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Citric acid production by Aspergillus niger immobilized on cellulose microfibrils: influence of morphology and fermenter conditions on productivity
Process Biochemistry 36 (2001) 1129– 1139 www.elsevier.com/locate/procbio
Citric acid production by Aspergillus niger immobilized on cellulose microfibrils: influence of morphology and fermenter conditions on productivity N.V. Sankpal, A.P. Joshi, B.D. Kulkarni * Chemical Engineering Di6ision, National Chemical Laboratory, Pune 411 008, India Received 22 February 2000; received in revised form 25 January 2001; accepted 12 February 2001
Abstract Continuous and batch production of citric acid from sucrose has been investigated using Aspergillus niger NCIM 588. Mycelia of A. niger grown on cellulose microfibril forms a uniform and thin mycelial proliferation under controlled conditions of cultivation rich in oxygen. In the fed batch mode using a recycle reactor, the DO of the system was maintained at 20 mg l − 1 using oxygen enriched air. This improved volumetric productivity to 1.85 g l − 1 h − 1 of citric acid, representing an increase of at least 15-fold over results obtained simultaneously using shake-flasks and 1.6-fold over a conventional aerated batch reactor. It was possible to substitute sucrose with sugarcane juice as a carbon source in a fed batch recycle system. An overall specific production rate of citric acid of 0.147 and 0.208 g g − 1 h − 1 was achieved using cane juice and sucrose, respectively. In continuous fermentation, a medium containing 50 g l − 1 of sucrose was allowed to drip through the fabric support at a residence time of 20 h. As a result of interface interaction, a citric acid volumetric productivity of 2.08 g l − 1 h − 1 was achieved for 26 days without any significant loss of productivity. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus niger; Citric acid; Immobilization; Cellulose microfibrils; Morphology; Batch and continuous fermentation
1. Introduction In the past few years, significant studies have been carried out on citric acid production and efforts to achieve higher volumetric productivity under conditions of submerged and surface modes of operation continue [1 – 5]. Implementation of these processes has many biological and operational hurdles. For filamentous fungi, when compared to single cell fermentations, the complex environment in the bioreactor and heterogeneity under agitated conditions could be observed [6]. Fungal morphology changes the physical properties of broth which causes numerous problems in industrial fermenters with respect to gas dispersion, mass and heat transfer and homogenization [6]. Further, in conventional bioreactors, oxygen transfer and mixing are rather inefficient, with a high apparent viscosity of the culture broth [7,8]. The viscosity related problem may * Corresponding author. Tel./fax: + 91-20-5893041. E-mail address: [email protected] (B.D. Kulkarni).
be overcome by ensuring that the mycelial growth is mainly in pellet form rather than in filamentous form [9], as the suspension of pelleted mycelia is usually less viscous than that of filamentous mycelia [10,11]. The size of the pellet and the relative amount of filamentous mycelia varies with fermentation time and intensity of agitation. Mechanical forces induced by turbulent flow chip off the outer mycelia of pellets and the extent of this chipping is a function of the hyphal length and the magnitude of the mechanical force [12]. The study of Wittler et al. [13] on Penicillium chrysogenum shows partial lysis and disintegrated pellet. Mass transfer limitations have been observed in alginate bead entrapped cells [14]. When compared to surface processes, biomass recycle as a means of medium replacement is generally more complicated in submerged fermentation. The possibility of the repeated use of Aspergillus niger biomass was demonstrated using an exchange filtration technique [15]. Anderson et al. [16] have used a disk fermenter for extended and continuous production of citric acid. Sev-
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 1 5 5 - 8
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eral attempts have been reported for continuous production of citric acid using magnetic drum contactors [17], reciprocating-jet-bioreactors [18], hollow fibre reactors [19] and continuous cultures [20]. In addition, several studies report the use of A. niger immobilized on various kinds of solid supports, viz. glass carrier [21], polyurethane foam [22], entrapment using calcium alginate [23,24], entrapment in polyacrylamide gels, [25,26], polypropylene hollow fibers [19], entrapment in agar [27], agarose [28] and cellulose carriers [29]. Citric acid production is an aerobic fermentation process. In submerged mode, the dissolved oxygen level is maintained at : 50% saturation using air as oxygen source. Partial or total lack of aeration, even for a short duration is detrimental and leads to delay in the fermentation period [30]. Tower fermenters are increasingly preferred because of ease of scaling up and the possibility to attain higher dissolved oxygen (DO) levels in the lower segment due to increased hydrostatic pressure [4]. In some reports, oxygen availability has been accomplished by using pure oxygen or using high pressure. At 60 mg l − 1 level of DO, better yields of citric acid are reported by Sato [31]. This was attributed to reduced biomass formation and less substrate diversion for this purpose. The aim of this work was to explore the possibility of achieving high volumetric productivity of citric acid using novel fermentation systems in batch and continuous modes. The study includes controlled cell immobilization and changes in reactor configuration to obtain minimum shear stress. This allows a considerable increase in interfacial area between the medium and mycelium, as well as mycelium and air. Surface fermentation for continuous operation and a recycle continuous flow reactor for fed batch operating conditions have been used to improve the specific rate of citric acid production.
2. Materials and methods
2.1. Micro-organism A. niger NCIM 588 (ATCC 1015) was the best strain screened from the culture bank of the National Collection of Industrial Microorganism (NCL, Pune). The strain was maintained on potato dextrose agar slants at 4°C and subcultured at intervals from 1 to 2 months
2.2. Immobilization of A. niger The cellulosic support and continuous fermentation (method of mycelial immobilization and unit operation) has been described earlier [32]. For batch fermentation, fully entangled mycelia were used directly.
2.3. Fermentation medium Surface and submerged flask cultures: 50–100 g sucrose purified, 0.25 g MgSO4·7H2O, 0.75 g KH2PO4 and 2.0 g NH4NO3 in 1.0 l of distilled water. Trace element solution containing Cu2 + , 0.00006; Zn2 + , 0.00025; Fe3 + , 0.0013 and Mn2 + 0.001 g l − 1 was incorporated, pH was adjusted to 5.5 using 1 M H2SO4. Immobilized mycelia: 100–180 g sucrose, 0.025 g MgSO4·7H2O, 0.075 g KH2PO4 and 0.1 g NH4NO3 in 1 l water was used. Before sterilization, pH of the medium was adjusted to 6.0 using 1 M H2SO4.
