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Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum
Bioresource Technology 94 (2004) 99–105
Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum Ning He 1, Yin Li, Jian Chen
*
The Key Laboratory of Industrial Biotechnology, Ministry of Education; School of Biotechnology, Southern Yangtze University, Huihe Road 170, Wuxi 214036, PR China
Abstract The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated. Sucrose was chosen as a carbon source for REA-11 production. Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination. A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ ml. The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best. Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control. In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kL a of 100 h1 . REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0–6.5. Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants. In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Corynebacterium glutamicum; Bioflocculant; Polygalacturonic acid; Fermentation conditions; Flocculating activity; Stability; Application
1. Introduction Organic synthetic polymer flocculants, such as polyacrylamide and its derivatives, are widely used in industries nowadays. However, considering the fact that acrylamide monomer is not only neurotoxic and carcinogenic but also non-degradable in nature (Kwon et al., 1996), bioflocculants are attracting great research interest due to their safety and biodegradable properties (Salehizadeh and Shojaosadati, 2001). In recent 25 years, more than 50 microorganisms, ranging from prokaryotic to eukaryotic cells, have been found to produce extracellular bioflocculants (Salehizadeh and Shojaosadati, 2001). However, none of the bioflocculants has been commercially produced or practically *
Corresponding author. Tel.: +86-510-587-7592; fax: +86-510-5807976. E-mail address: [email protected] (J. Chen). 1 Present address: Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China. 0960-8524/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2003.11.013
applied so far. The reason for which is, to a large extent, due to the low yields and the high costs in producing bioflocculants. Screening for microorganisms that are able to produce novel flocculants with high flocculating activities (Fujita et al., 2000; He et al., 2002; Salehizadeh and Shojaosadati, 2002; Shih et al., 2001; Zhang et al., 2002) and seeking for low-cost substrates for producing bioflocculants (Fujita et al., 2001) have, therefore, become an emphasis in this area recently. In our previous study, a novel polygalacturonic acid bioflocculant, REA-11, was purified from the culture broth of a newly isolated strain Corynebacterium glutamicum CCTCC M201005 and characterized (He et al., 2002). A biosynthetic pathway of REA-11 was proposed and experimentally confirmed (Li et al., 2003), based on which, a metabolic engineering approach is anticipated to engineer the production of REA-11. To achieve this objective the knowledge of genes encoding the enzymes involved in the biosynthesis of REA-11 is necessary. However, analysis of the genome sequence of C. glutamicum ATCC 13032 revealed the absence of gene encoding UDP-galactose dehydrogenase
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(http://www.genome.ad.jp/), one of the key enzymes involved in the production of REA-11 in C. glutamicum CCTCC M201005. This indicates that a comprehensive understanding of the molecular biology of strain CCTCC M201005, e.g. characterization of genes involved in the production of REA-11, is a prerequisite before starting engineering the production of REA-11. Although metabolic engineering is a powerful approach to improve metabolite productivity (Nielsen, 2001), traditional approaches such as optimization of fermentation conditions, are still useful to enhance the production of metabolite, especially for those nonconventional and physiology-unknown microorganisms. C. glutamicum CCTCC M201005 is different from the well-known amino acid producer C. glutamicum strains. It is, therefore, necessary to study the effect of nutritional factors on REA-11 production and optimize the fermentation conditions. To assess the application potential of REA-11, some characteristics of REA-11 raw product, and its preliminary application in both clarification and decoloration of wastewater, were also presented.
flasks were done on a reciprocal shaker with a rotation speed of 120 rpm. Fermentations in 10 l jar fermentor were operated at an aeration rate of 1 l/l/min throughout the whole fermentation process. Different levels of dissolved oxygen tension in the culture broth (expressed by the volumetric oxygen-transfer coefficient kL a) were obtained by changing the agitation speed of the fermentor automatically. All cultivations were done at 28 °C. Results represent the mean of three independent experiments and error bars indicate the standard deviation.
2. Methods
2.5. Determination of flocculating activity
2.1. Microorganism
Since the flocculating activity of REA-11 was proportional to the polygalacturonic acid produced (Li et al., 2003), the flocculating activity was, therefore, used to quantify the production of REA-11. Flocculating activity was measured using Kaolin clay suspension as an indicator (Kurane et al., 1994). One milliliter sample and 2.5 ml CaCl2 solution (10 g/l) were mixed with 40 ml 1% (w/v) Kaolin clay suspension, gently shaken, and left to stand still for 5 min at room temperature. By measuring the decrease of turbidity in upper phase, flocculating activity was expressed as flocculating rate (FR) calculated by FR ðU=mlÞ ¼ ðA BÞ=A 100 D, where A and B are the optical densities of the control and the sample at 550 nm, respectively. D is the dilution times of the fermentation broth free of cells.
Corynebacterium glutamicum CCTCC M201005, screened by our lab (He et al., 2002) and preserved at the China Center for Type Culture Collection (CCTCC, Wuhan, China), was used in this study. 2.2. Media and cultivation conditions The medium for slant consisted of (per liter): 1 g yeast extract, 1 g beef extract, 2 g tryptone, 10 g glucose, 0.005 g FeSO4 , and 20 g agar. The preculture medium consisted of (per liter): 10 g glucose, 0.5 g yeast extract, 0.5 g urea, 0.1 g NaCl, and 0.2 g MgSO4 Æ 7H2 O. The optimized fermentation medium consisted of (per liter): 17 g sucrose, 5 ml corn steep liquor (nitrogen concentration 3 mol/l), 0.45 g urea, 0.1 g KH2 PO4 , and 0.1 g MgSO4 Æ 7H2 O. The initial pH of all media was adjusted to 7.8 except the slant medium of which the initial pH was adjusted to 7.2. All media were prepared with tap water. 2.3. Culture conditions The culture from a slant was inoculated into a 250 ml flask containing 50 ml preculture medium and incubated for 18 h on a reciprocal shaker at 120 rpm. The preculture was inoculated into 250 ml flasks containing 100 ml fermentation medium or a 10 l jar fermentor containing 7 l culture medium. Cultivations in shaking
2.4. Preparation of REA-11 raw product Thousand milliliter of 48 h culture broth was centrifuged at 10,000g for 20 min to remove cell pellets. Three volumes of ethanol was added to the supernatant, stirred and left to stand still at 4 °C for 20 h, then centrifuged at 10,000g for 20 min. The precipitate was dissolved in water, centrifuged at 10,000g for 15 min to remove indiscerptible pellets. The supernatant was dialyzed against water for 24 h, freeze dried to get REA-11 raw product.
