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Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation
Bioresource Technology 102 (2011) 9345–9349
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Case Study
Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation Zhengxiong Zhou a,b, Guocheng Du b, Zhaozhe Hua b, Jingwen Zhou a,b,⇑, Jian Chen a,b,⇑ a b
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China
a r t i c l e
i n f o
Article history: Received 3 May 2011 Received in revised form 25 July 2011 Accepted 28 July 2011 Available online 10 August 2011 Keywords: Organic acids Fumarate Moulds Pellet diameter Morphology
a b s t r a c t The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fumaric acid, one of the top 12 building-block organic acids, has important roles as an acidulant and an additive. Also, fumaric acid derivation is widely used to provide feedstock for polymerization, esterification reactions and for medicines (Jones et al., 2010). Currently, fumaric acid is produced in two ways, chemical conversion from maleic anhydride or biological conversion by fungi (Roa Engel et al., 2008). However, as petroleum is depleting, there is increasing interest in fumaric acid production by fermentation, which was started in the 1940s but was discontinued. Rhizopus sp. were identified as the best fumarate-producing strains among different microorganisms tested, with Rhizopus delemar (Rhizopus oryzae) NRRL1526 one of the best strains (Oda et al., 2003). Different process optimization means were attempted to improve the fumaric acid production. Among of the former reports, the highest fumaric acid production by R. delemar was 85 g/L in submerged fermentation with an integrated system of simultaneous fermentation–adsorption and recovery of fumaric acid by a coupled adsorption column (Cao et al., 1996). During submerged growth, R. delemar may grow as suspended mycelia, clumps or pellets forms (Teng et al., 2009). The factors that affect fungal ⇑ Corresponding authors at: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Tel.: +86 510 85329031; fax: +86 510 85918309. E-mail addresses: [email protected] (J. Zhou), jchen@jiangnan. edu.cn (J. Chen). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.120
morphology include the properties of the individual hyphal elements and environmental conditions. However, as the complicated of R. delemar genetic manipulation, there was no interpretation about pellet formation in genetics to date. Nielsen et al. (1995) showed that the morphology of fungi germinated from noncoagulated spores is influenced by initial spore concentration and agitation rate on agglomeration. van Suijdam et al. (2003) found that the influences on formation of pellets were inoculum concentration, polymer additives and shearing forces in a fermentor. Engel et al. (2011) indicated that decreasing pH but increasing shaking frequency and flask size resulted in smaller average pellet diameter. Liao et al. (2008) found that adding CaCO3 into the medium could affect the pellet diameter of R. oryzae, but pH of culture medium had no significant influence on pellet formation (Bai et al., 2003). Others found that the factors influencing fungal morphology included medium composition (e.g. carbon source, nitrogen source and metal ions – Mg2+, Zn2+, K+ and Na+), pH, additives and culture temperature (Zhou et al., 2000). These reports indicated that the morphology formation of R. delemar should be a significant factor for the fumaric acid production. However, a comprehensive investigation of pellet formation and growth is lacking, and there is no relationship known between macroscopic morphology and fumaric acid production by R. delemar. To our knowledge, there is no agreement in the literature about the influence of pellet size on production of fumaric acid. Zhou et al. (2000) insisted that almost all fumaric acid is produced by the external zone of pellets, which means low viability zones exist within large pellets. Yang et al. (1995) showed one advantage
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of pellets is that they are easily separated from the fermentation broth and can be recycled. Karube et al. (1977) showed that the mycelia pellets used as carrier material for immobilized enzymes, which can provide a better starting material for production of immobilized mycelial particles, resulted in a stable continuous process. Others believe that fungal growth in pellet-form benefits industrial fermentation, as it reduces the viscosity of the medium and the possibility of wrapping around the impellers of a stirredtank bioreactor (Li et al., 2000; Liu et al., 2006; McIntyre et al., 2001); furthermore, it allows for fungal biomass reuse and improves mass and oxygen transfers (Rodriguez Porcel et al., 2005; Tixier et al., 2003). However, other factors may not benefit from such morphology, mycelium in a large pellet was associated with lower specific protease activities but higher specific glucoamylase activities, and also that Pleurotus ostreatus in smaller pellets was associated with lower laccase activity but higher biodegradation rates (Kim et al., 2002; Kim and Song, 2009). Although the factors that affect pellet formation have been investigated and the advantages of application of pellets have been reviewed in a qualitative fashion, no quantitative relationships among them have been discovered. This article focuses on not only the simple relationship among major kinds of factors and fungal morphology, but also how the relationship between pellet diameter and approximate yield can enhance fumaric acid production. Morphology and pellet size were manipulated by means of pre-culture composition, and fungal morphology was characterized and quantified using an image processing system.
2. Methods 2.1. Microorganisms R. delemar NRRL1526 was obtained from the National Center for Agricultural Utilization Research (Peoria, Illinois, USA). The strain was first cultured on sporulation medium (SM) slants, and further propagated on SM at 30 °C in 90-mm dishes to form spores. The spores were washed from the agar with sterile distilled water. The spore concentration of the suspension was controlled to 1 108 spores/mL.
