusses bioprocess engineering from the perspective of biology students. It includes a great deal of new material and has been extensively revised and expanded. These updates strengthen the book and maintain its position as the book of choice for senior undergraduates and graduates seeking to move from biochemistry/microbiology/molecular biology to bioprocess engineering.
Chapter
Mass transfer occurs in mixtures containing local concentration variations. For example, when dye is dropped into a pail of water, mass-transfer processes are responsible for movement of dye molecules through the water until equilibrium is established and the concentration is uniform. Mass is transferred from one location to another under the influence of a concentration difference or concentration gradient in the system.
Article
Recently, the production of furan derivatives from sugars has become exciting in chemistry and in catalysis studies, because it aids one of the major routes for achieving sustainable energy supply and chemicals production. 5-Hydroxymethylfurfural (5-HMF), 2,5-furan-dicarboxylic acid (2,5-FDCA) and 2,5-dimethylfuran (2,5-DMF) have been called the “sleeping giants” of renewable intermediate chemicals. 5-HMF is a dehydration product of hexoses and a potential substitute of petroleum-based building blocks of various polymers. 2,5-FDCA is derived from oxidative dehydration of hexoses and is considered as one of the top 12 compounds made from a sugar into a value-added chemical [T. Werpy, G. Petersen, Top Value Added Chemicals From Biomass, 2004. Available electronically at http://www.osti.gov/bridge]. 2,5-DMF is produced through hydrogenation of HMF and is less volatile and of 40% higher energy density than ethanol. This review discusses mainly the catalytic routes for the synthesis of 5-HMF, 2,5-FDCA, 2,5-DMF and other furanic derivatives from sugars. Meanwhile, the possible reaction mechanism for the conversion of hexoses is discussed, and furthermore, some promising research orientations and advantageous catalysts are suggested based on the major problems encountered in the recent research.
Article
The microbial polysaccharide xanthan was produced by cultivation of Xanthomonas campestris in four stirred tanks with volumes ranging from 0.072 to 3 m3. Many of the previously suggested correlations for the volumetric mass transfer coefficient described the present data very unsatisfactorily. When Rushton turbines were employed as stirrers, the experimental results agreed well with a correlation suggested by Kawase and Moo-Young; for the more energy-efficient INTERMIG impellers, this correlation was modified by a factor of three. Constant specific oxygen transfer rate was found to be the appropriate scale-up criterion for constant space-time yield and constant product quality, specifically, the molar mass which determines the viscosity yield.
Article
A Perkin Elmer MGA-1200 mass spectrometer has been coupled with a mini-computer and a sampling manifold to analyze up to 8 components in the exhaust gases of fermentors. Carbon dioxide, oxygen, and nitrogen are typically analyzed, but ethanol for yeast fermentations can also be tested by heating the line from the fermentor to the sampling manifold. Specifications, operation, and performance of the system are described. The system has been used for process control, the study of fermentation kinetics, and process development. 8 references, 7 figures, 1 table.
Article
Microbial polysaccharides are rapidly emerging as a new and important source of polymeric materials. These biopolymers have novel and unique properties and already have found a wide range of applications in the food, pharmaceutical, and other industries. In view of the impending importance of polysaccharides as an industrial commodity, there is renewed interest in the area of product and process development. This paper summarizes the state-of-the art in polysaccharide fermentations. An attempt is being made to review the following areas: rheological characteristics of polysaccharide solutions, mixing and power requirements of polysaccharides and other highly viscous non-Newtonian systems, oxygen mass transfer, and scale-up problems encountered in polysaccharide fermentations.