2.4. Fermentation procedure 2.4.1. Flask fermentations Surface and submerged culture fermentation was carried out in 1-l Erlenmeyer flasks containing 150 ml and 500-ml Erlenmeyer flasks containing 75 ml of sterile medium, respectively. Spores of a 7-day-old culture of A. niger grown on PDA slope were harvested and suspended in distilled water to obtain a spore density of 3–5 ×107 ml − 1. A 5 ml spore suspension was transferred to each flask and incubated at 28°C. For surface culture, the mycelial mat was allowed to grow for the first 2 days and thereafter, medium samples were collected for analyses at 24-h intervals until 12 days. For shake flask fermentations, spore germination was allowed to take place for the first 12 h and the contents of the flasks were agitated at 120 rpm. Samples were collected thereafter, for analyses at intervals of 24-h until 12 days. 2.4.2. Batch fermentation in recycle reactor and shake flask The support (8 × 70 cm) holding immobilized mycelia was rolled into a spiral and loaded into the reactor. A thin polythene mesh was put in between the folds to avoid mycelial adhesion between two adjacent layers. Fermenter design and operation is shown in Fig. 1. Aeration was carried out using a mixture of air (25 ml min − 1) and oxygen (5 ml min − 1) to attain a level of 20 mg l − 1 of dissolved oxygen. For shake flask fermentation biomass holding support was cut into 2×2 cm pieces and used. 2.4.3. Continuous fermentation Details regarding support and immobilization protocols for continuous operation has been described in an earlier publication [32]. Fresh sugarcane juice was purchased from a local vendor and was used without any additions. It was diluted to a suitable nitrogen level, steam sterilized and used within 2 days.
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2.5. Surface area analysis
3. Experimental results
The surface area of the cellulose microfibrils was analyzed by a single point BET method (Monosorb surface area analyzer, Quanta Chrome Corp., USA). This gave an average value of 11 m2 g − 1 microfibrils after three sets of adsorption and desorption of nitrogen. 2.6. Sugar and acid analyses
3.1. Surface and shake flask fermentation using free mycelia
Feed and unconverted sucrose was hydrolyzed using 2 N HCL at boiling temperature for 15 min into glucose and fructose and analyzed by a di-nitrosalicyclic acid method [33]. Citric acid was analyzed by a pyridine–acetic anhydride method [34] and simple titration against 1 N NaOH. Unreacted sugar was monitored usually by TLC using ethyl acetate, 1-propanol and water in a ratio of 8:7:2 as a mobile phase. The spray reagent used for visualization contained 4%-naphthol in 20:80, ethanol: 2 N sulfuric acid. This method helped visualization of sugars down to 0.05% in the medium. Nitrogen present at different stages of fermentation was analyzed by a micro-Kjeldahl method [35]. Phosphate was determined by a molybdate method [36]. Biomass analysis and scanning electron microscopy protocol has been described earlier [32].
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Experiments were conducted using surface culture and shake flask submerged cultures to provide a baseline for comparing results. Fig. 2 shows typical results during the progress of fermentation. Mycelia of A. niger grew well over a period of 48 h in shake flasks to form pellets of size 2–3 mm. It was also observed that the nitrogen in the medium becomes exhausted and citric acid accumulation occurs. The maximum specific growth rate (vmax) of A. niger was 0.0184 g h − 1 and a fermentation time of 9 days was required to utilize \70% sucrose. In this period, 35 g l − 1 citric acid was accumulated, giving an overall volumetric productivity of 0.125 g l − 1 h − 1 corresponding to a maximum specific production rate of 0.016 g g − 1 h − 1. An extension of fermentation to 13 days did not result in any improvement in specific production rate. These are in good agreement with earlier studies [37]. In surface culture, a thick mat was formed at the end of the 48 h, which gave 62 g l − 1 of citric acid at the termination of fermentation (thirteenth day). Under optimized conditions, the maximum specific growth rate (vmax) of A. niger observed was 0.02 g h − 1. At this
Fig. 1. Schematic diagram of fermentation setup for a recycle bioreactor.
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Fig. 2. Comparison between batch fermentations of citric acid by A. niger. Immobilized cells (IC); free cells (FC). Table 1 Comparison between different modes of fermentation Volumetric productivity (g l−1 h−1) Citric acid yield (g l−1)
Fermentation type Mycelia
Aeration type
Batch, shake flask Batch, shake flask Batch, surface culture Batch, recycle reactor Batch, recycle reactor Batch, recycle reactor Continuous Continuousa
Free Immobilized Free
Air Air Air
0.125 0.43 0.223
35 55 62
65 90 92
Free
Air+O2
1.16
50
78
Immobilized
Air
1.4
52
85
Immobilized
Air+O2
1.85
64
92
Immobilized Immobilized
Air Air
2.08 0.8–2.1b
65 58
96 90
a b
Sugar utilization (%)
Use of cane juice as carbon sources. Values decreased over a period of 10 days.
stage, the overall volumetric productivity was 0.235 g l − 1 h − 1 corresponding to a maximum specific production rate of 0.0176 g g − 1 h − 1. In this culture, a thick mat and hyphal structures protruding above the surface provided greater opportunities for air access. During this experiment, the surface depth ratio of the medium was 1:10 that is reported to be important for this fermentation [38].
sugar was exhausted. The volumetric productivity achieved was 0.43 g g − 1 h − 1 corresponding to a maximum specific production rate of 0.071 g g − 1 h − 1. The results are shown in Fig. 2 and Table 1. At this time the concentration of free citric acid accumulated was 55 g l − 1.
3.3. Batch recycle bioreactor with recirculation of the fermentation broth o6er immobilized mycelia
3.2. Immobilized mycelia in shake flask In another set of experiments, small strips of cellulose support on which mycelia were pregrown (0.62 g per flask) were placed in the shake flask. Citric acid accumulation began at :6 h. The experiment was continued for 11 days at which time almost all the available
Recirculation of the fermentation broth has been reported earlier [18] in order to obtain continuous citric acid production. In the present studies, free mycelia (FM) using oxygen enriched air and immobilized mycelia (IM) with oxygen enriched air were used to check reactor performance and efficiency. In the FM set,
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mycelial pellets were pregrown in shake flasks for 48 h and used with fresh medium. In the immobilized setup the fabric support (8×70 cm) holding 1.1 g biomass was located below the surface of the medium and was placed on a perforated glass base (Fig. 1), which served as an inlet for the circulating medium. This minimizes mycelial detachment from microfibrils. Sterile oxygen enriched air was let into the circulating medium prior to its entry into the reactor by inserting a venturi. This ensured air access throughout the packed bed. During recycle, oxygen enriched medium traverses upward through the capillaries of the fabric. The low flow rate
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ensured a mild current of flowing broth and prevented shear stress. As a result, the cells were not detached from the microfibrils. These immobilized mycelia utilize \ 90% sucrose and pH falls to 1.96 to give 64 g l − 1 citric acid over a period of 70 h (Fig. 3). It can be seen from Fig. 4 that oxygen enriched air reduces the fermentation period by 20 h compared to air and shake flask mode of immobilized cell systems. This improved productivity was reproducible in four subsequent recycles in respect of yield and productivity. Citric acid between 62 and 65 g l − 1 was obtained at the end of every cycle (Fig. 5). The overall productivity
Fig. 3. Batch production of citric acid using sucrose by immobilized A. niger in recycle bioreactor at partial pressure of oxygen at 20 mg l − 1.