2.2. Media Sporulation medium was used for R. delemar to form spores. It consisted of (g/L): glucose, 4.0; lactose, 6.0; glycerol, 10.0; corn steep liquor, 1.0; urea, 0.6; tryptone, 1.6; MgSO47H2O, 0.3; ZnSO47H2O, 0.088; FeSO47H2O, 0.25; CuSO4, 0.005; KH2PO4, 0.4; MnSO44H2O, 0.05; KCl, 0.4; NaCl, 40; and agar, 20. In spore-germination experiments, medium (SG) used for pellet formation, and consisted of (g/L): glucose, 20; soybean peptone, 6; and CaCO3, 6. The medium used for suspend mycelia fungi consisted of (g/L): glucose, 20; soybean peptone, 6; inorganic ions; CaCO3, 6. The medium used for clump fungi consisted of (g/L): glucose, 20; CaCO3, 6; inorganic ions. The mineral ions included (g/L): KH2PO4, 1; MgSO47H2O, 0.25; ZnSO47H2O, 0.066; FeSO47H2O, 0.01. Also, diverse size of pellet were prepared via culturing in different pre-culture medium. The medium consisted of: glucose, soybean peptone, CaCO3. The concentrations of glucose were 10–30 g/L, soybean peptone were 2–10 g/L, CaCO3 was 6 g/L. In fungal-fermentation experiments, acid production medium (AP) was used, and consisted of (g/L): glucose, 100; (NH4)2SO4, 2; KH2PO4, 0.3; MgSO47H2O, 0.4; ZnSO47H2O, 0.044; FeSO47H2O, 0.01; CaCO3, 80; and methanol, 15. All medium were sterilized by autoclaving at 115 °C for 15 min before the methanol being added to the AP under sterilized conditions.
2.3. Culture conditions Spores were inoculated 4% (v/v) into 250-mL Erlenmeyer flasks containing 50 mL of culture medium. Cultivation was carried out at 30 °C, 200 rpm in rotary shakers for 30 h. Fungal with diverse morphology or different size of pellet were obtained for fermentation, and transferred 10% (v/v) into another 250-mL flask, with 50 mL of AP, or transferred 10% (v/v) into 7 L stirred-tank bioreactor, with 4 L of AP, cultured at 30 °C, 200 rpm, 1 vvm air, and maintained pH 5.0 for 3 days. 2.4. Analytical methods 2.4.1. HPLC assay A high-performance liquid chromatograph (HPLC) with a refractive index detector and UV detector at 210 nm, an automatic injector, and an integrator was used to analyze fumaric acid, and the major byproduct concentrations. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.5 mL/min though a Bio-Rad Aminex HPX-87 H ion exclusion column at 35 °C. 2.4.2. Final fumaric acid concentration Owing to the low solubility of calcium fumarate, the final culture broth was diluted by addition of distilled water to dissolve the potential calcium fumarate precipitate, and then HCl to neutralize the excess CaCO3. The final culture broth was heated at 80 °C until the broth was clear in order to help fumarate dissolve. Samples were collected for HPLC analysis. 2.4.3. Dry weight Due to of the low solubility of calcium fumarate, the broth was heated until it was clear. Once the broth was cooled, it was centrifuged at 8000 g for 10 min to recover the biomass. Then the solid fraction was washed with deionized water completely, dried at 105 °C for 24 h, and weighed to yield the final dry weight. 2.4.4. Morphologies When pre-culture was finished, samples with different morphologies were taken from the culture, and quantified using a manual image analysis system consisting of a microphotograph, a CCD camera, a PC with a frame-grabber, and image analysis software. Pellet diameter was the average size of pellets. 2.5. Statistical analysis Parametric statistical tests were used to determine significant correlations between parameters (Pearson correlation analysis). Correlations were considered significant at P < 0.05. Statistical analyses were using by SPSS 16.0 software. The degree of variability was estimated by the coefficient of variation (CV) for different samples. 3. Results and discussion 3.1. Effects of culture medium on pellet formation Only soybean peptone and inorganic ions had significant effects on pellet formation; however, both initial glucose and soybean concentrations had direct impacts on pellet diameter and cell dry mass (DCW). Different sized pellets were observed during the cultivation of R. delemar at different initial glucose and soybean concentrations, but with fixed culture temperature and shaking frequency (Fig. 1A and B). The pellet diameters decreased range from 0.75 to 0.55 mm and 0.69 to 0.55 mm, respectively, when glucose and soybean concentrations were separately increased. When
Z. Zhou et al. / Bioresource Technology 102 (2011) 9345–9349
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A
B
Fig. 1. Effects of substance concentrations on pellet diameter and dry cell mass during pre-culture: (A) glucose concentrations of 10–30 g/L; (B) soybean peptone concentrations of 2–10 g/L; pellet diameter (h), DCW (j).
Fig. 3. Effects of culture temperature on pellet diameter and dry cell mass during pre-culture with the same medium compositions used at 30 °C and 200 rpm: pellet diameter (h), DCW (j).
Fungal morphology in submerged culture is influenced by medium composition, pH, the size of the inoculum and other physical environmental factors. Teng et al. (2009) concluded that different inocula and shear stresses resulted in distinctive morphologies. Liao et al. (2007) reported that pellet formation occurred when R. oryzae ATCC20344 culture contained soybean peptone, but no inorganic ions, which is consistent with the present experiment (Fig. 2). Fungal morphology varied during pre-culture with different combinations of factors (Fig. 2). The morphology was: A, clumps; B, pellets; and C, suspended mycelia. When soybean peptone as nitrogen resource in the medium, pellet formed, but no pellet formed when there were inorganic ions in the medium. Thus, soybean peptone in the medium has a positive effect on pellet formation, and inorganic ions have a negative effect. 3.2. Effects of temperature on pellet formation
the initial glucose and soybean concentrations increased beyond a certain range this resulted in an overall increase in pellet diameter; however, the largest pellet diameter was 0.63 mm. The DCW profiles illustrated the reciprocal tendency of DCW to that of pellet diameter (Fig. 1A and B). Therefore, it was beneficial to have appropriate initial substrate concentrations to produce smaller pellets and so greater biomass. We obtained the minimum diameter and maximum biomass when the initial glucose and soybean peptone concentrations were 20 and 6 g/L, respectively.