2.6. Analyses Cell growth was measured by dry cell weight (DCW). Five milliliter culture was centrifuged at 10,000g for 15 min, washed twice with distilled water, dried at 105 °C until constant weight was achieved. Nitrogen concentration was measured by Micro-Kjeldahl method (Chen et al., 1994). 2.7. pH stability of REA-11 raw product REA-11 raw product was dissolved into a suitable volume of water to achieve an initial flocculating activity of 600 U/ml and divided into eight aliquots afterwards.
N. He et al. / Bioresource Technology 94 (2004) 99–105
The pH of aliquots were adjusted to different values with 1 M NaOH or 1 M HCl solution. Aliquots were put at 4 °C for 24 h after which the residual flocculating activities were measured at room temperature. 2.8. Thermo-stability of REA-11 raw product REA-11 raw product was dissolved into a suitable volume of water to achieve an initial flocculating activity of 600 U/ml, and divided into six aliquots of which the pH were adjusted to different values. Sample with the same pH was subdivided into another five aliquots and treated at different temperatures for 1 h, the residual flocculating activities were measured at room temperature.
3. Results 3.1. Effects of carbon and nitrogen sources on REA-11 production The effect of various carbon sources on the production of REA-11 by C. glutamicum CCTCC M201005 was investigated (Fig. 1). Glucose, fructose and sucrose are favorable substrates for REA-11 production. Cells grew well on the medium with sodium acetate or ethanol as carbon sources but the REA-11 production was relatively low. Cells hardly could grow on lactose or starch, which led to a poor or none production of REA-11. Considering the highest REA-11 production was achieved on the sucrose medium, and sucrose is a relatively cheap substrate, this substance was, therefore, chosen as the sole carbon source in the following study. The effect of single nitrogen source on the production of REA-11 was studied (Fig. 2A). Relatively high productivity of REA-11 was observed when urea, powdered
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bean cake or corn steep liquor (CSL) was used as a sole nitrogen source. Notably, the cell growth is poor but the REA-11 productivity is quite high when CSL was used as a nitrogen source. Since urea is the only inorganic nitrogen source that provides moderate cell growth and REA-11 production, the effect of combining urea with other organic nitrogen source on the production of REA-11 was further investigated (Fig. 2B). Enhanced cell growth and REA-11 production was observed when complex nitrogen source was used. Strikingly, the use of a complex nitrogen source consisting of urea and CSL (total nitrogen concentration 30 mM) resulted in an approximately 2.7- and 3.3-fold increase in flocculating activity, and 1.6- and 4-fold increase in cell growth, compared to that using urea or CSL alone, respectively (Fig. 2A and B). Further increase in CSL concentration (urea concentration was fixed at 0.45 g/l) did not lead to an improved REA-11 production. On the contrary, CSL concentration of higher than 10 ml/l significantly diminished REA-11 production (data not shown). The effect of increasing urea concentration on REA-11 production (with CSL concentration fixed at 5 ml/l) showed a similar tendency to that of CSL: further increase of urea concentration gave no increase of FR while excessive presence of urea (concentration of higher than 2 g/l) led to a dramatic decrease of REA-11 production (data not shown). Therefore, a complex nitrogen source comprising 5 ml/l CSL and 0.45 g/l urea was used as an initial condition in following the study. 3.2. Effect of the molar ratio of carbon to nitrogen (C/N) on REA-11 production The fact that further increase in nitrogen concentration did not result in an increased REA-11 production made us consider the effect of C/N ratio, one of the most
Fig. 1. Effect of carbon sources on REA-11 production (grid columns) and cell growth (open columns) of C. glutamicum CCTCC M201005. The carbon concentration of each carbon source was 0.3 M. Urea (0.45 g/l) was used as a nitrogen source, the nitrogen concentration of which was 15 mM.
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Fig. 2. Effect of nitrogen sources on cell growth (open columns) and REA-11 production (grid columns) of C. glutamicum CCTCC M201005. Sucrose was used as the sole carbon source, the carbon concentration of which was 0.3 M. Panel A: effect of single nitrogen source. Panel B: effect of complex nitrogen sources: 1 (yeast extract + urea), 2 (beef extract + urea), 3 (tryptone + urea), 4 (peptone + urea), 5 (powered bean cake + urea), 6 (corn steep liquor + urea). The nitrogen concentration of each nitrogen source was 15 mM except that in panel B the nitrogen concentration of urea was 30 mM.
important parameters to assess the balance of carbon to nitrogen in batch fermentation. A dramatic increase on REA-11 production was observed when C/N increased up to 20:1 (Fig. 3). Further increase on C/N ratio did not contribute to, and even led to a decrease on REA-11 production, while the residual sucrose concentration
also increased (data not shown). This indicates that an excessive supply of sucrose would lead to a waste of carbon source, a sucrose concentration of 17 g/l coincide with the C/N ratio of 20:1 is favorable for REA-11 production. 3.3. Effect of feeding carbon and/or nitrogen on REA-11 production
500
FR (U/ml)
400 300 200 100 0 0
10
20 30 40 C/N (Molar ratio)
50
60
Fig. 3. Effect of molar ratio of C/N on REA-11 production. 0.45 g/l urea and 5 ml/l CSL was used as the nitrogen source (total nitrogen concentration 30 mM). Various molar ratio of C/N was realized by increasing sucrose concentration.