Fig. 4. Citric acid production using sucrose by immobilized A. niger in recycle bioreactor. Air and oxygen enriched air has been used at 7 and 20 mg l − 1 of oxygen, respectively.
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Fig. 5. Repeated batch production of citric acid using sucrose by immobilized A. niger in recycle bioreactor. ( points at which medium is replaced with fresh lot).
observed in these cycles was between 1.82 and 1.88 g l − 1 h − 1.
3.5. Continuous production of citric acid using immobilized mycelia
3.4. Utilization of cane juice in batch fermentation
Results obtained using the fed batch system with immobilized mycelia point to the potential benefits of this system in terms of cell recovery and reuse. A support having dimensions of 70× 8× 0.5 cm holds a 105 ml void volume and 1.13 g pregrown biomass. Medium containing 50 g l − 1 of sucrose was allowed to flow through the capillaries of the support. The residence time (RT) to achieve a near complete utilization of sucrose to form citric acid was 20 h. Volumetric productivity observed for this set was 2.08 g l − 1 h − 1. Fermented broth contained 65 g l − 1 of citric acid. This was more than the expected values of citric acid as a result of evaporative concentration (20%). Cane juice was also used to study whether on not it can replace sucrose. On the pregrown mycelia, continuous feeding of cane juice was carried out over a period of 10 days. The productivity declined over a period (Fig. 7). The biomass on the support slowly increased from 1.2 to 2.6 g, giving a visibly thick appearance and limiting air access to the inner layers of mycelia on the fabric. Under these conditions, experimental difficulties such as irregular medium flow and drops dripping through microfibrils were observed.
Replacement of sucrose with cane juice will reduce the cost of the starting material, sucrose to 1/3. Cane juice contains 100– 160 g l − 1 (average 120 g l − 1) of sucrose with other fermentable mono-, di- and polysaccharides. On average, the juice shows a ‘brick value’ (total solid content) ranging between 13 and 20 [39]. This raw material has several advantages as a fermentation feedstock [40]. One of the major hurdles found in the use of sugarcane juice is high levels of metabolizable nitrogen. The content of nitrogen in juice is dependent on the age of the cane; matured cane may have 2/3 N compared to younger cane (:800 mg l − 1). Thus, selecting low nitrogen juice or diluting the available juice are the options used for citric acid production [41]. Table 1 gives data on citric acid production using sucrose and diluted sugarcane juice. The yields were comparable and lead to a considerable saving on raw material costs. In fed batch culture, citric acid production activity was delayed presumably due to initial high levels of nitrogen. In this period, biomass builds up at the cost of nitrogen and as the level of nitrogen reaches 50 mg l − 1, citric acid production starts. In subsequent recycles up to the fifth, there was a biomass build up in every recycle to reach a level of 2.4 g from the starting level of 0.95 g. In the experiments using sucrose, the build up was marginal, reaching a level of 1.3 g from 1.1 g. The data are presented in Fig. 6 and shows that after every cycle, biomass per support rises.
3.6. Effect of water acti6ity on continuous operations Water is required for microbial growth and metabolism of the cell. The term water activity (aw) refers to the unbound water in the environment, around the mycelia. The water activity of the environment is related to osmolarity, which is an important variable
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affecting microbial metabolism. The aw influences microbial growth and biochemical activities and is an important parameter to be considered in surface culture (present continuous mode) and solid state fermentation [42]. To check the effect of aw, five sets of experiments, at aw of 0.25–0.4, 0.5– 0.6, 0.7– 0.8, 0.85– 0.9 and 0.95– 0.98 were carried out under conditions of constant temperature (Table 2). At aw B0.4, the fabric support shrinks disturbing the porosity and consequent decreasing productivity to 0.96 g l − 1 h − 1. Under such condi-
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tions, the air current removes the water contained in the medium as water vapor while moving past the high surface area of the support (11 m2 g − 1). This dries the microenvironment around the mycelia affecting cellular metabolism. Even after decreasing the residence time (increasing medium flow rate) to half (6 h), there was no noticeable improvement. As shown in Table 2, an aw of 0.85 was the minimum necessary for maximum productivity of 2.08 g l − 1 h − 1 citric acid that continued till the end of fermentation.
Fig. 6. Repeated batch production of citric acid using cane juice by immobilized A. niger in recycle bioreactor. In successive cycles, biomass increased to show decreased productivity.
Fig. 7. Continuous production of citric acid using sugarcane juice by immobilized A. niger.
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Table 2 Effect of water activity (aw) on continuous fermentation of citric acida aw
Sucrose utilization (%)
Productivity (g l−1 h−1)
Broth concentration (%)
Citric acid (g l−1)
Running time (days)
0.25–0.40 0.5–0.6 0.7–0.8 0.85–0.9 0.95–0.98
40 9 2 80 95 98 9 2 98 9 2 98 9 2
0.96 1.6 1.92 2.08 2.08
35 28 25 22 20
40 45 60 65 62
4 20 20 26 26
a At residence time 20 h; sucrose, 50 g l−1. The data shown was measured between the third and fifth day when citric acid production stabilized. Evaporation rate has S.E. of 0.12.