A
B
The effects of six different pre-culture temperatures on fungal pellet diameter were studied (Fig. 3). Smaller pellet diameter (0.57 mm) but greater biomass (7.58 g/L) was obtained at 30 °C; however, corresponding values at 37 °C were 0.87 mm and 6.82 g/L. Thus, pellet diameter became smaller with higher temperature at <30 °C, but became larger at >30 °C. Furthermore, there was a reciprocal relationship between pellet diameter and biomass. Our results suggested that the temperature was also a
C
Fig. 2. Effects of different medium components on fungal morphology during pre-culture. Medium components: A, glucose, inorganic ions and CaCO3; B, glucose, soybean peptone and CaCO3; C, glucose, inorganic ions, soybean peptone and CaCO3.
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Z. Zhou et al. / Bioresource Technology 102 (2011) 9345–9349 Table 1 Distribution of fumaric acid yield with different fungi morphology at 30 °C and 200 rpm in a flask. Spores were inoculated into 250-mL Erlenmeyer flasks containing 50 mL of different pre-culture mediums, cultured at 30 °C, 200 rpm for 30 h. Fungal with different morphology were obtained, and transferred into another 250-mL flask, with 50 mL of AP, cultured at 30 °C, 200 rpm for 3 d. Pre-culture medium components: clump (glucose, inorganic ions and CaCO3); suspend mycelia (glucose, inorganic ions, soybean peptone and CaCO3); pellet (glucose, soybean peptone and CaCO3). The mineral ions included (g/L): KH2PO4, 1; MgSO47H2O, 0.25; ZnSO47H2O, 0.066; FeSO47H2O, 0.01.
a
Fungi morphology
Fumarate (g/L)a
Clump Suspend mycelia Pellet
26.64 ± 2.54 31.85 ± 2.03 38.93 ± 1.57
The mean value of parallel determination.
significant factor for the pellet formation. A slight change in temperature could result in different morphology.
Fig. 4. Effects of pellet size on fumaric acid production at 30 °C and 200 rpm in a flask. Different size of pellet obtained with culturing in diverse substrates concentration of pre-culture (e.g., glucose, 10–30 g/L; soybean peptone, 2–10 g/L) or diverse pre-culture temperatures (28–37 °C).
3.3. Fumaric acid production based on morphologies optimization The effect of different morphologies on the yield of fumaric acid was investigated. In the present study, the fumaric acid concentration of pellet fermentation reached 38.93 g/L in 72 h of culture compared to clump fermentation that produced only 26.64 g/L (Table 1). Fumaric acid production by R. delemar first appeared at 16 h regardless of morphology (Table 1), from thereon both rapidly increased until 64 h and then only increased slightly until the end of fermentation. The results further demonstrated that fungal culture as pellets can yield fumaric acid more quickly and increase the total yield. Therefore, morphology was a very important factor in fungal fermentation. This may be interpreted that most fermentation broths show non-Newtonian flow behavior while cultures with the desired pellet morphology show the lowest suspension viscosity and broth rheology close to Newtonian flow behavior (Gogus et al., 2006). 3.4. Effect of pellet size on fumaric acid production Influence of pellet diameter on fumaric acid production was observed in flask fermentations. As previously reported, suitable pellet size benefits yield (Feng et al., 2004). We also found that R. delemar in submerged fermentation yielded more fumaric acid when pellet diameter was small, with a lower yield for larger pellets, after 72 h of cultivation in a flask (Fig. 3). For pellet diameter of 0.55 mm, the fumaric acid production was the highest (39.56 g/ L), with shaking rate of 200 rpm. The larger pellet (0.78 mm) resulted in less fumaric acid produced (30.35 g/L). The highest yield (Fig. 4) of fumaric acid was obtained for pellets of smaller diameter (Table 2), for mass transfer at the liquid– solid interface between the cell surface and medium is easier than for large pellets. Evermore, the inner zone of pellets was relatively inactive with active growth of fungi focused on their surface. Effective fungal fermentation needs more active zones to secrete more fumaric acid. All the above demonstrated that with more active zones, there was higher fumaric acid yields. 3.5. Process optimization of fungi related products based on morphology High-viscosity fermentation can lead to limitations in mixing of nutrients and in oxygen mass transfer, which can result in low
Table 2 The schema of pellet size distribution. Different size of pellet obtained with culturing in diverse substrates concentration of pre-culture (e.g., glucose, 10–30 g/L; soybean peptone, 2–10 g/L) or diverse pre-culture temperatures (28–37 °C). Culture conditions Concentration (g/L)
Temperature (°C)
G, G, G, G, G,
30 30 30 30 35
20; 20; 30; 20; 20;
S, S, S, S, S,
6 8 6 2 6
Peller diameter (mm)
Standard deviation
0.55 0.58 0.63 0.69 0.78
0.11 0.12 0.15 0.13 0.15
G: glucose. S: soybean peptone.