Since the structural and functional unit of REA-11 is a polygalacturonic acid (He et al., 2002), increasing sucrose concentration to a certain level would be helpful for REA-11 production. Considering a high level production of REA-11 is dependent on a suitable C/N ratio (Fig. 3), the carbon and nitrogen concentrations, ideally, should be increased proportionally. However, inhibition on REA-11 production occurred at CSL concentration of higher than 10 ml/l or urea concentration of higher than 2 g/l, suggesting that the initial nitrogen concentration should be kept at a low level to avoid inhibition. The effect of feeding carbon and/or nitrogen source on REA-11 production was then examined (Table 1). An optimized carbon and nitrogen composition (as shown
N. He et al. / Bioresource Technology 94 (2004) 99–105 Table 1 Effect of feeding carbon and/or nitrogen source on REA-11 production a Strategy
FR (U/ml)
Increment (%)
Control (no feeding)b +0.45 g/l ureac +5 ml/l CSLd +8.5 g/l sucrosee +0.45 g/l urea and 8.5 g/l sucrosef
390 ± 13 402 ± 12 87 ± 8 380 ± 12 458 ± 14
– 3 )78 )3 17
a
Initial concentration of carbon and nitrogen source: sucrose 17 g/l, CSL 5 ml/l, urea 0.45 g/l. Different carbon and/or nitrogen source solutions were added to 100 ml culture at 24 h. b 10 ml sterile water was added. c 5 ml sterile urea solution (9 g/l) and 5 ml sterile water were added. d 0.5 ml sterile CSL and 9.5 ml sterile water were added. e 5 ml sterile sucrose solution (170 g/l) and 5 ml sterile water were added. f 5 ml concentrated sterile urea (9 g/l) and 5 ml sterile sucrose solution (170 g/l) were added.
in the footnote of Table 1) was chosen as an initial condition. Table 1 shows that feeding carbon or nitrogen separately could not enhance REA-11 production (and negative effect was observed when feeding additional 5 ml/l CSL), while feeding sucrose and urea simultaneously with a C/N ratio of 20:1 increased REA11 production by 17%.
3.5. pH stability and thermo-stability of REA-11 raw product REA-11 raw product is relatively stable at moderate acidic conditions (pH 3.0–6.0). pH of lower than 3.0 or higher than 6.0 resulted in a significant decrease of flocculating activity (data not shown). Fig. 5 shows that REA-11 is relatively thermo-stable if the pH of the sample is in the range of 3.0–6.0, at which points there are no significant decreases in flocculating activity even when heated at 80 °C for 1 h. Increasing the temperature up to 100 °C makes the stability of REA-11 collapsed. When the pH of REA-11 solution was adjusted to an unstable pH, the heat-stability also decreased. 3.6. Application of REA-11 The flocculating capability of REA-11 was compared with several commonly used chemically synthetic flocculants by using Kaolin clay suspension as an indicator, to investigate the industrial application potential of REA-11 (Table 2). The dosage of REA-11 raw product was only 12%, 16%, and 19% that of polyaluminium chloride, aluminium sulfate, and polyacrylamide, respectively, to achieve the same flocculating efficiency in the clarification of Kaolin clay suspension. REA-11
3.4. Effect of dissolved oxygen tension on REA-11 production
700 600 Residual FR (U/ml)
As a key parameter in submerged fermentations, dissolved oxygen tension (DOT), very often, significantly influences the fermentation process. Since DOT can be easily controlled in fermentor, experiments were performed in a 10 l jar fermentor by using the optimized conditions generated from shaking flasks studies. Fig. 4 shows that a kL a of 100 h1 is favorable for REA-11 production (FR reached 520 U/ml). The increase of kL a resulted in a decrease in flocculating activity.
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500 400 300 200 100 0 0
600 500
20
40
60 T (°C)
80
Fig. 5. Effect of temperature on the stability of REA-11 raw product at different pH: 3.0 (r), 4.0 (j), 5.0 (N) 6.5 (M), 8.5 ( ), and 10.0 ( ).
FR (U/ml)
100
400 Table 2 Comparison of flocculating capability of chemically synthetic flocculants and REA-11
300 200 100 0
0
10
20
30
40
50
t (h) Fig. 4. Effect of kL a in the fermentation system on REA-11 production. kL a: 100 h1 (N), 160 h1 (r), 200 h1 (}), 300 h1 (M).
Flocculant
Dosage (mg/l)a
Al2 (SO4 )3 Polyaluminium chloride Polyacrylamide Polyacrylamide + Al2 (SO4 )3 REA-11
50 70 45 44b 8.2
a The dosage needed to achieve 90% flocculation of 10 g/l Kaolin clay suspension. b Total concentration of polyacrylamide and Al2 (SO4 )3 .
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also had a significant effect on ink decoloration, which inspired us to explore its potential decoloration capacity further. A decolorization rate of 48.9%, 31.2%, 30.6%, and 26.0% was achieved when mixing REA-11 with 0.5%, 1%, 3%, and 5% of molasses wastewater with 2 mM of CaCl2 solution as a co-flocculant, while polyaluminium chloride, aluminium sulfate, and polyacrylamide did not show any effect on the decolorization of the same molasses wastewater.