4. Discussion The present study had three primary objectives in mind; reducing fermentation time for bioconversion, reuse of same biomass and operating the system under less stringent conditions of sterility. Numbers of experiments, in different modes of reactor operation, were carried out to ascertain the best operating conditions to meet these objectives. In fed batch and continuous modes of fermentation, the productivity values of immobilized cells were higher when compared to those reported in the literature [14,19,22,43]. The maximum volumetric productivity of 2.08 and 1.85 g l − 1 h − 1 was achieved in continuous surface and continuous flow recycle reactor, respectively. The differences in productivity (surface and submerged modes) can be attributed to different morphology and its effects on apparent viscosity that alters mass transfer and mixing characteristics [44]. Fungal morphology has a strong influence on the physical properties of cultivation broth, which cause numerous problems in industrial fermenters with respect to gas dispersion, mass and heat transfer and homogenization [6]. Papagianni et al. [45] have shown that the length of the filaments is the only parameter that could be related to citric acid production. These authors also observed that, for similar yields of citric acid, the morphology of the organism differed in stirred tank and the loop reactor. The support used here for immobilization of A. niger and the mode of fermentation have played important roles in enhancing productivity of citric acid. This is due to different morphology achieved under conditions of submerged and surface modes of fermentations. In submerged fermentation, the mycelia form a thick mat of microfibrils (Fig. 8b), whereas in the surface mode, a protruding mycelial structure was seen in a cobweb like network of free filaments between the microfibrils of fabric (Fig. 8c). A typical microfibril of the support is shown in Fig. 8(a) and has a favorable shape. Convolutions with sharp folds along the microfibrils can also be seen. These microfibril also shows lumens, which may play an important role in medium flow and interfacial interactions during medium movement. The support
used for recycle reactor and continuous fermentation had dimensions of 8× 70 cm. It had an interfacial surface area of 0.53 m2 cm − 2. On such a strip, biomass immobilized was between 1.1 and 2.0 g. This biomass was spread over the support in the form of a thin mycelial film and in-between spaces of microfibrils. Considering the morphology, the surface culture mycelia may have been subjected to less shear stress when compared to the submerged mycelia. Further, the interfacial interactions during surface fermentation may be better due to free access to air exhibiting more productivity. In addition to this, the controlled growth of mycelia over microfibrils under oxygen enriched atmosphere can be advantageous. Using air grown mycelia, many fold increased productivity in submerged fermentation has been reported [19,43,46]. Several studies using fungi report on enhanced formation of organic acids under conditions of strong aeration. Aeration fulfills two functions — oxygenation and heat removal. The oxygen requirement varies with the stage of growth. In submerged fermentations, agitation influences the size of pellets and structure of the pellet’s surface. Stronger agitation in stirred tank reactor results in higher dissolved oxygen tension and more branching of hyphae. The pellets formed could be denser, stronger and exhibit higher tensile strength. In addition to this, the size of the pellet and the relative amount of filamentous mycelia vary with fermentation time and intensity of agitation [12]. Mechanical forces induced by turbulent flow chip off the outer mycelia of pellets and show partially lysed or disintegration of pellet [13]. Poor mixing in conventional bioreactors causes inefficient oxygen transfer [7,8]. Thus, for example, calcium alginate bead immobilized (entrapped) cells have shown limitations in mass transfer [14]. In such cases, the use of pure oxygen or oxygen enriched air can be a very effective way to reduce transport limitations and cell lysis. The viscosity related problem may be overcome by ensuring that mycelial growth is mainly in pellet form rather than in filamentous form. A suspension of pelleted mycelia is usually less viscous than that of filamentous mycelia [9–11]. The relation between the pellet size of A. niger and the oxygen transfer has been elucidated in a report considering the
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morphological characteristics of the pellet [47]. The optimum diameter of the pellet was found to be 2.2 mm which was quite stable in the bubble column bioreactor, leading to significant improvement in citric acid production.
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Our earlier report has shown that increased interfacial interactions lead to a high specific production rate of gluconic acid [32], which was comparable to that obtained using high dissolved oxygen levels at relatively high pressure [46]. In the case of di- and tricarboxylic acids, a high oxygen tension induces an alternate respiratory pathway required for re-oxidation of the glycolytically produced coenzyme, NADH [30,48,49]. An unidentified component of this pathway seems to be stringently dependent on the maintenance of a high oxygen tension [50], as even short interruptions of aeration impair the productivity [30].
5. Conclusion Mycelia of A. niger NCIM 588 were immobilized by adsorption onto cellulose microfibrils of a porous fabric and used in batch and continuous production of citric acid. The rise in the specific productivity of citric acid fermentation either in submerged or surface mode can be a result of reduced stress conditions on mycelia and less shear stress during operating conditions as described here as against their high levels in conventional aerated or agitated bioreactors. The data presented in this work suggests the possibility of improving the overall productivity of citric acid fermentation using a novel system. The individual parameters contributing to such improvement have been discussed. The results obtained have been compared to data available in the literature using related systems. Fungal pellet and immobilized cells show a great variability in morphology, which is an important consideration in heat and mass transfer. Apart from this, interface interaction and metabolite excretion are the major advantages in immobilization techniques. Highly improved interface interaction between mycelia and oxygen, as well as substrate, improves the diffusion of substrate; continuous removal of the metabolite formed also contributes to improved productivity.
Acknowledgements The authors acknowledge financial support provided by the Council of Scientific and Industrial Research (CSIR) for carrying out these investigations.
References Fig. 8. Scanning electron micrographs (SEM) of (a) a microfibril on which A. niger NCIM 588 immobilized; (b) SEM of immobilized mycelia at submerged mode of fermentation, magnification (1 K × ); (c) SEM of immobilized mycelia at surface mode of fermentation, magnification (300 × ).
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[25] Horitsu H, Adachi S, Takahashi Y, Kawai K, Kawano Y. Production of citric acid by A. niger immobilized in polyacrylamide gels. Appl Microbiol Biotechnol 1985;22:8 – 12. [26] Garg K, Sharma CB. Continuous production of citric acid by immobilized whole cells of Aspergillus niger. J Gen Appl Microbiol 1992;38:605 – 15. [27] Borglum GB, Marshall JJ. The potential of immobilized biocatalysts for production of industrial chemicals. Appl Microbiol Biotechnol 1984;9:117 – 230. [28] Khare SK, Jha K, Gandhi AP. Use of agarose-entrapped Aspergillus niger cells for the production of citric acid from soy whey. Appl Microbiol Biotechnol 1994;41:571 – 3. [29] Fujii N, Yasuda K, Sakakibara M. Effect of volume ratio of cellulose carriers and time interval of repeated batch culture on citric acid productivity by immobilized A. niger. J Ferment Bioeng 1994;78:389 – 93. [30] Kubicek CP, Zehentgruber M, El-Kalak H, Roehr M. Regulation of citric acid production by oxygen: effect of dissolved oxygen tension on adenylate levels and respiration in A. niger. Eur J Appl Microbiol Biotechnol 1980;9:101 – 16. [31] Sato S. Microbial production and control of cellular growth under high dissolved oxygen concentration. Hakko Kogaku Kaishi 1990;68:411 – 21. [32] Sankpal NV, Joshi AP, Sutar II, Kulkarni BD. Continuous production of gluconic acid by Aspergillus niger immobilized on cellulosic support: study of low pH fermentative behaviour of A. niger. Process Biochem 1999;35:317 – 25. [33] Miller GL. Use of di-nitrosalicyclic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 – 9. [34] Marier JR, Boulet M. Direct determination of citric acid in milk with an improved pyridine – acetic anhydride method. J Dairy Sci 1958;41:1683 – 92. [35] Lowenheim FA. Kjeldahl method. In: Foster DS, Leslic SE, editors. Encyclopedia of Industrial Chemical Analysis, vol. 16. London: Interscience, 1972:458 – 66. [36] Fiske CH, Subba Rao Y. The colorimetric determination of phosphorous. J Biol Chem 1925;66:375 – 400. [37] Sankpal NV, Joshi AP, Kulkarni BD. Nitrogen-dependant regulation of gluconic acid and/or citric acid production by Aspergillus niger. J Microbiol Biotechnol 2000;10:15 – 55. [38] Matteau PP. Enhanced performance of immobilized bioreactors using variable surface bioreactors. In: Moody GW, Baker PB, editors. Bioreaction Biotransformation. London: Elsevier, 1987:395 – 406. [39] Misra A, Shrivastava M. Mineral constituent in relation to sucrose in cane juice. Bharatiya Sugar 1989;14:39 – 45. [40] Qazi GN, Gaind CN, Chaturvedi SK, Chopra CL, Traeger M, Onken U. Pilot-scale citric acid production with Aspergillus niger under several conditions. J Ferment Bioeng 1990;69:72 –4. [41] Sankpal NV, Joshi AP, Kulkarni BD. A process for the simultaneous production of high fructose and gluconic acid using sugarcane juice. Indian Patent Application No. NCL/41/99, 1998. [42] Ramana Murthy MV, Karanth NG, Raghava Rao KSMS. Biochemical engineering aspects of solid-state fermentation. In: Neidleman S, Laskin AI, editors. Advances in Applied Microbiology, vol. 8. New York: Academic Press, 1993:99 – 144. [43] Gupta S, Sharma CB. Continuous production of citric acid from sugarcane molasses using a combination of submerged immobilized and surface stabilized cultures of Aspergillus niger KCU 520. Biotechnol Lett 1994;16:599 – 604. [44] Johansen CL, Coolen L, Hunik JH. Influence of morphology on product formation in Aspergillus awamori during submerged fermentations. Biotechnol Prog 1998;14:233 – 40. [45] Papagianni M, Mattey M, Kristiansen B. Morphology and citric acid production of Aspergillus niger PM 1. Biotechnol Lett 1994;16:929 – 34.