yields. Therefore, we could get the largest yield in a stirred bioreactor tank with R. delemar fermented in small pellets, which was consistent with the profile of process optimization of fungi related products based on morphology (Fig. 5). Glucose consumption and fumaric acid production were more rapid when R. delemar was fermented in pellets rather than in clumps. Even fungi fermented in suspended mycelia at the beginning reverted to clumps eventually. We obtained the highest yield of fumaric acid (35.42 g/L) when fungi were grown in pellets, and the lowest yield (26.87 g/L) when in clumps. The results illustrated the control of mycelial morphology is a prerequisite to ensure increased productivity in industry applications (Papagianni and Mattey, 2006). Fungi in large pellets cause decreased substrate consumption rate and led to lower biomass concentration compared to small pellets (Sitanggang et al., 2010). Furthermore, the proportion of metabolically active biomass was restricted to the outer zone of fungi for R. delemar (Papagianni and Moo-Young, 2002).
4. Conclusion R. delemar NRRL1526 was used to produce fumaric acid cultured in flask and fermentor systems. Soybean peptone and inorganic ions affected pellets formation, which had a significant influence on fumaric acid yield. The pellet diameter of 0.55 mm at 30 °C, 200 rpm and pH 5.0 yield highest fumaric acid (39.56 g/L) in flask
Z. Zhou et al. / Bioresource Technology 102 (2011) 9345–9349
Fig. 5. Time courses of fumarate production (solid) and glucose consumption (open) by R. delemar NRRL1526 of different morphology in a stirred-tank bioreactor operated at 30 °C, and 200 rpm, pH 5.0 and 1.0 vvm air: square, clump; triangle, pellet; spherical, suspended mycelia. Different morphology of fungi obtained with diverse medium compositions. Pre-culture medium components: clump (glucose, inorganic ions and CaCO3); suspend mycelia (glucose, inorganic ions, soybean peptone and CaCO3); pellet (glucose, soybean peptone and CaCO3). The mineral ions included (g/L): KH2PO4, 1; MgSO47H2O, 0.25; ZnSO47H2O, 0.066; FeSO47H2O, 0.01.
fermentation, and 35.42 g/L in stirred bioreactor tank fermentation. Although the reasons for pellets formation were found some years ago, they have not yet been implemented in fermentation. However, the macroscopic effects of pellet diameter on fumaric acid yield can be directly implemented in the fermentation industry.
Acknowledgements This work was supported by grants from the Key Program of National Natural Science Foundation of China (No. 20836003), the Major State Basic Research Development Program of China (973 Program, No. 2007CB714306, 2010CB535014), the Research Program of Sate Key Laboratory of Food Science and Technology, Jiangnan University (No. SKLF-TS-200901), the Priority Academic Program Development of Jiangsu Higher Education Institutions and the 111 Project (No. 111-2-06).
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Cao, N., Du, J., Gong, C., Tsao, G., 1996. Simultaneous production and recovery of fumaric acid from immobilized Rhizopus oryzae with a rotary biofilm contactor and an adsorption column. Appl. Environ. Microb. 62, 2926–2931. Engel, C., van Gulik, W., Marang, L., van der Wielen, L., Straathof, A., 2011. Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae. Enzyme Microb. Tech. 48, 39–47. Feng, K.C., Rou, T.M., Liu, B.L., Tzeng, Y.M., Chang, Y.N., 2004. Effect of fungal pellet size on the high yield production of destruxin B by Metarhizium anisopliae. Enzyme Microb. Tech. 34, 22–25. Gogus, N., Tari, C., Oncu, S., Unluturk, S., Tokatli, F., 2006. Relationship between morphology, rheology and polygalacturonase production by Aspergillus sojae ATCC20235 in submerged cultures. Biochem. Eng. J. 32, 171–178. Jones, J., Colquitt, J., Shepherd, J., Harris, P., Cooper, K., 2010. Tenofovir disoproxil fumarate for the treatment of chronic hepatitis B. Health Technol. Asses. (Suppl. 1). Karube, I., Hirano, K.I., Suzuki, S., 1977. Glucose oxidase pellets. Biotechnol. Bioeng. 19, 1233–1238. Kim, H.J., Kim, J.H., Oh, H.J., Shin, C.S., 2002. Morphology control of Monascus cells and scale-up of pigment fermentation. Process Biochem. 38, 649–655. Kim, Y.M., Song, H.G., 2009. Effect of fungal pellet morphology on enzyme activities involved in phthalate degradation. J. Microbiol. 47, 420–424. Li, Z., Shukla, V., Fordyce, A., Pedersen, A., Wenger, K., Marten, M., 2000. Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale. Biotechnol. Bioeng. 70, 300–312. Liao, W., Liu, Y., Frear, C., Chen, S., 2007. A new approach of pellet formation of a filamentous fungus-Rhizopus oryzae. Bioresource Technol. 98, 3415–3423. Liao, W., Liu, Y., Frear, C., Chen, S.L., 2008. Co-production of fumaric acid and chitin from a nitrogen-rich lignocellulosic material – dairy manure – using a pelletized filamentous fungus Rhizopus oryzae ATCC20344. Bioresource Technol. 