4. Discussion The effects of nutritional conditions on the production of bioflocculants have been extensively studied (Salehizadeh and Shojaosadati, 2001). Generally, a suitable medium for bioflocculant production comprises glucose or fructose as the carbon source (Kurane et al., 1991) and yeast extract or beef extract as the nitrogen source (Kurane et al., 1986), while moderate amounts of phosphate and ions are also necessary (Fujita et al., 2000). In this study, C. glutamicum CCTCC M201005 could use glucose, fructose, and sucrose to produce REA-11. Sucrose was chosen as the carbon source because of the cheap cost and the high productivity of bioflocculant. The ability to utilize sucrose makes it possible to use molasses as a carbon source industrially, which might be very important to realize a commercial production of REA-11. The role of CSL in REA-11 production is rather a source of stimulation factors than a source of nitrogen, since the use of CSL alone did not provide good cell growth (Fig. 2A), while the addition of CSL together with urea enhanced REA-11 production significantly (Fig. 2B). CSL is, usually, considered as a source of biotin and amino acids. Since the addition of biotin alone (ranging from 0.01 to 1 mg/l) did not affect either cell growth or REA-11 production (data not shown), the stimulation effect of CSL on cell growth and REA11 production might be due to the amino acids it contains. We detected the amino acids concentration of CSL (detailed data not shown) and found the concentrations of most amino acids were below 0.5 mg/ml, while the concentrations of threonine and glutamate were relatively high (3 mg/ml). Furthermore, the addition of threonine or glutamate (up to 15 mg/l) was found to stimulate REA-11 production, conceiving that threonine and glutamate are important factors for REA-11 production. The fact that high concentration of CSL dramatically abolished REA-11 production might be ascribed to the shift of metabolic pathways regulated by the high concentration of glutamate present in CSL, since the addition of glutamate (higher than 30 mg/l) also diminished REA-11 production. At present we do not know exactly the effect of glutamate on REA-11 production. With respect to the effect of
amino acids on the growth of C. glutamicum, threonine, leucine and methionine were reported essential for the growth of C. glutamicum ATCC 21253 (Vallino and Stephanopoulos, 1993). Moreover, CSL has been used as an amino acids source for lysine production by C. glutamicum (Van Walsem and Thompson, 1997). We have tested different CSLs originated from five companies (both CSLs contained relatively high concentration of glutamate and threonine) and obtained similar results. Further studies in a 10 l fermentor indicated that the optimized fermentation medium from shaking flasks was also favorable for larger scale fermentation. However, the final flocculating activity in 10 l fermentor was significantly affected by the DOT. Within the kL a range tested, the lower the kL a, the better the REA-11 production, indicating that high DOT is deleterious for REA-11 production. This conclusion can also be supported by the fermentations in shaking flasks, in which the flocculating activity decreased from 400 to 200 U/ml when the volume of medium reduced from 100 to 15 ml in 250 ml shaking flasks. The raw product of REA-11 was relatively thermostable at a wide pH range of 3.0–6.5. This is due to that the backbone of this bioflocculant is a polysaccharide but not a protein. It has been noticed that the flocculants with protein or peptide backbone in the structure are generally thermally labile, but those made of sugars are thermo-stable (Salehizadeh and Shojaosadati, 2001). The comparison of the clarifying ability between REA11 and chemically synthetic flocculants reveals that REA-11 has stronger flocculating activity and the capability to decolorize the molasses wastewater. This Corynebacterium flocculant is, therefore, very unique in many aspects: strong flocculating activity, cost-effective fermentation medium, easy-control fermentation process, stable quality in practical ranges of pH and temperature, especially the capacity to decolorize the molasses wastewater. These unique characteristics of this novel bioflocculant indicate the application potential in industry. Acknowledgements This work was supported by the Natural Science Foundation of China (contract no. 20176042). References Chen, Z.X., Liu, J., Luo, D., 1994. Experiments of Biochemistry. China Science and Technology University Press, Hefei. pp. 7–11 (in Chinese). Fujita, M., Ike, M., Tachibana, S., Kitada, G., Kim, S.M., Inoue, Z., 2000. Characterization of a bioflocculant produced by Citrobacter sp. TKF04 from acetic and propionic acids. J. Biosci. Bioeng. 89, 40–46.