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[48] Zehentgruber O, Kubicek CP, Roehr M. Alternative respiration of A. niger. FEMS Microbiol Lett 1980;8:71 – 4. [49] Kirimarra K, Hirowateri Y, Usami S. Alterations of respiratory systems in A. niger under the condition of citric acid fermentation. Agri Biol Chem 1987;51:1299 – 303. [50] Bennett JW, Klich MA editors. Aspergillus: Biology and Industrial Applications. Stoneham: Butterworth-Heinemann, 1992.
Citric acid production by Aspergillus niger immobilized on cellulose microfibrils: influence of morphology and fermenter conditions on productivity N.V. Sankpal, A.P. Joshi, B.D. Kulkarni * Chemical Engineering Di6ision, National Chemical Laboratory, Pune 411 008, India Received 22 February 2000; received in revised form 25 January 2001; accepted 12 February 2001
Abstract Continuous and batch production of citric acid from sucrose has been investigated using Aspergillus niger NCIM 588. Mycelia of A. niger grown on cellulose microfibril forms a uniform and thin mycelial proliferation under controlled conditions of cultivation rich in oxygen. In the fed batch mode using a recycle reactor, the DO of the system was maintained at 20 mg l − 1 using oxygen enriched air. This improved volumetric productivity to 1.85 g l − 1 h − 1 of citric acid, representing an increase of at least 15-fold over results obtained simultaneously using shake-flasks and 1.6-fold over a conventional aerated batch reactor. It was possible to substitute sucrose with sugarcane juice as a carbon source in a fed batch recycle system. An overall specific production rate of citric acid of 0.147 and 0.208 g g − 1 h − 1 was achieved using cane juice and sucrose, respectively. In continuous fermentation, a medium containing 50 g l − 1 of sucrose was allowed to drip through the fabric support at a residence time of 20 h. As a result of interface interaction, a citric acid volumetric productivity of 2.08 g l − 1 h − 1 was achieved for 26 days without any significant loss of productivity. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus niger; Citric acid; Immobilization; Cellulose microfibrils; Morphology; Batch and continuous fermentation
1. Introduction In the past few years, significant studies have been carried out on citric acid production and efforts to achieve higher volumetric productivity under conditions of submerged and surface modes of operation continue [1 – 5]. Implementation of these processes has many biological and operational hurdles. For filamentous fungi, when compared to single cell fermentations, the complex environment in the bioreactor and heterogeneity under agitated conditions could be observed [6]. Fungal morphology changes the physical properties of broth which causes numerous problems in industrial fermenters with respect to gas dispersion, mass and heat transfer and homogenization [6]. Further, in conventional bioreactors, oxygen transfer and mixing are rather inefficient, with a high apparent viscosity of the culture broth [7,8]. The viscosity related problem may * Corresponding author. Tel./fax: + 91-20-5893041. E-mail address: [email protected] (B.D. Kulkarni).
be overcome by ensuring that the mycelial growth is mainly in pellet form rather than in filamentous form [9], as the suspension of pelleted mycelia is usually less viscous than that of filamentous mycelia [10,11]. The size of the pellet and the relative amount of filamentous mycelia varies with fermentation time and intensity of agitation. Mechanical forces induced by turbulent flow chip off the outer mycelia of pellets and the extent of this chipping is a function of the hyphal length and the magnitude of the mechanical force [12]. The study of Wittler et al. [13] on Penicillium chrysogenum shows partial lysis and disintegrated pellet. Mass transfer limitations have been observed in alginate bead entrapped cells [14]. When compared to surface processes, biomass recycle as a means of medium replacement is generally more complicated in submerged fermentation. The possibility of the repeated use of Aspergillus niger biomass was demonstrated using an exchange filtration technique [15]. Anderson et al. [16] have used a disk fermenter for extended and continuous production of citric acid. Sev-
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 1 5 5 - 8
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eral attempts have been reported for continuous production of citric acid using magnetic drum contactors [17], reciprocating-jet-bioreactors [18], hollow fibre reactors [19] and continuous cultures [20]. In addition, several studies report the use of A. niger immobilized on various kinds of solid supports, viz. glass carrier [21], polyurethane foam [22], entrapment using calcium alginate [23,24], entrapment in polyacrylamide gels, [25,26], polypropylene hollow fibers [19], entrapment in agar [27], agarose [28] and cellulose carriers [29]. Citric acid production is an aerobic fermentation process. In submerged mode, the dissolved oxygen level is maintained at : 50% saturation using air as oxygen source. Partial or total lack of aeration, even for a short duration is detrimental and leads to delay in the fermentation period [30]. Tower fermenters are increasingly preferred because of ease of scaling up and the possibility to attain higher dissolved oxygen (DO) levels in the lower segment due to increased hydrostatic pressure [4]. In some reports, oxygen availability has been accomplished by using pure oxygen or using high pressure. At 60 mg l − 1 level of DO, better yields of citric acid are reported by Sato [31]. This was attributed to reduced biomass formation and less substrate diversion for this purpose. The aim of this work was to explore the possibility of achieving high volumetric productivity of citric acid using novel fermentation systems in batch and continuous modes. The study includes controlled cell immobilization and changes in reactor configuration to obtain minimum shear stress. This allows a considerable increase in interfacial area between the medium and mycelium, as well as mycelium and air. Surface fermentation for continuous operation and a recycle continuous flow reactor for fed batch operating conditions have been used to improve the specific rate of citric acid production.