99, 5859– 5866. Liu, Y., Liao, W., Liu, C., Chen, S., 2006. Optimization of L-(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL395. Appl. Biochem. Biotech. 129–132, 844–853. McIntyre, M., Mller, C., Dynesen, J., Nielsen, J., 2001. Metabolic engineering of the morphology of Aspergillus. Metab. Eng. 77, 103–128. Nielsen, J., Johansen, C.L., Jacobsen, M., Krabben, P., Villadsen, J., 1995. Pellet formation and fragmentation in submerged cultures of Penicillium chrysogenum and its relation to penicillin production. Biotechnol. Progr. 11, 93–98. Oda, Y., Yajima, Y., Kinoshita, M., Ohnishi, M., 2003. Differences of Rhizopus oryzae strains in organic acid synthesis and fatty acid composition. Food Microbiol. 20, 371–375. Papagianni, M., Mattey, M., 2006. Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology. Microb. Cell Fact. 5, 3. Papagianni, M., Moo-Young, M., 2002. Protease secretion in glucoamylase producer Aspergillus niger cultures: fungal morphology and inoculum effects. Process Biochem. 37, 1271–1278. Roa Engel, C.A., Straathof, A.J.J., Zijlmans, T.W., van Gulik, W.M., van der Wielen, L.A.M., 2008. Fumaric acid production by fermentation. Appl. Microbiol. Biot. 78, 379–389. Rodriguez Porcel, E., Casas Lopez, J., Sanchez Perez, J., Fernandez Sevilla, J., Chisti, Y., 2005. Effects of pellet morphology on broth rheology in fermentations of Aspergillus terreus. Biochem. Eng. J. 26, 139–144. Sitanggang, A., Wu, H., Wang, S., Ho, Y., 2010. Effect of pellet size and stimulating factor on the glucosamine production using Aspergillus sp. BCRC31742. Bioresource Technol. 101, 3595–3601. Teng, Y., Xu, Y., Wang, D., 2009. Changes in morphology of Rhizopus chinensis in submerged fermentation and their effect on production of mycelium-bound lipase. Bioproc. Biosyst. Eng. 32, 397–405. Tixier, N., Guibaud, G., Baudu, M., 2003. Determination of some rheological parameters for the characterization of activated sludge. Bioresource Technol. 90, 215–220. Yang, C., Lu, Z., Tsao, G.T., 1995. Lactic acid production by pellet-form Rhizopus oryzae in a submerged system. Appl. Biochem. Biotech. 51, 57–71. Zhou, Y., Du, J., Tsao, G., 2000. Mycelial pellet formation by Rhizopus oryzae ATCC20344. Appl. Biochem. Biotech. 84, 779–789.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Case Study
Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation Zhengxiong Zhou a,b, Guocheng Du b, Zhaozhe Hua b, Jingwen Zhou a,b,⇑, Jian Chen a,b,⇑ a b
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China
a r t i c l e
i n f o
Article history: Received 3 May 2011 Received in revised form 25 July 2011 Accepted 28 July 2011 Available online 10 August 2011 Keywords: Organic acids Fumarate Moulds Pellet diameter Morphology
a b s t r a c t The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Fumaric acid, one of the top 12 building-block organic acids, has important roles as an acidulant and an additive. Also, fumaric acid derivation is widely used to provide feedstock for polymerization, esterification reactions and for medicines (Jones et al., 2010). Currently, fumaric acid is produced in two ways, chemical conversion from maleic anhydride or biological conversion by fungi (Roa Engel et al., 2008). However, as petroleum is depleting, there is increasing interest in fumaric acid production by fermentation, which was started in the 1940s but was discontinued. Rhizopus sp. were identified as the best fumarate-producing strains among different microorganisms tested, with Rhizopus delemar (Rhizopus oryzae) NRRL1526 one of the best strains (Oda et al., 2003). Different process optimization means were attempted to improve the fumaric acid production. Among of the former reports, the highest fumaric acid production by R. delemar was 85 g/L in submerged fermentation with an integrated system of simultaneous fermentation–adsorption and recovery of fumaric acid by a coupled adsorption column (Cao et al., 1996). During submerged growth, R. delemar may grow as suspended mycelia, clumps or pellets forms (Teng et al., 2009). The factors that affect fungal ⇑ Corresponding authors at: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Tel.: +86 510 85329031; fax: +86 510 85918309. E-mail addresses: [email protected] (J. Zhou), jchen@jiangnan. edu.cn (J. Chen). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.120
