Please wait a minute...
Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

Postal Subscription Code 80-969

2018 Impact Factor: 2.809

Front. Chem. Sci. Eng.    2015, Vol. 9 Issue (3) : 369-375    https://doi.org/10.1007/s11705-015-1525-3
RESEARCH ARTICLE
High production of butyric acid by Clostridium tyrobutyricum mutant? ?
Chao Ma,Jianfa Ou,Matthew Miller,Sarah McFann,Xiaoguang (Margaret) Liu()
Department of Chemical and Biological Engineering, The University of Alabama, 245 7th Avenue, Tuscaloosa, AL 35401, USA
 Download: PDF(713 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

The objective of this study was to improve the production of butyric acid by process optimization using the metabolically engineered mutant of Clostridium tyrobutyricum (PAK-Em). First, the free-cell fermentation at pH 6.0 produced butyric acid with concentration of 38.44 g/L and yield of 0.42 g/g. Second, the immobilized-cell fermentations using fibrous-bed bioreactor (FBB) were run at pHs of 5.0, 5.5, 6.0, 6.5 and 7.0 to optimize fermentation process and improve the butyric acid production. It was found that the highest titer of butyric acid, 63.02 g/L, was achieved at pH 6.5. Finally, the metabolic flux balance analysis was performed to investigate the carbon rebalance in C. tyrobutyricum. The results show both gene manipulation and fermentation pH change redistribute carbon between biomass, acetic acid and butyric acid. This study demonstrated that high butyric acid production could be obtained by integrating metabolic engineering and fermentation process optimization.

Keywords Clostridium tyrobutyricum      butyric acid production      fermentation      mutant      pH      flux balance analysis     
Corresponding Author(s): Xiaoguang (Margaret) Liu   
Online First Date: 10 September 2015    Issue Date: 30 September 2015
 Cite this article:   
Chao Ma,Jianfa Ou,Matthew Miller, et al. High production of butyric acid by Clostridium tyrobutyricum mutant? ?[J]. Front. Chem. Sci. Eng., 2015, 9(3): 369-375.
 URL:  
https://academic.hep.com.cn/fcse/EN/10.1007/s11705-015-1525-3
https://academic.hep.com.cn/fcse/EN/Y2015/V9/I3/369
Strains Fermentation mode Sugar Concentration /g·L−1 Ref.
C. populeti Batch, free-cell Glucose 6.30 [11]
C. butyricum ZJUCB Batch, free-cell Glucose 12.25 [12]
Fed-batch, free-cell 16.74
C. butyricum S21 Batch, free-cell Lactose 18.60 [13]
C. beijerinckii Batch, free-cell Lactose 12.00 [14]
C. thermobutyricum Fed-batch, free-cell Glucose 19.40 [15]
C. tyrobutyricum JM1 Batch, free-cell Glucose 13.76 [16]
C. tyrobutyricum, wild type Fed-batch, free-cell Glucose 24.88 [17]
Continuous, free-cell Glucose 33.00 [18]
C. tyrobutyricum, mutant Fed-batch, immobilized-cell Glucose 49.90 [19]
Repeated fed-batch, immobilized-cell Glucose 86.9 [20]
E. coli Batch, free-cell Glucose 10.00 [21]
Tab.1  Recent progresses of butyric acid production from sugar
Reaction No. Biological function Stoichiometric equation a,b)
(1) Biomass formation 2 Glucose+ 1.75 NADH+ 1.75 H+ + 29.7 ATP →3 C4H6.4O1.72N+ 1.75 NAD+ + 29.7 ADP+ 29.7 Pi
(2) Formation of pyruvate (glycolysis) Glucose+ 2 NAD+ + 2 ADP+ 2 Pi → 2 Pyruvate+ 2 NADH+ 2 H+ + 2 ATP
(3) Formation of AcCoAand CO2 Pyruvate+ CoA+ Fdox → AcCoA+ Fdred + CO2
(4) Formation of H2 Fdred+ 2 H+ → H2+ Fdox
(5) Formation of NADH Fdred+ NAD+ NADH+ H+ + Fdox
(6) Formation of acetate AcCoA+ ADP+ Pi Acetate+ CoA+ ATP
(7) Formation of BuCoAand water 2 AcCoA+ 2 NADH+ 2H+ → BuCoA+ 2 NAD+ + CoA+ H2O
(8) Formation of butyrateor acetate BuCoA+ Acetate Butyrate+ AcCoA
(9) Formation of butyrate BuCoA+ ADP+ Pi Butyrate+ CoA+ ATP
Tab.2  Stoichiometric equations used in the FBA modeling of C. tyrobutyricum