2. Materials and methods
2.1. Micro-organism A. niger NCIM 588 (ATCC 1015) was the best strain screened from the culture bank of the National Collection of Industrial Microorganism (NCL, Pune). The strain was maintained on potato dextrose agar slants at 4°C and subcultured at intervals from 1 to 2 months
2.2. Immobilization of A. niger The cellulosic support and continuous fermentation (method of mycelial immobilization and unit operation) has been described earlier [32]. For batch fermentation, fully entangled mycelia were used directly.
2.3. Fermentation medium Surface and submerged flask cultures: 50–100 g sucrose purified, 0.25 g MgSO4·7H2O, 0.75 g KH2PO4 and 2.0 g NH4NO3 in 1.0 l of distilled water. Trace element solution containing Cu2 + , 0.00006; Zn2 + , 0.00025; Fe3 + , 0.0013 and Mn2 + 0.001 g l − 1 was incorporated, pH was adjusted to 5.5 using 1 M H2SO4. Immobilized mycelia: 100–180 g sucrose, 0.025 g MgSO4·7H2O, 0.075 g KH2PO4 and 0.1 g NH4NO3 in 1 l water was used. Before sterilization, pH of the medium was adjusted to 6.0 using 1 M H2SO4.
2.4. Fermentation procedure 2.4.1. Flask fermentations Surface and submerged culture fermentation was carried out in 1-l Erlenmeyer flasks containing 150 ml and 500-ml Erlenmeyer flasks containing 75 ml of sterile medium, respectively. Spores of a 7-day-old culture of A. niger grown on PDA slope were harvested and suspended in distilled water to obtain a spore density of 3–5 ×107 ml − 1. A 5 ml spore suspension was transferred to each flask and incubated at 28°C. For surface culture, the mycelial mat was allowed to grow for the first 2 days and thereafter, medium samples were collected for analyses at 24-h intervals until 12 days. For shake flask fermentations, spore germination was allowed to take place for the first 12 h and the contents of the flasks were agitated at 120 rpm. Samples were collected thereafter, for analyses at intervals of 24-h until 12 days. 2.4.2. Batch fermentation in recycle reactor and shake flask The support (8 × 70 cm) holding immobilized mycelia was rolled into a spiral and loaded into the reactor. A thin polythene mesh was put in between the folds to avoid mycelial adhesion between two adjacent layers. Fermenter design and operation is shown in Fig. 1. Aeration was carried out using a mixture of air (25 ml min − 1) and oxygen (5 ml min − 1) to attain a level of 20 mg l − 1 of dissolved oxygen. For shake flask fermentation biomass holding support was cut into 2×2 cm pieces and used. 2.4.3. Continuous fermentation Details regarding support and immobilization protocols for continuous operation has been described in an earlier publication [32]. Fresh sugarcane juice was purchased from a local vendor and was used without any additions. It was diluted to a suitable nitrogen level, steam sterilized and used within 2 days.
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2.5. Surface area analysis
3. Experimental results
The surface area of the cellulose microfibrils was analyzed by a single point BET method (Monosorb surface area analyzer, Quanta Chrome Corp., USA). This gave an average value of 11 m2 g − 1 microfibrils after three sets of adsorption and desorption of nitrogen. 2.6. Sugar and acid analyses
3.1. Surface and shake flask fermentation using free mycelia
Feed and unconverted sucrose was hydrolyzed using 2 N HCL at boiling temperature for 15 min into glucose and fructose and analyzed by a di-nitrosalicyclic acid method [33]. Citric acid was analyzed by a pyridine–acetic anhydride method [34] and simple titration against 1 N NaOH. Unreacted sugar was monitored usually by TLC using ethyl acetate, 1-propanol and water in a ratio of 8:7:2 as a mobile phase. The spray reagent used for visualization contained 4%-naphthol in 20:80, ethanol: 2 N sulfuric acid. This method helped visualization of sugars down to 0.05% in the medium. Nitrogen present at different stages of fermentation was analyzed by a micro-Kjeldahl method [35]. Phosphate was determined by a molybdate method [36]. Biomass analysis and scanning electron microscopy protocol has been described earlier [32].
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Experiments were conducted using surface culture and shake flask submerged cultures to provide a baseline for comparing results. Fig. 2 shows typical results during the progress of fermentation. Mycelia of A. niger grew well over a period of 48 h in shake flasks to form pellets of size 2–3 mm. It was also observed that the nitrogen in the medium becomes exhausted and citric acid accumulation occurs. The maximum specific growth rate (vmax) of A. niger was 0.0184 g h − 1 and a fermentation time of 9 days was required to utilize \70% sucrose. In this period, 35 g l − 1 citric acid was accumulated, giving an overall volumetric productivity of 0.125 g l − 1 h − 1 corresponding to a maximum specific production rate of 0.016 g g − 1 h − 1. An extension of fermentation to 13 days did not result in any improvement in specific production rate. These are in good agreement with earlier studies [37]. In surface culture, a thick mat was formed at the end of the 48 h, which gave 62 g l − 1 of citric acid at the termination of fermentation (thirteenth day). Under optimized conditions, the maximum specific growth rate (vmax) of A. niger observed was 0.02 g h − 1. At this
Fig. 1. Schematic diagram of fermentation setup for a recycle bioreactor.
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Fig. 2. Comparison between batch fermentations of citric acid by A. niger. Immobilized cells (IC); free cells (FC). Table 1 Comparison between different modes of fermentation Volumetric productivity (g l−1 h−1) Citric acid yield (g l−1)
Fermentation type Mycelia
Aeration type
Batch, shake flask Batch, shake flask Batch, surface culture Batch, recycle reactor Batch, recycle reactor Batch, recycle reactor Continuous Continuousa
Free Immobilized Free
Air Air Air
0.125 0.43 0.223
35 55 62
65 90 92
Free
Air+O2
1.16
50
78
Immobilized
Air
1.4
52
85
Immobilized
Air+O2
1.85
64
92
Immobilized Immobilized
Air Air
2.08 0.8–2.1b
65 58
96 90
a b
Sugar utilization (%)
Use of cane juice as carbon sources. Values decreased over a period of 10 days.
stage, the overall volumetric productivity was 0.235 g l − 1 h − 1 corresponding to a maximum specific production rate of 0.0176 g g − 1 h − 1. In this culture, a thick mat and hyphal structures protruding above the surface provided greater opportunities for air access. During this experiment, the surface depth ratio of the medium was 1:10 that is reported to be important for this fermentation [38].
sugar was exhausted. The volumetric productivity achieved was 0.43 g g − 1 h − 1 corresponding to a maximum specific production rate of 0.071 g g − 1 h − 1. The results are shown in Fig. 2 and Table 1. At this time the concentration of free citric acid accumulated was 55 g l − 1.