morphology include the properties of the individual hyphal elements and environmental conditions. However, as the complicated of R. delemar genetic manipulation, there was no interpretation about pellet formation in genetics to date. Nielsen et al. (1995) showed that the morphology of fungi germinated from noncoagulated spores is influenced by initial spore concentration and agitation rate on agglomeration. van Suijdam et al. (2003) found that the influences on formation of pellets were inoculum concentration, polymer additives and shearing forces in a fermentor. Engel et al. (2011) indicated that decreasing pH but increasing shaking frequency and flask size resulted in smaller average pellet diameter. Liao et al. (2008) found that adding CaCO3 into the medium could affect the pellet diameter of R. oryzae, but pH of culture medium had no significant influence on pellet formation (Bai et al., 2003). Others found that the factors influencing fungal morphology included medium composition (e.g. carbon source, nitrogen source and metal ions – Mg2+, Zn2+, K+ and Na+), pH, additives and culture temperature (Zhou et al., 2000). These reports indicated that the morphology formation of R. delemar should be a significant factor for the fumaric acid production. However, a comprehensive investigation of pellet formation and growth is lacking, and there is no relationship known between macroscopic morphology and fumaric acid production by R. delemar. To our knowledge, there is no agreement in the literature about the influence of pellet size on production of fumaric acid. Zhou et al. (2000) insisted that almost all fumaric acid is produced by the external zone of pellets, which means low viability zones exist within large pellets. Yang et al. (1995) showed one advantage
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of pellets is that they are easily separated from the fermentation broth and can be recycled. Karube et al. (1977) showed that the mycelia pellets used as carrier material for immobilized enzymes, which can provide a better starting material for production of immobilized mycelial particles, resulted in a stable continuous process. Others believe that fungal growth in pellet-form benefits industrial fermentation, as it reduces the viscosity of the medium and the possibility of wrapping around the impellers of a stirredtank bioreactor (Li et al., 2000; Liu et al., 2006; McIntyre et al., 2001); furthermore, it allows for fungal biomass reuse and improves mass and oxygen transfers (Rodriguez Porcel et al., 2005; Tixier et al., 2003). However, other factors may not benefit from such morphology, mycelium in a large pellet was associated with lower specific protease activities but higher specific glucoamylase activities, and also that Pleurotus ostreatus in smaller pellets was associated with lower laccase activity but higher biodegradation rates (Kim et al., 2002; Kim and Song, 2009). Although the factors that affect pellet formation have been investigated and the advantages of application of pellets have been reviewed in a qualitative fashion, no quantitative relationships among them have been discovered. This article focuses on not only the simple relationship among major kinds of factors and fungal morphology, but also how the relationship between pellet diameter and approximate yield can enhance fumaric acid production. Morphology and pellet size were manipulated by means of pre-culture composition, and fungal morphology was characterized and quantified using an image processing system.
2. Methods 2.1. Microorganisms R. delemar NRRL1526 was obtained from the National Center for Agricultural Utilization Research (Peoria, Illinois, USA). The strain was first cultured on sporulation medium (SM) slants, and further propagated on SM at 30 °C in 90-mm dishes to form spores. The spores were washed from the agar with sterile distilled water. The spore concentration of the suspension was controlled to 1 108 spores/mL.
2.2. Media Sporulation medium was used for R. delemar to form spores. It consisted of (g/L): glucose, 4.0; lactose, 6.0; glycerol, 10.0; corn steep liquor, 1.0; urea, 0.6; tryptone, 1.6; MgSO47H2O, 0.3; ZnSO47H2O, 0.088; FeSO47H2O, 0.25; CuSO4, 0.005; KH2PO4, 0.4; MnSO44H2O, 0.05; KCl, 0.4; NaCl, 40; and agar, 20. In spore-germination experiments, medium (SG) used for pellet formation, and consisted of (g/L): glucose, 20; soybean peptone, 6; and CaCO3, 6. The medium used for suspend mycelia fungi consisted of (g/L): glucose, 20; soybean peptone, 6; inorganic ions; CaCO3, 6. The medium used for clump fungi consisted of (g/L): glucose, 20; CaCO3, 6; inorganic ions. The mineral ions included (g/L): KH2PO4, 1; MgSO47H2O, 0.25; ZnSO47H2O, 0.066; FeSO47H2O, 0.01. Also, diverse size of pellet were prepared via culturing in different pre-culture medium. The medium consisted of: glucose, soybean peptone, CaCO3. The concentrations of glucose were 10–30 g/L, soybean peptone were 2–10 g/L, CaCO3 was 6 g/L. In fungal-fermentation experiments, acid production medium (AP) was used, and consisted of (g/L): glucose, 100; (NH4)2SO4, 2; KH2PO4, 0.3; MgSO47H2O, 0.4; ZnSO47H2O, 0.044; FeSO47H2O, 0.01; CaCO3, 80; and methanol, 15. All medium were sterilized by autoclaving at 115 °C for 15 min before the methanol being added to the AP under sterilized conditions.