Fig.1  Kinetics of free-cell fermentations by (a) C. tyrobutyricum ATCC 25755 wild type and (b) PAK-Em mutant at pH 6.0 and 37 °C. ○: Glucose, ■: Butyric acid, △: Acetic acid, ×: OD
Products Wild type (control) PAK-Em
Cell growth Growth rate µ /h−1 0.21±0.01 0.14±0.01
Biomass yield /(g·g−1) 0.06±0.01 0.04±0.01
Butyric acid Concentration /(g·L−1) 19.24±0.05 38.44±0.03
Yield /(g·g−1) 0.34±0.02 0.42±0.01
Acetic acid Concentration /(g·L−1) 4.22±0.002 7.16±0.002
Yield /(g·g−1) 0.07±0.001 0.07±0.01
C4/C2 B/A ratio /(g·g−1) 4.56±0.85 5.36±0.61
Tab.3  Comparison of wild type (control) with PAK-Em in free-cell fermentation products a,b,c)
Fig.2  Kinetics of immobilized-cell fermentations by PAK-Em at different pHs under 37 °C
Products pH
5.0 5.5 6.0 6.5 7.0
Butyrate Conc. /(g·L−1) 14.79±0.99 23.18±0.78 50.11±2.42 63.02±1.54 61.01±0.78
Yield /(g·g−1) 0.37±0.03 0.38±0.01 0.45±0.02 0.45±0.01 0.42±0.01
Acetate Conc. /(g·L−1) 2.11±0.02 3.13±0.02 7.03±0.01 7.26±0.03 7.09±0.02
Yield /(g·g−1) 0.03±0.004 0.03±0.006 0.08±0.01 0.05±0.01 0.04±0.01
C4/C2 Ratio /(g·g−1) 6.53 6.77 7.12 8.60 8.60
Tab.4  Effect of pH on acid production in immobilized-cell fermentations by PAK-Em
Fig.3  C. tyrobutyricum metabolic flux distribution on a basis of 1 mole glucose consumed by wild type and PAK-Em at 37 °C and pH 5.0?7.0
1 Wei  D, Liu  X, Yang  S T. Butyric acid production from sugarcane bagasse hydrolysate by Clostridium tyrobutyricum immobilized in a fibrous-bed bioreactor. Bioresource Technology, 2013, 129: 553–560
2 Dwidar  M, Park  J Y, Mitchell  R J, Sang  B I. The future of butyric acid in industry. The Scientific World Journal, 2012, 471417
3 Atweh  G  F,  DeSimone  J,  Saunthararajah  Y,  Fathallah  H, Weinberg  R S, Nagel  R L, Fabry  M E, Adams  R J. Hemoglobinopathies. American Society of Hematology Education Program, 2003: 14–39
4 Canani  R B, Costanzo  M D, Leone  L, Pedata  M, Meli  R, Calignano  A. Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World Journal of Gastroenterology, 2011, 17(12): 1519–1528
5 Lazarova  D L, Chiaro  C, Bordonaro  M. Butyrate induced changes in Wnt-signaling specific gene expression in colorectal cancer cells. BMC Research Notes, 2014, 7(1): 226
6 Huang  J, Cai  J, Wang  J, Zhu  X, Huang  L, Yang  S T, Xua  Z. Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor. Bioresource Technology, 2011, 102(4): 3923–3926
7 Kong  Q, He  G Q, Chen  F, Ruan  H. Studies on a kinetic model for butyric acid bioproduction by Clostridium butyricum. Letters in Applied Microbiology, 2006, 43(1): 71–77
8 Zhang  C, Yang  H, Yang  F, Ma  Y. Current progress on butyric acid production by fermentation. Current Microbiology, 2009, 59(6): 656–663
9 Canganella  F, Wiegel  J. Continuous cultivation of Clostridium thermobutyricum in a rotary fermentor system. Journal of Industrial Microbiology & Biotechnology, 2000, 24(1): 7–13
10 Ma  C, Kojima  K, Xu  N, Mobley  J, Zhou  L, Yang  S T, Liu  X M. Comparative proteomics analysis of high n-butanol producing metabolically engineered Clostridium tyrobutyricum. Journal of Biotechnology, 2015, 193: 108–119
11 Patel  G B, Agnew  B J. Growth and butyric acid production by Clostridium populeti. Archives of Microbiology, 1988, 150(3): 267–271
12 He  G Q, Kong  Q, Chen  Q H, Ruan  H. Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB. Journal of Zhejiang University, 2005, 6(11): 1076–1080
13 Cascone  R. Biobutanol—A replacement for bioethanol. Chemical Engineering Progress, 2008, 104(8): 4–9
14 Alam  S, Stevens  D, Bajpai  R. Production of butyric acid by batch fermentation of cheese whey with Clostridium beijerinckii. Journal of Industrial Microbiology, 1988, 2(6): 359–364