Article
Process Biochemistry j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o Relation between pristinamycins production by Streptomyces pristinaespiralis, power dissipation and volumetric gas–liquid mass transfer coefficient, k L a a b s t r a c t During bioreactor cultures, microorganisms are submitted to non-optimal conditions such as nutri-tional and hydrodynamic stresses which may lead to modifications of the physiological cell response; this is especially true for filamentous microorganisms like Streptomycetes also subjected to significant morphological changes. In the present work, growth and production of pristinamycins by Streptomyces pristinaespiralis in shaking flasks have been related to power dissipation. The filamentous bacteria were grown in different flask conditions with various total and working volumes and at two agitation rates, to test the influence of power dissipation and gas–liquid mass transfer coefficient on growth and antibi-otics production. As a first step, computational fluid dynamics–volume of fluid (CFD–VOF) calculations were shown to be able to predict power dissipations for the various operating conditions in Newtonian flow conditions. Then, in non-Newtonian flow conditions (biomass concentration superior to 14 g L −1), the rheological model of Sisko was implemented in CFD simulations for the calculation of the fluid vis-cosity and then of power dissipation. Whereas microbial growth was correlated to k L a, the antibiotics production onset was linked to the volume mean power dissipation. Once a minimal cell concentration of 15 g L −1 was reached, the concentration of antibiotics was correlated to power dissipation with an optimal range of production, between 5.5 and 8.5 kW m −3 . Higher power dissipation entailed a drop in production which could be explained by hydrodynamic cell damages.
Article
The effects of reduced oxygen on growth and antibiotic production were studied in batch cultures ofStreptomyces clavuligerus in defined media. Antibiotic levels were unaffected by reduced oxygen for the first 50 hours of fermentation. After growth ceased, antibiotic concentrations dropped by a factor of about three under reduced oxygen, while the antibiotic concentration was stable when air was used. This suggests that enzymes for hydrolyzing antibiotics may be regulated by the aeration conditions.
Article
The effect of medium composition on the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation has been studied. Four types of mycelial pellets were obtained under the conditions used and may be classified as (a) frayed and loose with 0.1-0.5 mm diameter (b) compact with 0.1-0.5 mm diameter (c) loose with 0.5-2.0 mm diameter and (d) compact with 0.5-2.0 mm diameter. Their respective maximum specific rates of formation and yields of itaconic acid, based on 100 g sucrose supplied, were (a) 1.25 μmol mg-1h-1 and 55-59 g, (b) 0.27-0.43 μmol mg-1 h-1 and 26-38 g, (c) 0.75-0.90 μmol mg-1 h-1 and 45-51 g and (d) 0.12 μmol mg-1 h-1 and 10 g. The presence of Ca2+, Zn2+ and Fe2+ in the basal medium at concentrations of 23.3 mg/100 ml, 0.01 mg/100 ml and 0.006 mg/100 ml respectively were found to be adequate and crucial in obtaining the desired outgrowth for both high production rates and consistent yields of itaconic acid. The further addition of either commercial plaster of Paris or analytical-reagent-grade CaSO4, especially when activated by heating to 530°C and present in excess of solubility, results in small and frayed pellets, which lead to itaconic acid yields of 55-59 g acid/100 g sugar supplied.
Chapter
Highly viscous media often occur in biotechnology, fermentation and food processing industries as well as in chemical and pharmaceutical industries. The present paper considers the behavior of aerated highly viscous media employing stirred tank reactors, sparged single and multistage tower reactors. Measuring methods to determine rheological properties, hydrodynamical behavior, power input, and mass transfer as well as behavior of single bubbles and bubble swarm are reviewed. Hydrodynamical properties, power inputs, oxygen transfer rates, volumetric mass transfer coefficients and heat transfer coefficients are considered as a function of the mean operating parameters employing stirred tank reactors with different impellers. Also sparged single and multistage tower reactors are treated, especially hydrodynamical properties, oxygen transfer rates and volumetric mass transfer coefficients. Relationships are given to lay out stirred tank and tower reactors with highly viscous media.
Article
Oxygen uptake rate and oxygen mass transfer rate have been studied during xanthan gum production process in stirred tank bioreactor. Empirical equations for the oxygen mass transfer coefficient have been obtained taking into account several variables such as air flow rate, stirrer speed and apparent viscosity. Oxygen uptake rate evolution in the course fermentation has been measured, obtaining an equation as a function of biomass concentration, including overall growth and non growth-associated oxygen uptake. A metabolic kinetic model has been employed for xanthan gum production description including oxygen mass transfer and uptake rates. The results point out that this model is able to describe adequately not only oxygen dissolved evolution, but also of the production of xanthan and substrate consumption. Also, the influence of several parameters (kLa, air flow rate and dissolved oxygen) in the evolution of the key compounds of the system have been studied. The results of the simulation shown that an increasing of dissolved oxygen concentration favor the xanthan gum production.