2.3. Culture conditions Spores were inoculated 4% (v/v) into 250-mL Erlenmeyer flasks containing 50 mL of culture medium. Cultivation was carried out at 30 °C, 200 rpm in rotary shakers for 30 h. Fungal with diverse morphology or different size of pellet were obtained for fermentation, and transferred 10% (v/v) into another 250-mL flask, with 50 mL of AP, or transferred 10% (v/v) into 7 L stirred-tank bioreactor, with 4 L of AP, cultured at 30 °C, 200 rpm, 1 vvm air, and maintained pH 5.0 for 3 days. 2.4. Analytical methods 2.4.1. HPLC assay A high-performance liquid chromatograph (HPLC) with a refractive index detector and UV detector at 210 nm, an automatic injector, and an integrator was used to analyze fumaric acid, and the major byproduct concentrations. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.5 mL/min though a Bio-Rad Aminex HPX-87 H ion exclusion column at 35 °C. 2.4.2. Final fumaric acid concentration Owing to the low solubility of calcium fumarate, the final culture broth was diluted by addition of distilled water to dissolve the potential calcium fumarate precipitate, and then HCl to neutralize the excess CaCO3. The final culture broth was heated at 80 °C until the broth was clear in order to help fumarate dissolve. Samples were collected for HPLC analysis. 2.4.3. Dry weight Due to of the low solubility of calcium fumarate, the broth was heated until it was clear. Once the broth was cooled, it was centrifuged at 8000 g for 10 min to recover the biomass. Then the solid fraction was washed with deionized water completely, dried at 105 °C for 24 h, and weighed to yield the final dry weight. 2.4.4. Morphologies When pre-culture was finished, samples with different morphologies were taken from the culture, and quantified using a manual image analysis system consisting of a microphotograph, a CCD camera, a PC with a frame-grabber, and image analysis software. Pellet diameter was the average size of pellets. 2.5. Statistical analysis Parametric statistical tests were used to determine significant correlations between parameters (Pearson correlation analysis). Correlations were considered significant at P < 0.05. Statistical analyses were using by SPSS 16.0 software. The degree of variability was estimated by the coefficient of variation (CV) for different samples. 3. Results and discussion 3.1. Effects of culture medium on pellet formation Only soybean peptone and inorganic ions had significant effects on pellet formation; however, both initial glucose and soybean concentrations had direct impacts on pellet diameter and cell dry mass (DCW). Different sized pellets were observed during the cultivation of R. delemar at different initial glucose and soybean concentrations, but with fixed culture temperature and shaking frequency (Fig. 1A and B). The pellet diameters decreased range from 0.75 to 0.55 mm and 0.69 to 0.55 mm, respectively, when glucose and soybean concentrations were separately increased. When
Z. Zhou et al. / Bioresource Technology 102 (2011) 9345–9349
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A
B
Fig. 1. Effects of substance concentrations on pellet diameter and dry cell mass during pre-culture: (A) glucose concentrations of 10–30 g/L; (B) soybean peptone concentrations of 2–10 g/L; pellet diameter (h), DCW (j).
Fig. 3. Effects of culture temperature on pellet diameter and dry cell mass during pre-culture with the same medium compositions used at 30 °C and 200 rpm: pellet diameter (h), DCW (j).
Fungal morphology in submerged culture is influenced by medium composition, pH, the size of the inoculum and other physical environmental factors. Teng et al. (2009) concluded that different inocula and shear stresses resulted in distinctive morphologies. Liao et al. (2007) reported that pellet formation occurred when R. oryzae ATCC20344 culture contained soybean peptone, but no inorganic ions, which is consistent with the present experiment (Fig. 2). Fungal morphology varied during pre-culture with different combinations of factors (Fig. 2). The morphology was: A, clumps; B, pellets; and C, suspended mycelia. When soybean peptone as nitrogen resource in the medium, pellet formed, but no pellet formed when there were inorganic ions in the medium. Thus, soybean peptone in the medium has a positive effect on pellet formation, and inorganic ions have a negative effect. 3.2. Effects of temperature on pellet formation
the initial glucose and soybean concentrations increased beyond a certain range this resulted in an overall increase in pellet diameter; however, the largest pellet diameter was 0.63 mm. The DCW profiles illustrated the reciprocal tendency of DCW to that of pellet diameter (Fig. 1A and B). Therefore, it was beneficial to have appropriate initial substrate concentrations to produce smaller pellets and so greater biomass. We obtained the minimum diameter and maximum biomass when the initial glucose and soybean peptone concentrations were 20 and 6 g/L, respectively.
A
B
The effects of six different pre-culture temperatures on fungal pellet diameter were studied (Fig. 3). Smaller pellet diameter (0.57 mm) but greater biomass (7.58 g/L) was obtained at 30 °C; however, corresponding values at 37 °C were 0.87 mm and 6.82 g/L. Thus, pellet diameter became smaller with higher temperature at <30 °C, but became larger at >30 °C. Furthermore, there was a reciprocal relationship between pellet diameter and biomass. Our results suggested that the temperature was also a
C
Fig. 2. Effects of different medium components on fungal morphology during pre-culture. Medium components: A, glucose, inorganic ions and CaCO3; B, glucose, soybean peptone and CaCO3; C, glucose, inorganic ions, soybean peptone and CaCO3.
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Z. Zhou et al. / Bioresource Technology 102 (2011) 9345–9349 Table 1 Distribution of fumaric acid yield with different fungi morphology at 30 °C and 200 rpm in a flask. Spores were inoculated into 250-mL Erlenmeyer flasks containing 50 mL of different pre-culture mediums, cultured at 30 °C, 200 rpm for 30 h. Fungal with different morphology were obtained, and transferred into another 250-mL flask, with 50 mL of AP, cultured at 30 °C, 200 rpm for 3 d. Pre-culture medium components: clump (glucose, inorganic ions and CaCO3); suspend mycelia (glucose, inorganic ions, soybean peptone and CaCO3); pellet (glucose, soybean peptone and CaCO3). The mineral ions included (g/L): KH2PO4, 1; MgSO47H2O, 0.25; ZnSO47H2O, 0.066; FeSO47H2O, 0.01.
a
Fungi morphology
Fumarate (g/L)a
Clump Suspend mycelia Pellet
26.64 ± 2.54 31.85 ± 2.03 38.93 ± 1.57
The mean value of parallel determination.
significant factor for the pellet formation. A slight change in temperature could result in different morphology.
Fig. 4. Effects of pellet size on fumaric acid production at 30 °C and 200 rpm in a flask. Different size of pellet obtained with culturing in diverse substrates concentration of pre-culture (e.g., glucose, 10–30 g/L; soybean peptone, 2–10 g/L) or diverse pre-culture temperatures (28–37 °C).