N. He et al. / Bioresource Technology 94 (2004) 99–105 Fujita, M., Ike, M., Jang, J.H., Kim, S.M., Hirao, T., 2001. Bioflocculant production from lower-molecular fatty acids as a novel strategy for utilization of sludge digestion liquor. Water Sci. Technol. 44, 237–243. He, N., Li, Y., Chen, J., Lun, S.-Y., 2002. Identification of a novel bioflocculant from a newly isolated Corynebacterium glutamicum. Biochem. Eng. J. 11, 137–148. Kurane, R., Toeda, K., Takeda, K., Suzuki, T., 1986. Culture condition for production of microbial flocculant by Rhodococcus erythropolis. Agric. Biol. Chem. 50, 2309–2313. Kurane, R., Hatakeyama, S., Tsugeno, H., 1991. Correlation between flocculant production and morphological changes in Rhodococcus erythropolis S-1. J. Ferment. Bioeng. 72, 498–500. Kurane, R., Hatamochi, K., Kakuno, T., Kiyohara, M., Hirano, M., Taniguchi, Y., 1994. Production of a bioflocculant by Rhodococcus erythropolis S-1 grown on alcohols. Biosci. Biotechnol. Biochem. 58, 428–429. Kwon, G.S., Moon, S.H., Hong, S.D., Lee, H.M., Kim, H.S., Oh, H.M., Yoon, B.D., 1996. A novel flocculant biopolymer produced by Pestalotiopsis sp. KCTC 8637P. Biotechnol. Lett. 18, 1459–1464. Li, Y., He, N., Guan, H.-Q., Du, G.-C., Chen, J., 2003. A novel polygalacturonic acid bioflocculant REA-11 produced by Coryne-
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bacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation. Appl. Microbiol. Biotechnol. 63, 200– 206. Nielsen, J., 2001. Metabolic engineering. Appl. Microbiol. Biotechnol. 55, 263–283. Salehizadeh, H., Shojaosadati, S.A., 2001. Extracellular biopolymeric flocculants: recent trends and biotechnological importance. Biotechnol. Adv. 19, 371–385. Salehizadeh, H., Shojaosadati, S.A., 2002. Isolation and characterisation of a bioflocculant produced by Bacillus firmus. Biotechnol. Lett. 24, 35–40. Shih, I.L., Van, Y.T., Yeh, L.C., Lin, H.G., Chang, Y.N., 2001. Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties. Bioresour. Technol. 78, 267–272. Vallino, J.J., Stephanopoulos, G., 1993. Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol. Bioeng. 41, 633–646. Van Walsem, H.J., Thompson, M.C., 1997. Simulated moving bed in the production of lysine. J. Biotechnol. 59, 127–132. Zhang, J., Wang, R., Jiang, P., Liu, Z., 2002. Production of an exopolysaccharide bioflocculant by Sorangium cellulosum. Lett. Appl. Microbiol. 34, 178–181.
Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum Ning He 1, Yin Li, Jian Chen
*
The Key Laboratory of Industrial Biotechnology, Ministry of Education; School of Biotechnology, Southern Yangtze University, Huihe Road 170, Wuxi 214036, PR China
Abstract The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated. Sucrose was chosen as a carbon source for REA-11 production. Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination. A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ ml. The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best. Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control. In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kL a of 100 h1 . REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0–6.5. Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants. In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Corynebacterium glutamicum; Bioflocculant; Polygalacturonic acid; Fermentation conditions; Flocculating activity; Stability; Application
1. Introduction Organic synthetic polymer flocculants, such as polyacrylamide and its derivatives, are widely used in industries nowadays. However, considering the fact that acrylamide monomer is not only neurotoxic and carcinogenic but also non-degradable in nature (Kwon et al., 1996), bioflocculants are attracting great research interest due to their safety and biodegradable properties (Salehizadeh and Shojaosadati, 2001). In recent 25 years, more than 50 microorganisms, ranging from prokaryotic to eukaryotic cells, have been found to produce extracellular bioflocculants (Salehizadeh and Shojaosadati, 2001). However, none of the bioflocculants has been commercially produced or practically *
Corresponding author. Tel.: +86-510-587-7592; fax: +86-510-5807976. E-mail address: [email protected] (J. Chen). 1 Present address: Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China. 0960-8524/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2003.11.013
applied so far. The reason for which is, to a large extent, due to the low yields and the high costs in producing bioflocculants. Screening for microorganisms that are able to produce novel flocculants with high flocculating activities (Fujita et al., 2000; He et al., 2002; Salehizadeh and Shojaosadati, 2002; Shih et al., 2001; Zhang et al., 2002) and seeking for low-cost substrates for producing bioflocculants (Fujita et al., 2001) have, therefore, become an emphasis in this area recently. In our previous study, a novel polygalacturonic acid bioflocculant, REA-11, was purified from the culture broth of a newly isolated strain Corynebacterium glutamicum CCTCC M201005 and characterized (He et al., 2002). A biosynthetic pathway of REA-11 was proposed and experimentally confirmed (Li et al., 2003), based on which, a metabolic engineering approach is anticipated to engineer the production of REA-11. To achieve this objective the knowledge of genes encoding the enzymes involved in the biosynthesis of REA-11 is necessary. However, analysis of the genome sequence of C. glutamicum ATCC 13032 revealed the absence of gene encoding UDP-galactose dehydrogenase
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(http://www.genome.ad.jp/), one of the key enzymes involved in the production of REA-11 in C. glutamicum CCTCC M201005. This indicates that a comprehensive understanding of the molecular biology of strain CCTCC M201005, e.g. characterization of genes involved in the production of REA-11, is a prerequisite before starting engineering the production of REA-11. Although metabolic engineering is a powerful approach to improve metabolite productivity (Nielsen, 2001), traditional approaches such as optimization of fermentation conditions, are still useful to enhance the production of metabolite, especially for those nonconventional and physiology-unknown microorganisms. C. glutamicum CCTCC M201005 is different from the well-known amino acid producer C. glutamicum strains. It is, therefore, necessary to study the effect of nutritional factors on REA-11 production and optimize the fermentation conditions. To assess the application potential of REA-11, some characteristics of REA-11 raw product, and its preliminary application in both clarification and decoloration of wastewater, were also presented.
flasks were done on a reciprocal shaker with a rotation speed of 120 rpm. Fermentations in 10 l jar fermentor were operated at an aeration rate of 1 l/l/min throughout the whole fermentation process. Different levels of dissolved oxygen tension in the culture broth (expressed by the volumetric oxygen-transfer coefficient kL a) were obtained by changing the agitation speed of the fermentor automatically. All cultivations were done at 28 °C. Results represent the mean of three independent experiments and error bars indicate the standard deviation.