Article
Mycelial microorganisms are exploited extensively in the commercial production of a wide range of secondary metabolites. They can be cultured as free mycelia, as aggregated forms (pellets/flocs), or as artificially bound/entrapped cells, though problems are associated with the culture of each morphological type. Since the morphological type can strongly influence metabolite production, the methodology for inducing pellet formation, and the type of pellets produced are an important consideration for effective metabolite production.
Article
Industrial filamentous fungal fermentations are typically operated in fed- batch mode. Oxygen control represents an important operational challenge due to the varying biomass concentration. In this study, oxygen control is implemented by manipulating the substrate feed rate, i.e. the rate of oxygen consumption. It turns out that the setpoint for dissolved oxygen represents a trade-off since a low dissolved oxygen value favors productivity but can also induce oxygen limitation. This paper addresses the regulation of dissolved oxygen using a cascade control scheme that incorporates auxiliary measurements to improve the control performance. The computation of an appropriate setpoint profile for dissolved oxygen is solved via process optimization. For that purpose, an existing morphologically structured model is extended to include the effects of both low levels of oxygen on growth and medium rheological properties on oxygen transfer. Experimental results obtained at the industrial pilot-scale level confirm the efficiency of the proposed control strategy but also illustrate the shortcomings of the process model at hand for optimizing the dissolved oxygen setpoints.
Article
In this work, the agitation and aeration effects in the maximization of the beta-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2(2) trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL(-1) for enzymatic activity, 1.2 U mL(-1) h(-1) for productivity in 14 h of process, a cellular concentration of 11 mg mL(-1), and a 167.2 h(-1) volumetric oxygen transfer coefficient.
Article
Modern (bio)pharmaceutical process development requires thorough investigation of all process parameters that are critical to product quality. The impact of a disturbance of such a parameter during processing needs to be known so that a rational decision can be made about the release of the product. In cultivation processes the dissolved oxygen (DO) concentration is generally accepted as being a critical parameter. In this article the impact of a 90 min period of oxygen limitation during the cultivation of the strictly aerobic Bordetella pertussis bacterium is investigated. The cultivation is the most important process step for the manufacturing of a vaccine against whooping cough disease. Samples were taken immediately before and after oxygen limitation and at the end of cultivation of four oxygen limited and three control cultivations. DNA microarray analysis of the full transcriptome of the B. pertussis bacterium revealed that a 90 min period of oxygen limitation has a substantial effect on overall gene expression patterns. In total 104 genes were identified as a significant hit at any of the sample points, of which 58 were directly related to oxygen limitation. The other genes were mainly affected towards the end of cultivation. Of all genes involved in oxygen limitation none were identified to show a significant difference between the oxygen limited and control cultivations at the end of the batch. This indicates a fully reversible effect of oxygen limitation on gene expression. This finding has implications for the risk assessment of dissolved oxygen concentration as a critical process parameter.
Article
Streptomyces clavuligerus is a commercially important actinomycete that is used to produce clavulanic acid, a beta-lactamase inhibitor. Observations during 10 batch cultivations with S. clavuligerus on defined media have led to the finding that the organism is very sensitive to shear when grown in batch cultures with increasing stirrer speed. The stirrer speed was increased to keep the dissolved oxygen level above 50% air saturation. A quantitative approach based on the calculation of elemental balances and a simple mathematical model is proposed to characterize the biomass lysis. Finally, a linear relation between biomass yield and observed specific growth rate is determined. Results show that cell lysis occurs at a high degradation rate, e.g., mu(max) = 0.16 h(-1) and k(d) = 0.07 h(-1), when the gassed power input increases above 1.1, 1.7, or 2.0 kW/m(3), respectively, depending on the medium composition. The overall biomass yield on substrate is dramatically reduced in all experiments (>30%).