3.3. Fumaric acid production based on morphologies optimization The effect of different morphologies on the yield of fumaric acid was investigated. In the present study, the fumaric acid concentration of pellet fermentation reached 38.93 g/L in 72 h of culture compared to clump fermentation that produced only 26.64 g/L (Table 1). Fumaric acid production by R. delemar first appeared at 16 h regardless of morphology (Table 1), from thereon both rapidly increased until 64 h and then only increased slightly until the end of fermentation. The results further demonstrated that fungal culture as pellets can yield fumaric acid more quickly and increase the total yield. Therefore, morphology was a very important factor in fungal fermentation. This may be interpreted that most fermentation broths show non-Newtonian flow behavior while cultures with the desired pellet morphology show the lowest suspension viscosity and broth rheology close to Newtonian flow behavior (Gogus et al., 2006). 3.4. Effect of pellet size on fumaric acid production Influence of pellet diameter on fumaric acid production was observed in flask fermentations. As previously reported, suitable pellet size benefits yield (Feng et al., 2004). We also found that R. delemar in submerged fermentation yielded more fumaric acid when pellet diameter was small, with a lower yield for larger pellets, after 72 h of cultivation in a flask (Fig. 3). For pellet diameter of 0.55 mm, the fumaric acid production was the highest (39.56 g/ L), with shaking rate of 200 rpm. The larger pellet (0.78 mm) resulted in less fumaric acid produced (30.35 g/L). The highest yield (Fig. 4) of fumaric acid was obtained for pellets of smaller diameter (Table 2), for mass transfer at the liquid– solid interface between the cell surface and medium is easier than for large pellets. Evermore, the inner zone of pellets was relatively inactive with active growth of fungi focused on their surface. Effective fungal fermentation needs more active zones to secrete more fumaric acid. All the above demonstrated that with more active zones, there was higher fumaric acid yields. 3.5. Process optimization of fungi related products based on morphology High-viscosity fermentation can lead to limitations in mixing of nutrients and in oxygen mass transfer, which can result in low
Table 2 The schema of pellet size distribution. Different size of pellet obtained with culturing in diverse substrates concentration of pre-culture (e.g., glucose, 10–30 g/L; soybean peptone, 2–10 g/L) or diverse pre-culture temperatures (28–37 °C). Culture conditions Concentration (g/L)
Temperature (°C)
G, G, G, G, G,
30 30 30 30 35
20; 20; 30; 20; 20;
S, S, S, S, S,
6 8 6 2 6
Peller diameter (mm)
Standard deviation
0.55 0.58 0.63 0.69 0.78
0.11 0.12 0.15 0.13 0.15
G: glucose. S: soybean peptone.
yields. Therefore, we could get the largest yield in a stirred bioreactor tank with R. delemar fermented in small pellets, which was consistent with the profile of process optimization of fungi related products based on morphology (Fig. 5). Glucose consumption and fumaric acid production were more rapid when R. delemar was fermented in pellets rather than in clumps. Even fungi fermented in suspended mycelia at the beginning reverted to clumps eventually. We obtained the highest yield of fumaric acid (35.42 g/L) when fungi were grown in pellets, and the lowest yield (26.87 g/L) when in clumps. The results illustrated the control of mycelial morphology is a prerequisite to ensure increased productivity in industry applications (Papagianni and Mattey, 2006). Fungi in large pellets cause decreased substrate consumption rate and led to lower biomass concentration compared to small pellets (Sitanggang et al., 2010). Furthermore, the proportion of metabolically active biomass was restricted to the outer zone of fungi for R. delemar (Papagianni and Moo-Young, 2002).
4. Conclusion R. delemar NRRL1526 was used to produce fumaric acid cultured in flask and fermentor systems. Soybean peptone and inorganic ions affected pellets formation, which had a significant influence on fumaric acid yield. The pellet diameter of 0.55 mm at 30 °C, 200 rpm and pH 5.0 yield highest fumaric acid (39.56 g/L) in flask
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Fig. 5. Time courses of fumarate production (solid) and glucose consumption (open) by R. delemar NRRL1526 of different morphology in a stirred-tank bioreactor operated at 30 °C, and 200 rpm, pH 5.0 and 1.0 vvm air: square, clump; triangle, pellet; spherical, suspended mycelia. Different morphology of fungi obtained with diverse medium compositions. Pre-culture medium components: clump (glucose, inorganic ions and CaCO3); suspend mycelia (glucose, inorganic ions, soybean peptone and CaCO3); pellet (glucose, soybean peptone and CaCO3). The mineral ions included (g/L): KH2PO4, 1; MgSO47H2O, 0.25; ZnSO47H2O, 0.066; FeSO47H2O, 0.01.
fermentation, and 35.42 g/L in stirred bioreactor tank fermentation. Although the reasons for pellets formation were found some years ago, they have not yet been implemented in fermentation. However, the macroscopic effects of pellet diameter on fumaric acid yield can be directly implemented in the fermentation industry.
Acknowledgements This work was supported by grants from the Key Program of National Natural Science Foundation of China (No. 20836003), the Major State Basic Research Development Program of China (973 Program, No. 2007CB714306, 2010CB535014), the Research Program of Sate Key Laboratory of Food Science and Technology, Jiangnan University (No. SKLF-TS-200901), the Priority Academic Program Development of Jiangsu Higher Education Institutions and the 111 Project (No. 111-2-06).
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