2. Methods
2.5. Determination of flocculating activity
2.1. Microorganism
Since the flocculating activity of REA-11 was proportional to the polygalacturonic acid produced (Li et al., 2003), the flocculating activity was, therefore, used to quantify the production of REA-11. Flocculating activity was measured using Kaolin clay suspension as an indicator (Kurane et al., 1994). One milliliter sample and 2.5 ml CaCl2 solution (10 g/l) were mixed with 40 ml 1% (w/v) Kaolin clay suspension, gently shaken, and left to stand still for 5 min at room temperature. By measuring the decrease of turbidity in upper phase, flocculating activity was expressed as flocculating rate (FR) calculated by FR ðU=mlÞ ¼ ðA BÞ=A 100 D, where A and B are the optical densities of the control and the sample at 550 nm, respectively. D is the dilution times of the fermentation broth free of cells.
Corynebacterium glutamicum CCTCC M201005, screened by our lab (He et al., 2002) and preserved at the China Center for Type Culture Collection (CCTCC, Wuhan, China), was used in this study. 2.2. Media and cultivation conditions The medium for slant consisted of (per liter): 1 g yeast extract, 1 g beef extract, 2 g tryptone, 10 g glucose, 0.005 g FeSO4 , and 20 g agar. The preculture medium consisted of (per liter): 10 g glucose, 0.5 g yeast extract, 0.5 g urea, 0.1 g NaCl, and 0.2 g MgSO4 Æ 7H2 O. The optimized fermentation medium consisted of (per liter): 17 g sucrose, 5 ml corn steep liquor (nitrogen concentration 3 mol/l), 0.45 g urea, 0.1 g KH2 PO4 , and 0.1 g MgSO4 Æ 7H2 O. The initial pH of all media was adjusted to 7.8 except the slant medium of which the initial pH was adjusted to 7.2. All media were prepared with tap water. 2.3. Culture conditions The culture from a slant was inoculated into a 250 ml flask containing 50 ml preculture medium and incubated for 18 h on a reciprocal shaker at 120 rpm. The preculture was inoculated into 250 ml flasks containing 100 ml fermentation medium or a 10 l jar fermentor containing 7 l culture medium. Cultivations in shaking
2.4. Preparation of REA-11 raw product Thousand milliliter of 48 h culture broth was centrifuged at 10,000g for 20 min to remove cell pellets. Three volumes of ethanol was added to the supernatant, stirred and left to stand still at 4 °C for 20 h, then centrifuged at 10,000g for 20 min. The precipitate was dissolved in water, centrifuged at 10,000g for 15 min to remove indiscerptible pellets. The supernatant was dialyzed against water for 24 h, freeze dried to get REA-11 raw product.
2.6. Analyses Cell growth was measured by dry cell weight (DCW). Five milliliter culture was centrifuged at 10,000g for 15 min, washed twice with distilled water, dried at 105 °C until constant weight was achieved. Nitrogen concentration was measured by Micro-Kjeldahl method (Chen et al., 1994). 2.7. pH stability of REA-11 raw product REA-11 raw product was dissolved into a suitable volume of water to achieve an initial flocculating activity of 600 U/ml and divided into eight aliquots afterwards.
N. He et al. / Bioresource Technology 94 (2004) 99–105
The pH of aliquots were adjusted to different values with 1 M NaOH or 1 M HCl solution. Aliquots were put at 4 °C for 24 h after which the residual flocculating activities were measured at room temperature. 2.8. Thermo-stability of REA-11 raw product REA-11 raw product was dissolved into a suitable volume of water to achieve an initial flocculating activity of 600 U/ml, and divided into six aliquots of which the pH were adjusted to different values. Sample with the same pH was subdivided into another five aliquots and treated at different temperatures for 1 h, the residual flocculating activities were measured at room temperature.
3. Results 3.1. Effects of carbon and nitrogen sources on REA-11 production The effect of various carbon sources on the production of REA-11 by C. glutamicum CCTCC M201005 was investigated (Fig. 1). Glucose, fructose and sucrose are favorable substrates for REA-11 production. Cells grew well on the medium with sodium acetate or ethanol as carbon sources but the REA-11 production was relatively low. Cells hardly could grow on lactose or starch, which led to a poor or none production of REA-11. Considering the highest REA-11 production was achieved on the sucrose medium, and sucrose is a relatively cheap substrate, this substance was, therefore, chosen as the sole carbon source in the following study. The effect of single nitrogen source on the production of REA-11 was studied (Fig. 2A). Relatively high productivity of REA-11 was observed when urea, powdered
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bean cake or corn steep liquor (CSL) was used as a sole nitrogen source. Notably, the cell growth is poor but the REA-11 productivity is quite high when CSL was used as a nitrogen source. Since urea is the only inorganic nitrogen source that provides moderate cell growth and REA-11 production, the effect of combining urea with other organic nitrogen source on the production of REA-11 was further investigated (Fig. 2B). Enhanced cell growth and REA-11 production was observed when complex nitrogen source was used. Strikingly, the use of a complex nitrogen source consisting of urea and CSL (total nitrogen concentration 30 mM) resulted in an approximately 2.7- and 3.3-fold increase in flocculating activity, and 1.6- and 4-fold increase in cell growth, compared to that using urea or CSL alone, respectively (Fig. 2A and B). Further increase in CSL concentration (urea concentration was fixed at 0.45 g/l) did not lead to an improved REA-11 production. On the contrary, CSL concentration of higher than 10 ml/l significantly diminished REA-11 production (data not shown). The effect of increasing urea concentration on REA-11 production (with CSL concentration fixed at 5 ml/l) showed a similar tendency to that of CSL: further increase of urea concentration gave no increase of FR while excessive presence of urea (concentration of higher than 2 g/l) led to a dramatic decrease of REA-11 production (data not shown). Therefore, a complex nitrogen source comprising 5 ml/l CSL and 0.45 g/l urea was used as an initial condition in following the study. 3.2. Effect of the molar ratio of carbon to nitrogen (C/N) on REA-11 production The fact that further increase in nitrogen concentration did not result in an increased REA-11 production made us consider the effect of C/N ratio, one of the most
Fig. 1. Effect of carbon sources on REA-11 production (grid columns) and cell growth (open columns) of C. glutamicum CCTCC M201005. The carbon concentration of each carbon source was 0.3 M. Urea (0.45 g/l) was used as a nitrogen source, the nitrogen concentration of which was 15 mM.
