A fed‑batch culture is, in the broadest sense, defined as a bioprocessing operation in which one or more nutrients (substrates) are supplied to the bioreactor during cultivation of microorganisms or animal cells, while no culture broth (cells and medium) is taken out until the final harvest.Tsuneo Yamane, Shoichi Shimizu. (1984) "Fed-batch Techniques in Microbial Processes". Advances Biochem Eng./Biotechnol., 30:147-194. An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion". In some fed‑batch cultures, the entire culture medium or a precursor of the target metabolite serves as the feed substrate. It is also called semi-batch culture. While the term "semi-batch" is well-established in the field of chemical reaction engineering, "fed-batch" is considered the most appropriate term for microbial reactions, as the supplied substrates are typically nutrients consumed by the microorganisms. Indeed, this terminology is widely adopted in numerous English-language research papers and reviews. In modern bioprocess engineering, batch, fed-batch, and continuous cultures are typically described as the three fundamental modes of cultivation. Given these characteristics, fed-batch culture is essentially a variant of batch culture. The advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels (in many cases, at low levels). Generally speaking, fed-batch culture is superior to conventional batch culture when controlling concentrations of a nutrient (or nutrients) affects the yield or productivity of the desired metabolite. In other words, the primary advantage of fed-batch culture is the ability to arbitrarily control the concentration of the fed substrate within the culture broth. In batch culture, all necessary medium components are added at the outset, leaving their concentrations uncontrolled and subject to only the metabolic activity of the microorganisms. In contrast, fed-batch culture allows for the gradual supply of substrates, enabling the optimal maintenance of concentrations—most often at low levels—according to the specific objectives of the process. Meanwhile, in continuous culture (chemostat), all medium components, including the growth-limiting substrate, are maintained at constant values. Therefore, from the perspective of environmental control, fed-batch culture can be positioned as an intermediate between batch and continuous cultivations. Currently, due to risks such as bacterial or phage contamination and genetic mutation, industrial-scale continuous culture is restricted to a limited number of specific fermentations. Consequently, fed-batch culture is increasingly emphasized as an essential improvement over traditional batch culture. In industrial fed-batch processes, the specific substrates used and the feeding strategies employed are often treated as highly confidential proprietary know-how. Consequently, while it is difficult to obtain detailed information on industrial applications, a significant number of commercial fermentations are being conducted using this method.
History The technical term "Fed-batch" first appeared in the title of an original research paper in 1973.Fumitake Yoshida, Tsuneo Yamane and Ken-ichiro Nakamoto. (1973). "Fed-batch hydrocarbon fermentation with colloidal emulsion feed". Biotechnol. and Bioeng, 15(2): 257-270. While Reference is a monograph covering microbial and cell culture in general, Chapter 21 is dedicated to fed-batch cultivation.S. John Pirt. (1975). "Principles of Microbe and Cell Cultivation" Blackwell Scientific Publications, Chapter 21: 211-218. However, the discussion therein is limited to constant fed-batch culture (described later), where the substrate concentration in the feed is equal to the initial substrate concentration of the batch culture. This results in a significant increase in the culture volume within the bioreactor, making it less representative of discussions regarding industrial fed-batch processes. Reference is a review published in 1984, and Reference is a monograph published in 2013.;Henry C. Lim and Hwa Sung Shin. (2013). "Fed-Batch Cultures: Principles and Applications of Semi-Batch Bioreactors", Cambridge University Press, 32 Avenue of the Americas, New York, NY 10013-2473, USA. both provide a comprehensive overview of the literature related to fed-batch cultivation up to their respective years of publication. In practice, however, this method was implemented much earlier than its formal naming. The oldest and best-known example of this fermentation process or operation is in baker's yeast production. To minimize the formation of ethanol (a byproduct) and maximize the yeast yield on sugar, molasses is added intermittently and sequentially to maintain a low sugar concentration. A German patent for this method was filed in 1917 and published in 1919, shortly after World War I.Friedrich Hayduck (1919). "Verfahren der Hefefabrikation ohne order mit nur geringer Alkoholerzeugung", DE 300662. Furthermore, a patent for an improved fed-batch method for baker's yeast was accepted in 1933.Westfälische Hefefabrik G.m.b.H. (1933). "Verfahren zur Herstellung von Hefe nach dem Zulaufverfahren", DE 583760 C. Since then, this process has been known in Germany as "Zulaufverfahren". The production of baker's yeast via the fed-batch method has since undergone various improvements,Henry Y. Wang, Charles L. Cooney, Daniel I. C. Wang. (1979). "Computer control of bakers' yeast production". Biotechnol. Bioeng., 21(6), 975-995. and remains an industrially vital fed-batch fermentation process. Historically, the next application appeared in penicillin fermentation, which involves the sequential addition of energy sources (e.g., glucose) and penicillin precursors (e.g., phenylacetic acid).Marvin J. Johnson. (1952) "Recent advances in penicillin fermentation". Bull. World Helth Org., 6:99-121. Subsequently, in the field of amino acid fermentation—an industry pioneered by Japan starting with glutamate fermentation in 1956—the fed-batch method was adopted for the production of several amino acids (e.g., ReferenceHiromu Ohno, Eiji Nakanishi and Takeichiro Takamatsu. (1976) “Optimal Control of aSemibatch Fermentation". Biotechnol. Bioeng., 18: 847-864.). Furthermore, following the development of genetic engineering, the fed-batch method was adopted for the high-density cultivation of recombinants (primarily Escherichia coli).Joseph Shiloach, Rephael Fass.(2005) "Growing E. coli to high cell density--a historical perspective on method development". Biotechnol Adv 2005, 23:345-357. It has also been applied to heterologous protein production using recombinant yeast.O Mendoza-Vega, J. Sabatie, S. W. Brown. (1994) "Industrial-Production of Heterologous Proteins by Fed-Batch Cultures of the Yeast, Saccharomyces-cerevisiae". FEMS Microbiology Reviews, 15:369-410. More recently, the method has been utilized in the manufacture of antibody drugs via high-density liquid cultivation of animal cells, primarily Chinese Hamster Ovary (CHO) cells.Zhou Jiang, Kurt Droms, Zhaohui Geng, Susan Casnocha, Zhihua Xiao, Steve Gorfien and Scott J. Jacobia. (2012) "Fed-Batch Cell Culture, Process Optimization--A Rationally Integrated Approach--". BioProcess International, 10(3): 40-45.Wen‑Jing Xu, Yan Lin, Chun‑Liu Mi, Jing‑Ying Pang, Tian‑Yun Wang. (2023) "Progress in fed‑batch culture for recombinant protein production in CHO cells". Applied Microbiology and Biotechnology, 107: 1063-1075.