15 Canganella  F, Kuk  S U, Morgan  H, Wiegel  J. Clostridium thermobutyricum: Growth studies and stimulation of butyrate formation by acetate supplementation. Microbiological Research, 2002, 157(2): 149–156
16 Jo  J H, Lee  D S, Park  J M. The effects of pH on carbon material and energy balances in hydrogen-producing Clostridium tyrobutyricum JM1. Bioresource Technology, 2008, 99(17): 8485–8491
17 Huang  Y L, Wu  Z, Zhang  L, Cheung  C M, Yang  S T. Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor. Bioresource Technology, 2002, 82(1): 51–59
18 Michel-Savin  D, Marchal  R, Vandecasteele  J P. Butyric fermentation: Metabolic behavior and production performance of Clostridium tyrobutyricum in a continuous culture with cell recycle. Applied Microbiology and Biotechnology, 1990, 34(2): 172–177
19 Liu  X, Yang  S T. Kinetics of butyric acid fermentation of glucose and xylose by Clostridium tyrobutyricum wild type and mutant. Process Biochemistry, 2006, 41(4): 801–808
20 Jiang  L, Wang  J, Liang  S, Cai  J, Xu  Z, Cen  P, Yang  S T, Li  S. Enhanced butyric acid tolerance and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor. Biotechnology and Bioengineering, 2009, 108(1): 31–40
21 Saini  M, Wang  Z W, Chiang  C J, Chao  Y P. Metabolic engineering of Escherichia coli for production of butyric acid. Journal of Agricultural and Food Chemistry, 2014, 62(19): 4342–4348
22 Liu  X, Zhu  Y, Yang  S. Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme and Microbial Technology, 2006, 38(3–4): 521–528
23 Lewis  V P, Yang  S T. Continuous propionic acid fermentation by immobilized Propionibacterium acidipropionici in a novel packed-bed bioreactor. Biotechnology and Bioengineering, 1992, 40(4): 465–474
24 Huang  Y, Yang  S T. Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor. Biotechnology and Bioengineering, 1998, 60(4): 499–507
25 Silva  E M, Yang  S T. Kinetics and stability of a fibrous bed bioreactor for continuous production of lactic from unsupplemented acid whey. Journal of Biotechnology, 1995, 41(1): 59–70
26 Liu  X, Yang  S T. Kinetics of butyric acid fermentation of glucose and xylose by Clostridium tyrobutyricum wild type and mutant. Process Biochemistry, 2006, 41(4): 801–808
27 Yang  S T. Extractive fermentation using convoluted fibrous bed bioreactor. US Patent, 5563069, 1996-<month>10</month>-<day>08</day>
28 Zhu  Y. Enhanced butyric acid fermentation by Clostridium tyrobutyricum immobilized in a fibrous-bed bioreactor. Dissertation for the Doctoral Degree. Columbus: The Ohio State University, USA, 2003, 99–100
29 Zhu  Y, Yang  S T. Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum. Journal of Biotechnology, 2004, 110(2): 143–157
30 Zhu  Y, Liu  X, Yang  S T. Construction and characterization of pta gene deleted mutant of Clostridium tyrobutyricm for enhanced butyric acid fermentation. Biotechnology and Bioengineering, 2005, 90(2): 154–166
31 Du  Y, Jiang  W, Yu  M, Tang  I, Yang  S T. Metabolic process engineering of Clostridium tyrobutyricum ?ack-adhE2 for enhanced n-butanol production from glucose: Effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics. Biotechnology and Bioengineering, 2014, 112(4): 705–715
[1] Yonghyun Kim, Huiwen Liu, Yi Liu, Boa Jin, Hao Zhang, Wenjing Tian, Chan Im. Long-lasting photoluminescence quantum yield of cesium lead halide perovskite-type quantum dots[J]. Front. Chem. Sci. Eng., 2021, 15(1): 187-197.
[2] Boa Jin, Hyunmin Park, Yang Liu, Leijing Liu, Jongdeok An, Wenjing Tian, Chan Im. Charge-carrier photogeneration and extraction dynamics of polymer solar cells probed by a transient photocurrent nearby the regime of the space charge-limited current[J]. Front. Chem. Sci. Eng., 2021, 15(1): 164-179.