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Fig. 2. Effect of nitrogen sources on cell growth (open columns) and REA-11 production (grid columns) of C. glutamicum CCTCC M201005. Sucrose was used as the sole carbon source, the carbon concentration of which was 0.3 M. Panel A: effect of single nitrogen source. Panel B: effect of complex nitrogen sources: 1 (yeast extract + urea), 2 (beef extract + urea), 3 (tryptone + urea), 4 (peptone + urea), 5 (powered bean cake + urea), 6 (corn steep liquor + urea). The nitrogen concentration of each nitrogen source was 15 mM except that in panel B the nitrogen concentration of urea was 30 mM.
important parameters to assess the balance of carbon to nitrogen in batch fermentation. A dramatic increase on REA-11 production was observed when C/N increased up to 20:1 (Fig. 3). Further increase on C/N ratio did not contribute to, and even led to a decrease on REA-11 production, while the residual sucrose concentration
also increased (data not shown). This indicates that an excessive supply of sucrose would lead to a waste of carbon source, a sucrose concentration of 17 g/l coincide with the C/N ratio of 20:1 is favorable for REA-11 production. 3.3. Effect of feeding carbon and/or nitrogen on REA-11 production
500
FR (U/ml)
400 300 200 100 0 0
10
20 30 40 C/N (Molar ratio)
50
60
Fig. 3. Effect of molar ratio of C/N on REA-11 production. 0.45 g/l urea and 5 ml/l CSL was used as the nitrogen source (total nitrogen concentration 30 mM). Various molar ratio of C/N was realized by increasing sucrose concentration.
Since the structural and functional unit of REA-11 is a polygalacturonic acid (He et al., 2002), increasing sucrose concentration to a certain level would be helpful for REA-11 production. Considering a high level production of REA-11 is dependent on a suitable C/N ratio (Fig. 3), the carbon and nitrogen concentrations, ideally, should be increased proportionally. However, inhibition on REA-11 production occurred at CSL concentration of higher than 10 ml/l or urea concentration of higher than 2 g/l, suggesting that the initial nitrogen concentration should be kept at a low level to avoid inhibition. The effect of feeding carbon and/or nitrogen source on REA-11 production was then examined (Table 1). An optimized carbon and nitrogen composition (as shown
N. He et al. / Bioresource Technology 94 (2004) 99–105 Table 1 Effect of feeding carbon and/or nitrogen source on REA-11 production a Strategy
FR (U/ml)
Increment (%)
Control (no feeding)b +0.45 g/l ureac +5 ml/l CSLd +8.5 g/l sucrosee +0.45 g/l urea and 8.5 g/l sucrosef
390 ± 13 402 ± 12 87 ± 8 380 ± 12 458 ± 14
– 3 )78 )3 17
a
Initial concentration of carbon and nitrogen source: sucrose 17 g/l, CSL 5 ml/l, urea 0.45 g/l. Different carbon and/or nitrogen source solutions were added to 100 ml culture at 24 h. b 10 ml sterile water was added. c 5 ml sterile urea solution (9 g/l) and 5 ml sterile water were added. d 0.5 ml sterile CSL and 9.5 ml sterile water were added. e 5 ml sterile sucrose solution (170 g/l) and 5 ml sterile water were added. f 5 ml concentrated sterile urea (9 g/l) and 5 ml sterile sucrose solution (170 g/l) were added.
in the footnote of Table 1) was chosen as an initial condition. Table 1 shows that feeding carbon or nitrogen separately could not enhance REA-11 production (and negative effect was observed when feeding additional 5 ml/l CSL), while feeding sucrose and urea simultaneously with a C/N ratio of 20:1 increased REA11 production by 17%.
3.5. pH stability and thermo-stability of REA-11 raw product REA-11 raw product is relatively stable at moderate acidic conditions (pH 3.0–6.0). pH of lower than 3.0 or higher than 6.0 resulted in a significant decrease of flocculating activity (data not shown). Fig. 5 shows that REA-11 is relatively thermo-stable if the pH of the sample is in the range of 3.0–6.0, at which points there are no significant decreases in flocculating activity even when heated at 80 °C for 1 h. Increasing the temperature up to 100 °C makes the stability of REA-11 collapsed. When the pH of REA-11 solution was adjusted to an unstable pH, the heat-stability also decreased. 3.6. Application of REA-11 The flocculating capability of REA-11 was compared with several commonly used chemically synthetic flocculants by using Kaolin clay suspension as an indicator, to investigate the industrial application potential of REA-11 (Table 2). The dosage of REA-11 raw product was only 12%, 16%, and 19% that of polyaluminium chloride, aluminium sulfate, and polyacrylamide, respectively, to achieve the same flocculating efficiency in the clarification of Kaolin clay suspension. REA-11
3.4. Effect of dissolved oxygen tension on REA-11 production
700 600 Residual FR (U/ml)
As a key parameter in submerged fermentations, dissolved oxygen tension (DOT), very often, significantly influences the fermentation process. Since DOT can be easily controlled in fermentor, experiments were performed in a 10 l jar fermentor by using the optimized conditions generated from shaking flasks studies. Fig. 4 shows that a kL a of 100 h1 is favorable for REA-11 production (FR reached 520 U/ml). The increase of kL a resulted in a decrease in flocculating activity.