3.3. Batch recycle bioreactor with recirculation of the fermentation broth o6er immobilized mycelia
3.2. Immobilized mycelia in shake flask In another set of experiments, small strips of cellulose support on which mycelia were pregrown (0.62 g per flask) were placed in the shake flask. Citric acid accumulation began at :6 h. The experiment was continued for 11 days at which time almost all the available
Recirculation of the fermentation broth has been reported earlier [18] in order to obtain continuous citric acid production. In the present studies, free mycelia (FM) using oxygen enriched air and immobilized mycelia (IM) with oxygen enriched air were used to check reactor performance and efficiency. In the FM set,
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mycelial pellets were pregrown in shake flasks for 48 h and used with fresh medium. In the immobilized setup the fabric support (8×70 cm) holding 1.1 g biomass was located below the surface of the medium and was placed on a perforated glass base (Fig. 1), which served as an inlet for the circulating medium. This minimizes mycelial detachment from microfibrils. Sterile oxygen enriched air was let into the circulating medium prior to its entry into the reactor by inserting a venturi. This ensured air access throughout the packed bed. During recycle, oxygen enriched medium traverses upward through the capillaries of the fabric. The low flow rate
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ensured a mild current of flowing broth and prevented shear stress. As a result, the cells were not detached from the microfibrils. These immobilized mycelia utilize \ 90% sucrose and pH falls to 1.96 to give 64 g l − 1 citric acid over a period of 70 h (Fig. 3). It can be seen from Fig. 4 that oxygen enriched air reduces the fermentation period by 20 h compared to air and shake flask mode of immobilized cell systems. This improved productivity was reproducible in four subsequent recycles in respect of yield and productivity. Citric acid between 62 and 65 g l − 1 was obtained at the end of every cycle (Fig. 5). The overall productivity
Fig. 3. Batch production of citric acid using sucrose by immobilized A. niger in recycle bioreactor at partial pressure of oxygen at 20 mg l − 1.
Fig. 4. Citric acid production using sucrose by immobilized A. niger in recycle bioreactor. Air and oxygen enriched air has been used at 7 and 20 mg l − 1 of oxygen, respectively.
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Fig. 5. Repeated batch production of citric acid using sucrose by immobilized A. niger in recycle bioreactor. ( points at which medium is replaced with fresh lot).
observed in these cycles was between 1.82 and 1.88 g l − 1 h − 1.
3.5. Continuous production of citric acid using immobilized mycelia
3.4. Utilization of cane juice in batch fermentation
Results obtained using the fed batch system with immobilized mycelia point to the potential benefits of this system in terms of cell recovery and reuse. A support having dimensions of 70× 8× 0.5 cm holds a 105 ml void volume and 1.13 g pregrown biomass. Medium containing 50 g l − 1 of sucrose was allowed to flow through the capillaries of the support. The residence time (RT) to achieve a near complete utilization of sucrose to form citric acid was 20 h. Volumetric productivity observed for this set was 2.08 g l − 1 h − 1. Fermented broth contained 65 g l − 1 of citric acid. This was more than the expected values of citric acid as a result of evaporative concentration (20%). Cane juice was also used to study whether on not it can replace sucrose. On the pregrown mycelia, continuous feeding of cane juice was carried out over a period of 10 days. The productivity declined over a period (Fig. 7). The biomass on the support slowly increased from 1.2 to 2.6 g, giving a visibly thick appearance and limiting air access to the inner layers of mycelia on the fabric. Under these conditions, experimental difficulties such as irregular medium flow and drops dripping through microfibrils were observed.
Replacement of sucrose with cane juice will reduce the cost of the starting material, sucrose to 1/3. Cane juice contains 100– 160 g l − 1 (average 120 g l − 1) of sucrose with other fermentable mono-, di- and polysaccharides. On average, the juice shows a ‘brick value’ (total solid content) ranging between 13 and 20 [39]. This raw material has several advantages as a fermentation feedstock [40]. One of the major hurdles found in the use of sugarcane juice is high levels of metabolizable nitrogen. The content of nitrogen in juice is dependent on the age of the cane; matured cane may have 2/3 N compared to younger cane (:800 mg l − 1). Thus, selecting low nitrogen juice or diluting the available juice are the options used for citric acid production [41]. Table 1 gives data on citric acid production using sucrose and diluted sugarcane juice. The yields were comparable and lead to a considerable saving on raw material costs. In fed batch culture, citric acid production activity was delayed presumably due to initial high levels of nitrogen. In this period, biomass builds up at the cost of nitrogen and as the level of nitrogen reaches 50 mg l − 1, citric acid production starts. In subsequent recycles up to the fifth, there was a biomass build up in every recycle to reach a level of 2.4 g from the starting level of 0.95 g. In the experiments using sucrose, the build up was marginal, reaching a level of 1.3 g from 1.1 g. The data are presented in Fig. 6 and shows that after every cycle, biomass per support rises.
3.6. Effect of water acti6ity on continuous operations Water is required for microbial growth and metabolism of the cell. The term water activity (aw) refers to the unbound water in the environment, around the mycelia. The water activity of the environment is related to osmolarity, which is an important variable
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affecting microbial metabolism. The aw influences microbial growth and biochemical activities and is an important parameter to be considered in surface culture (present continuous mode) and solid state fermentation [42]. To check the effect of aw, five sets of experiments, at aw of 0.25–0.4, 0.5– 0.6, 0.7– 0.8, 0.85– 0.9 and 0.95– 0.98 were carried out under conditions of constant temperature (Table 2). At aw B0.4, the fabric support shrinks disturbing the porosity and consequent decreasing productivity to 0.96 g l − 1 h − 1. Under such condi-
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tions, the air current removes the water contained in the medium as water vapor while moving past the high surface area of the support (11 m2 g − 1). This dries the microenvironment around the mycelia affecting cellular metabolism. Even after decreasing the residence time (increasing medium flow rate) to half (6 h), there was no noticeable improvement. As shown in Table 2, an aw of 0.85 was the minimum necessary for maximum productivity of 2.08 g l − 1 h − 1 citric acid that continued till the end of fermentation.
Fig. 6. Repeated batch production of citric acid using cane juice by immobilized A. niger in recycle bioreactor. In successive cycles, biomass increased to show decreased productivity.
Fig. 7. Continuous production of citric acid using sugarcane juice by immobilized A. niger.
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Table 2 Effect of water activity (aw) on continuous fermentation of citric acida aw
Sucrose utilization (%)
Productivity (g l−1 h−1)
Broth concentration (%)
Citric acid (g l−1)
Running time (days)
0.25–0.40 0.5–0.6 0.7–0.8 0.85–0.9 0.95–0.98
40 9 2 80 95 98 9 2 98 9 2 98 9 2
0.96 1.6 1.92 2.08 2.08
35 28 25 22 20
40 45 60 65 62
4 20 20 26 26
a At residence time 20 h; sucrose, 50 g l−1. The data shown was measured between the third and fifth day when citric acid production stabilized. Evaporation rate has S.E. of 0.12.
4. Discussion The present study had three primary objectives in mind; reducing fermentation time for bioconversion, reuse of same biomass and operating the system under less stringent conditions of sterility. Numbers of experiments, in different modes of reactor operation, were carried out to ascertain the best operating conditions to meet these objectives. In fed batch and continuous modes of fermentation, the productivity values of immobilized cells were higher when compared to those reported in the literature [14,19,22,43]. The maximum volumetric productivity of 2.08 and 1.85 g l − 1 h − 1 was achieved in continuous surface and continuous flow recycle reactor, respectively. The differences in productivity (surface and submerged modes) can be attributed to different morphology and its effects on apparent viscosity that alters mass transfer and mixing characteristics [44]. Fungal morphology has a strong influence on the physical properties of cultivation broth, which cause numerous problems in industrial fermenters with respect to gas dispersion, mass and heat transfer and homogenization [6]. Papagianni et al. [45] have shown that the length of the filaments is the only parameter that could be related to citric acid production. These authors also observed that, for similar yields of citric acid, the morphology of the organism differed in stirred tank and the loop reactor. The support used here for immobilization of A. niger and the mode of fermentation have played important roles in enhancing productivity of citric acid. This is due to different morphology achieved under conditions of submerged and surface modes of fermentations. In submerged fermentation, the mycelia form a thick mat of microfibrils (Fig. 8b), whereas in the surface mode, a protruding mycelial structure was seen in a cobweb like network of free filaments between the microfibrils of fabric (Fig. 8c). A typical microfibril of the support is shown in Fig. 8(a) and has a favorable shape. Convolutions with sharp folds along the microfibrils can also be seen. These microfibril also shows lumens, which may play an important role in medium flow and interfacial interactions during medium movement. The support
used for recycle reactor and continuous fermentation had dimensions of 8× 70 cm. It had an interfacial surface area of 0.53 m2 cm − 2. On such a strip, biomass immobilized was between 1.1 and 2.0 g. This biomass was spread over the support in the form of a thin mycelial film and in-between spaces of microfibrils. Considering the morphology, the surface culture mycelia may have been subjected to less shear stress when compared to the submerged mycelia. Further, the interfacial interactions during surface fermentation may be better due to free access to air exhibiting more productivity. In addition to this, the controlled growth of mycelia over microfibrils under oxygen enriched atmosphere can be advantageous. Using air grown mycelia, many fold increased productivity in submerged fermentation has been reported [19,43,46]. Several studies using fungi report on enhanced formation of organic acids under conditions of strong aeration. Aeration fulfills two functions — oxygenation and heat removal. The oxygen requirement varies with the stage of growth. In submerged fermentations, agitation influences the size of pellets and structure of the pellet’s surface. Stronger agitation in stirred tank reactor results in higher dissolved oxygen tension and more branching of hyphae. The pellets formed could be denser, stronger and exhibit higher tensile strength. In addition to this, the size of the pellet and the relative amount of filamentous mycelia vary with fermentation time and intensity of agitation [12]. Mechanical forces induced by turbulent flow chip off the outer mycelia of pellets and show partially lysed or disintegration of pellet [13]. Poor mixing in conventional bioreactors causes inefficient oxygen transfer [7,8]. Thus, for example, calcium alginate bead immobilized (entrapped) cells have shown limitations in mass transfer [14]. In such cases, the use of pure oxygen or oxygen enriched air can be a very effective way to reduce transport limitations and cell lysis. The viscosity related problem may be overcome by ensuring that mycelial growth is mainly in pellet form rather than in filamentous form. A suspension of pelleted mycelia is usually less viscous than that of filamentous mycelia [9–11]. The relation between the pellet size of A. niger and the oxygen transfer has been elucidated in a report considering the
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morphological characteristics of the pellet [47]. The optimum diameter of the pellet was found to be 2.2 mm which was quite stable in the bubble column bioreactor, leading to significant improvement in citric acid production.
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Our earlier report has shown that increased interfacial interactions lead to a high specific production rate of gluconic acid [32], which was comparable to that obtained using high dissolved oxygen levels at relatively high pressure [46]. In the case of di- and tricarboxylic acids, a high oxygen tension induces an alternate respiratory pathway required for re-oxidation of the glycolytically produced coenzyme, NADH [30,48,49]. An unidentified component of this pathway seems to be stringently dependent on the maintenance of a high oxygen tension [50], as even short interruptions of aeration impair the productivity [30].
5. Conclusion Mycelia of A. niger NCIM 588 were immobilized by adsorption onto cellulose microfibrils of a porous fabric and used in batch and continuous production of citric acid. The rise in the specific productivity of citric acid fermentation either in submerged or surface mode can be a result of reduced stress conditions on mycelia and less shear stress during operating conditions as described here as against their high levels in conventional aerated or agitated bioreactors. The data presented in this work suggests the possibility of improving the overall productivity of citric acid fermentation using a novel system. The individual parameters contributing to such improvement have been discussed. The results obtained have been compared to data available in the literature using related systems. Fungal pellet and immobilized cells show a great variability in morphology, which is an important consideration in heat and mass transfer. Apart from this, interface interaction and metabolite excretion are the major advantages in immobilization techniques. Highly improved interface interaction between mycelia and oxygen, as well as substrate, improves the diffusion of substrate; continuous removal of the metabolite formed also contributes to improved productivity.
Acknowledgements The authors acknowledge financial support provided by the Council of Scientific and Industrial Research (CSIR) for carrying out these investigations.
References Fig. 8. Scanning electron micrographs (SEM) of (a) a microfibril on which A. niger NCIM 588 immobilized; (b) SEM of immobilized mycelia at submerged mode of fermentation, magnification (1 K × ); (c) SEM of immobilized mycelia at surface mode of fermentation, magnification (300 × ).
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