Cases where fed-batch cultures are advantageous The types of bioprocesses for which fed-batch culture is effective can be summarized as follows:
Substrate inhibition
Nutrients such as methanol, ethanol, acetic acid, and aromatic compounds inhibit the growth of microorganisms even at relatively low concentrations. By adding such substrates properly lag-time can be shortened and the inhibition of the cell growth markedly reduced.
High cell density (High cell concentration)
In a batch culture, to achieve very high cell concentrations, e.g. 50-100 g of dry cells/L, high initial concentrations of the nutrients in the medium are needed. At such high concentrations, the nutrients become inhibitory, even though they have no such effect at the normal concentrations used in batch cultures. The fed-batch strategy is typically used in bio-industrial processes to reach a high cell density in the bioreactor.Dieter Riesenberg.(1991) "High-cell-density cultivation of Escherichia coli". Curr Opin Biotechnol., 2:380-384.L. Yee, Harvey W. Blanch.(1992) "Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli". Bio/Technology (N Y ) 10:1550-1556.Michimasa Kishimoto and Hideyuki Suzuki (1995) "Application of an Expert System to High Cell Density Cultivation of Escherichia coli", J. Ferment. Bioeng., 80: 58-62.Sang Yup Lee.(1996) "High cell-density culture of Escherichia coli". Trends Biotechnol., 14:98-105.JosephShiloach, Rephael Fass.(2005) "Growing E. coli to high cell density--a historical perspective on method development". Biotechnol Adv 2005, 23:345-357. Mostly the feed solution is highly concentrated to avoid dilution of the bioreactor. Production of heterologous proteins by fed-batch cultures of recombinant microorganisms have been extensively studied.Paulina Balbás.(2001) "Understanding the art of producing protein and nonprotein molecules in Escherichia coli". Molecular Biotechnology, 19:251-267.Neubauer P, Winter J. (2001) "Expression and fermentation strategies for recombinant protein production in Escherichia coli". In: Merten OW et al. (Eds). Recombinant Protein Production with prokaryotic and eukaryotic cells. A comparative view on host physiology. Kluwer Academic Publisher, Dordrecht, The Netherlands. pp. 195-258.Amulya K. Panda. (2003) "Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli". Adv Biochem Eng Biotechnol, 85:43-93.
The controlled addition of the nutrient directly affects the growth rate of the culture and helps to avoid overflow metabolism (formation of side metabolites, such as acetate for Escherichia coli, lactic acid in mammalian cell cultures, ethanol in Saccharomyces cerevisiae), oxygen limitation (anaerobiosis).Jeongseok Lee, Sang Yup Lee, Suwon Park, Anton P. J. Middelberg. (1999) "Control of fed-batch fermentations". Biotechnol Adv, 17:29-48.Katie F. Wlaschin, Wei-Shou Hu. (2006) "Fed-batch culture and dynamic nutrient feeding". Adv Biochem Engin/Biotechnol, 101:43-74.
Glucose effect (Crabtree effect)
In the production of baker's yeast from malt wort or molasses it has been recognized since early 1900s that ethanol is produced even in the presence of sufficient dissolved oxygen (DO) if an excess of sugar is present in the culture liquid. Ethanol is a main cause of low cell yield. Aerobic ethanol formation in the presence of glucose concentration is known as glucose effect or Crabtree effect. To reduce this effect, a fed-batch process is generally employed for baker's yeast production. In aerobic cultures of Escherichia coli and Bacillus subtilis, organic acids such as acetic acid, (and in lesser amounts, lactic acid and formic acid), are produced as byproducts when sugar concentration is high, and these acids inhibit cell growth as well as show deteriorating effect on the metabolic activities. The formation of these acids are called bacterial Crabtree effects.
Catabolite repression