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500 400 300 200 100 0 0
600 500
20
40
60 T (°C)
80
Fig. 5. Effect of temperature on the stability of REA-11 raw product at different pH: 3.0 (r), 4.0 (j), 5.0 (N) 6.5 (M), 8.5 ( ), and 10.0 ( ).
FR (U/ml)
100
400 Table 2 Comparison of flocculating capability of chemically synthetic flocculants and REA-11
300 200 100 0
0
10
20
30
40
50
t (h) Fig. 4. Effect of kL a in the fermentation system on REA-11 production. kL a: 100 h1 (N), 160 h1 (r), 200 h1 (}), 300 h1 (M).
Flocculant
Dosage (mg/l)a
Al2 (SO4 )3 Polyaluminium chloride Polyacrylamide Polyacrylamide + Al2 (SO4 )3 REA-11
50 70 45 44b 8.2
a The dosage needed to achieve 90% flocculation of 10 g/l Kaolin clay suspension. b Total concentration of polyacrylamide and Al2 (SO4 )3 .
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also had a significant effect on ink decoloration, which inspired us to explore its potential decoloration capacity further. A decolorization rate of 48.9%, 31.2%, 30.6%, and 26.0% was achieved when mixing REA-11 with 0.5%, 1%, 3%, and 5% of molasses wastewater with 2 mM of CaCl2 solution as a co-flocculant, while polyaluminium chloride, aluminium sulfate, and polyacrylamide did not show any effect on the decolorization of the same molasses wastewater.
4. Discussion The effects of nutritional conditions on the production of bioflocculants have been extensively studied (Salehizadeh and Shojaosadati, 2001). Generally, a suitable medium for bioflocculant production comprises glucose or fructose as the carbon source (Kurane et al., 1991) and yeast extract or beef extract as the nitrogen source (Kurane et al., 1986), while moderate amounts of phosphate and ions are also necessary (Fujita et al., 2000). In this study, C. glutamicum CCTCC M201005 could use glucose, fructose, and sucrose to produce REA-11. Sucrose was chosen as the carbon source because of the cheap cost and the high productivity of bioflocculant. The ability to utilize sucrose makes it possible to use molasses as a carbon source industrially, which might be very important to realize a commercial production of REA-11. The role of CSL in REA-11 production is rather a source of stimulation factors than a source of nitrogen, since the use of CSL alone did not provide good cell growth (Fig. 2A), while the addition of CSL together with urea enhanced REA-11 production significantly (Fig. 2B). CSL is, usually, considered as a source of biotin and amino acids. Since the addition of biotin alone (ranging from 0.01 to 1 mg/l) did not affect either cell growth or REA-11 production (data not shown), the stimulation effect of CSL on cell growth and REA11 production might be due to the amino acids it contains. We detected the amino acids concentration of CSL (detailed data not shown) and found the concentrations of most amino acids were below 0.5 mg/ml, while the concentrations of threonine and glutamate were relatively high (3 mg/ml). Furthermore, the addition of threonine or glutamate (up to 15 mg/l) was found to stimulate REA-11 production, conceiving that threonine and glutamate are important factors for REA-11 production. The fact that high concentration of CSL dramatically abolished REA-11 production might be ascribed to the shift of metabolic pathways regulated by the high concentration of glutamate present in CSL, since the addition of glutamate (higher than 30 mg/l) also diminished REA-11 production. At present we do not know exactly the effect of glutamate on REA-11 production. With respect to the effect of
amino acids on the growth of C. glutamicum, threonine, leucine and methionine were reported essential for the growth of C. glutamicum ATCC 21253 (Vallino and Stephanopoulos, 1993). Moreover, CSL has been used as an amino acids source for lysine production by C. glutamicum (Van Walsem and Thompson, 1997). We have tested different CSLs originated from five companies (both CSLs contained relatively high concentration of glutamate and threonine) and obtained similar results. Further studies in a 10 l fermentor indicated that the optimized fermentation medium from shaking flasks was also favorable for larger scale fermentation. However, the final flocculating activity in 10 l fermentor was significantly affected by the DOT. Within the kL a range tested, the lower the kL a, the better the REA-11 production, indicating that high DOT is deleterious for REA-11 production. This conclusion can also be supported by the fermentations in shaking flasks, in which the flocculating activity decreased from 400 to 200 U/ml when the volume of medium reduced from 100 to 15 ml in 250 ml shaking flasks. The raw product of REA-11 was relatively thermostable at a wide pH range of 3.0–6.5. This is due to that the backbone of this bioflocculant is a polysaccharide but not a protein. It has been noticed that the flocculants with protein or peptide backbone in the structure are generally thermally labile, but those made of sugars are thermo-stable (Salehizadeh and Shojaosadati, 2001). The comparison of the clarifying ability between REA11 and chemically synthetic flocculants reveals that REA-11 has stronger flocculating activity and the capability to decolorize the molasses wastewater. This Corynebacterium flocculant is, therefore, very unique in many aspects: strong flocculating activity, cost-effective fermentation medium, easy-control fermentation process, stable quality in practical ranges of pH and temperature, especially the capacity to decolorize the molasses wastewater. These unique characteristics of this novel bioflocculant indicate the application potential in industry. Acknowledgements This work was supported by the Natural Science Foundation of China (contract no. 20176042). References Chen, Z.X., Liu, J., Luo, D., 1994. Experiments of Biochemistry. China Science and Technology University Press, Hefei. pp. 7–11 (in Chinese). Fujita, M., Ike, M., Tachibana, S., Kitada, G., Kim, S.M., Inoue, Z., 2000. Characterization of a bioflocculant produced by Citrobacter sp. TKF04 from acetic and propionic acids. J. Biosci. Bioeng. 89, 40–46.
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