Article
The gas-liquid mass transfer coefficient K(L)a in the fermenter is a strong function of mode of energy dissipation and physico-chemical properties of the liquid media. A combination of disc turbine (DT) and pitched blade turbine down flow (PTD) impellers has been tested in laboratory bioreactor for gas hold-up and gas-liquid mass transfer performance for the growth and biotransformation medium for an yeast isolate VS1 capable of biotransforming benzaldehyde to L-phenyl acetyl carbinol (L-PAC) and compared with those in water.Correlations have been developed for the prediction of the fractional gas hold-up and gas-liquid mass transfer coefficient for the above media. The mass transfer coefficient and respiration rate have been determined in the shake flask for the growth as well as for biotransformation medium. These results, then have been used to optimize the operating parameters (impeller speed and aeration) for growth and biotransformation in a laboratory bioreactor. The comparison of cell mass production and L-PAC production in the bioreactor has been done with that obtained in shake flask studies.
Article
A continuous supply of O(2) is important for itaconic acid production in Aspergillus terreus. Any interruption of aeration significantly reduces itaconic acid production. To overcome this effect, A. terreus M8 was transformed with the Vitreoscilla hemoglobin gene (vgb) which, as shown by Southern hybridization, was integrated into the recipient chromosome. The activity of the expressed hemoglobin was confirmed by a CO-difference spectrum. During itaconic acid production, the effect of a break in aeration during cultivation in the transformant with the vgb gene is alleviated. Additionally, the transformant shows improved itaconic acid production.
Article
A new inhibitor of p53-HDM2 interaction was isolated from a culture of marine-derived fungus, Arthrinium sp. The structure was identified to be (-)-hexylitaconic acid (1) by spectroscopic analysis. The inhibition of p53-HDM2 binding was tested by the ELISA method, and 1 inhibited the binding with an IC50 value of 50 mu g/mL. Although a number of synthetic inhibitors of p53-HDM2 interaction have been reported so far, 1 is the second inhibitor isolated from natural resources. (c) 2005 Elsevier Ltd. All rights reserved.
Article
The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO(2)) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO(2) concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of [see text]. HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.
Article
Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.
Article
Filamentous fungi are metabolically versatile organisms with a very wide distribution in nature. They exist in association with other species, e.g. as lichens or mycorrhiza, as pathogens of animals and plants or as free-living species. Many are regarded as nature's primary degraders because they secrete a wide variety of hydrolytic enzymes that degrade waste organic materials. Many species produce secondary metabolites such as polyketides or peptides and an increasing range of fungal species is exploited commercially as sources of enzymes and metabolites for food or pharmaceutical applications. The recent availability of fungal genome sequences has provided a major opportunity to explore and further exploit fungi as sources of enzymes and metabolites. In this review chapter we focus on the use of fungi in the production of food additives but take a largely pre-genomic, albeit a mainly molecular, view of the topic.
Article
The performance of a penicillin fermentation was assessed in a laboratory-scale bubble column fermentor, with mycelial growth confined to the pore matrix of celite beads. Final cell densities of 29 g/L and penicillin titres of 5.5 g/L were obtained in the confined cell cultures. In comparison, cultures of free mycelial cells grown in the absence of beads experienced dissolved oxygen limitations in the bubble column, giving only 17 g/L final cell concentrations with equally low penicillin titres of 2 g/L. The better performance of the confined cell cultures was attributed to enhanced gas liquid mass transfer rates, with mass transfer coefficients (k(L)a) two to three times higher than those determined in the free cell cultures. Furthermore, the confined cell cultures showed more efficient utilization of power input for mass transfer, providing up to 50% reduction in energy requirements for aeration.
Enhanced production of itaconic acid by transformed fungal cells of Aspergillus terreus harboring Vitreoscilla hemoglobin gene
  • W S Shin
  • Y H Kim
  • Y S Jeong
  • C Park
  • S Y Kim
  • D Lee
Shin WS, Kim YH, Jeong YS, Park C, Kim SY, Lee D, et al. 2009. Enhanced production of itaconic acid by transformed fungal cells of Aspergillus terreus harboring Vitreoscilla hemoglobin gene. J. Biosci. Bioeng. 108: S123.