[3] Lifeng Zhang, Yifei Song, Weiping Wu, Robert Bradley, Yue Hu, Yi Liu, Shouwu Guo. Fe2Mo3O8 nanoparticles self-assembling 3D mesoporous hollow spheres toward superior lithium storage properties[J]. Front. Chem. Sci. Eng., 2021, 15(1): 156-163.
[4] Wenqian Chen, Vikram Karde, Thomas N. H. Cheng, Siti S. Ramli, Jerry Y. Y. Heng. Surface hydrophobicity: effect of alkyl chain length and network homogeneity[J]. Front. Chem. Sci. Eng., 2021, 15(1): 90-98.
[5] Njud S. Alharbi, Baowei Hu, Tasawar Hayat, Samar Omar Rabah, Ahmed Alsaedi, Li Zhuang, Xiangke Wang. Efficient elimination of environmental pollutants through sorption-reduction and photocatalytic degradation using nanomaterials[J]. Front. Chem. Sci. Eng., 2020, 14(6): 1124-1135.
[6] Uthen Thubsuang, Suphawadee Chotirut, Apisit Thongnok, Archw Promraksa, Mudtorlep Nisoa, Nicharat Manmuanpom, Sujitra Wongkasemjit, Thanyalak Chaisuwan. Facile preparation of polybenzoxazine-based carbon microspheres with nitrogen functionalities: effects of mixed solvents on pore structure and supercapacitive performance[J]. Front. Chem. Sci. Eng., 2020, 14(6): 1072-1086.
[7] Xuewen Hu, Yun Wang, Jinbo Ou Yang, Yang Li, Peng Wu, Hengju Zhang, Dingzhong Yuan, Yan Liu, Zhenyu Wu, Zhirong Liu. Synthesis of graphene oxide nanoribbons/chitosan composite membranes for the removal of uranium from aqueous solutions[J]. Front. Chem. Sci. Eng., 2020, 14(6): 1029-1038.
[8] Yu Cao, Xinyun Zhu, Xingyu Tong, Jing Zhou, Jian Ni, Jianjun Zhang, Jinbo Pang. Ultrathin microcrystalline hydrogenated Si/Ge alloyed tandem solar cells towards full solar spectrum conversion[J]. Front. Chem. Sci. Eng., 2020, 14(6): 997-1005.
[9] Krishnaveni Kalaiappan, Subadevi Rengapillai, Sivakumar Marimuthu, Raja Murugan, Premkumar Thiru. Kombucha SCOBY-based carbon and graphene oxide wrapped sulfur/polyacrylonitrile as a high-capacity cathode in lithium-sulfur batteries[J]. Front. Chem. Sci. Eng., 2020, 14(6): 976-987.
[10] Qingzhuo Ni, Hao Cheng, Jianfeng Ma, Yong Kong, Sridhar Komarneni. Efficient degradation of orange II by ZnMn2O4 in a novel photo-chemical catalysis system[J]. Front. Chem. Sci. Eng., 2020, 14(6): 956-966.
[11] Eniko Haaz, Botond Szilagyi, Daniel Fozer, Andras Jozsef Toth. Combining extractive heterogeneous-azeotropic distillation and hydrophilic pervaporation for enhanced separation of non-ideal ternary mixtures[J]. Front. Chem. Sci. Eng., 2020, 14(5): 913-927.
[12] Yang An, Chao Chen, Jundong Zhu, Pankaj Dwivedi, Yanjun Zhao, Zheng Wang. Hypoxia-induced activity loss of a photo-responsive microtubule inhibitor azobenzene combretastatin A4[J]. Front. Chem. Sci. Eng., 2020, 14(5): 880-888.
[13] Yifei Wang, Shouying Huang, Xinsheng Teng, Hongyu Wang, Jian Wang, Qiao Zhao, Yue Wang, Xinbin Ma. Controllable Fe/HCS catalysts in the Fischer-Tropsch synthesis: Effects of crystallization time[J]. Front. Chem. Sci. Eng., 2020, 14(5): 802-812.
[14] Chenggang Qiu, Alei Zhang, Sha Tao, Kang Li, Kequan Chen, Pingkai Ouyang. Combination of ARTP mutagenesis and color-mediated high-throughput screening to enhance 1-naphthol yield from microbial oxidation of naphthalene in aqueous system[J]. Front. Chem. Sci. Eng., 2020, 14(5): 793-801.
[15] Feichao Wu, Yanling Wang, Xiongfu Zhang. Flow synthesis of a novel zirconium-based UiO-66 nanofiltration membrane and its performance in the removal of p-nitrophenol from water[J]. Front. Chem. Sci. Eng., 2020, 14(4): 651-660.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed