Rumen-mimetic continuous cultivation system

07/06/2026

The book “Rumen-Mimetic Continuous Cultivation System: In vitro Rumen Fermentation” is published.

Ruminal bacteria form a diverse community composed of many members that enables efficient and accelerated growth under anaerobic conditions. This community is the result of evolutionary processes that have occurred over billions of years. Their strategy for rapid growth involves cross-feeding and mutualistic interactions without competitive elimination. Consequently, the community can accommodate nutritional disturbances. This ecological adaptability is one of the reasons why we were able to develop the RMS. The remarkable adaptability of ruminal bacteria provides a significant opportunity to engineer or guide microbial communities toward optimized biotechnological production of VFAs from lignocellulosic biomass, as well as toward accurate feed evaluation for raising livestock healthily.

This book introduces a rumen‑mimetic continuous cultivation system (RMS) that enables long‑term, active cultivation of ruminal bacteria and the production of volatile fatty acids (VFAs)—including acetic, propionic, and butyric acids—from lignocellulosic biomass. It also presents a novel proc...

01/06/2026

Rumen-mimetic continuous cultivation system (RMS)

The Kindle e-Book has been published by Amazon.
The paperback will be published soon.

This book describes a rumen mimetic continuous cultivation system (RMS) that enables long term active cultivation of ruminal bacteria and the production of volatile fatty acids (VFAs), such as acetic, propionic, and butyric acids, from lignocellulosic biomass. It also presents a novel in vitro procedure, based on the RMS platform, that allows accurate evaluation of fermentation dynamics and feed quality. Ruminants such as cattle and sheep offer a compelling biological model for these challenges.
We developed an RMS bioreactor specifically designed for the utilization of lignocellulosic biomass to produce VFAs. Human intestinal bacteria influence host physiology and pathophysiology through several pathways, one of which is the microbial production of chemical metabolites, namely VFAs, also known as short chain fatty acids (SCFAs). Ruminal bacteria likewise produce SCFAs in the rumen of cattle. SCFA supplementation has been widely explored as a therapeutic approach, and various health effects of SCFAs have been reported. Therefore, the VFA mixture produced using the RMS bioreactor, with defined purity and impurity profiles, is expected to be suitable for use as food additives and active pharmaceutical ingredients.
We also developed a novel in vitro procedure to elucidate the nutritional value of feedstuffs. This method enables us to measure not only the amount of VFA produced from feed but also the digestion time of the feed. To increase productivity and reduce feed costs per unit of milk or meat in modern cattle production systems, it is important to estimate VFA production rates from feed and to use alternative economical roughage sources. Reliable in vitro evaluation techniques are therefore essential for assessing the fermentation activity of ruminal bacteria and determining the nutritional value of economical feed resources.
Ruminal bacteria form a diverse community composed of many members that enables efficient and accelerated growth under anaerobic conditions. This community is the result of evolutionary processes that have occurred over billions of years. Their strategy for rapid growth involves cross-feeding and mutualistic interactions without competitive elimination. Consequently, the community can accommodate nutritional disturbances. This ecological adaptability is one of the reasons why we were able to develop the RMS. The remarkable adaptability of ruminal bacteria provides a significant opportunity to engineer or guide microbial communities toward optimized biotechnological production of VFAs from lignocellulosic biomass, as well as toward accurate feed evaluation for raising livestock healthily.

21/05/2026

My Kindle e-Book has been published by Amazon.
Its contents are as follows:

INTRODUCTION
Chapter 1: Design of the Rumen-Mimetic Continuous Cultivation System (RMS)
1-1. Collection of rumen fluid from cattle
1-2. Materials and methods used in the RMS
1-3. Operational principles and workflow of the RMS
Chapter 2: Operational Procedures and Functional Behavior of the RMS
2-1. Elimination of ruminal protozoa
2-2. Induction of cellulosomes
2-3. Quantification of volatile fatty acids (VFAs)
2-4. Pulverization of roughage
2-5. Supplementation of alfalfa hay (hay cubes)
2-6. Formation of bacterial flocs and biofilms
2-7. pH dynamics during substrate digestion
2-8. Influence of cultivation pH on bacterial activity
2-9. Nitrogen supply via artificial saliva
2-10. Digestion of cellulose and starch
2-11. Differences in rumen fluid collected from different donor cattle
Chapter 3: Microbial and Physiological Characteristics of the RMS Ecosystem
3-1. PCR-DGGE analysis of microbial community structure
3-2. PCR assays of RMS culture broth
3-3. Digestion of Avicel and feedstuffs within flocs and biofilms
3-4. Carbon-flux estimation during Avicel digestion
3-5. Coupling of catabolism and anabolism and the “catabolic pH shift”
3-6. Catabolic pH shift during carbohydrate digestion
3-7. Kinetic analysis of substrate digestion to measure bacterial activity
3-8. Specific growth rate of ruminal bacteria
Chapter 4: Metabolic Flux Analysis of Substrate Digestion by Ruminal Bacteria
4-1. Stoichiometric metabolic flux analysis of rumen fermentation
4-2. Metabolic flux analysis of alfalfa hay digestion
4-3. Metabolic flux analysis of cellulose and starch digestion
4-4. Effect of rice straw particle size on digestion efficiency
Chapter 5: Application of the RMS for VFA (SCFA) Production from Lignocellulosic Biomass
5-1. Long term cultivation for VFA production
5-2. Effect of pH on metabolic activity
5-3. Fermentation under nitrogen-limited conditions
5-4. VFA (SCFA) production from lignocellulosic biomass using the RMS bioreactor
5-5. Health effects of VFAs (SCFAs): A review
Chapter 6: Application of the RMS in Animal Nutrition and Husbandry
6-1. Monitoring ruminal pH of cattle using a wireless pH electrode
6-2. Nutritional evaluation of feedstuffs based on VFA production
6-3. VFA production from mixed cellulose–starch substrates
6-4. Accessibility of cellulolytic bacteria to roughage cellulose
Acknowledgements
References

This book introduces a rumen‑mimetic continuous cultivation system (RMS) that enables long‑term, active cultivation of ruminal bacteria and the production of volatile fatty acids (VFAs)—including acetic, propionic, and butyric acids—from lignocellulosic biomass. It also presents a novel proc...

10/05/2026

My Kindle e-Book is published by Amazon.

This book introduces a rumen‑mimetic continuous cultivation system (RMS) that enables long‑term, active cultivation of ruminal bacteria and the production of volatile fatty acids (VFAs)—including acetic, propionic, and butyric acids—from lignocellulosic biomass. It also presents a novel proc...

10/05/2026

Our research article published in a scientific journal.

Individual differences in gut microbiota composition highlight the need for methods capable of selectively enriching host-associated bacteria from complex microbial communities. Conventional cultiv...

18/03/2026

3-6. Catabolic pH shift
Catabolism and anabolism are coupled via ATP and ADP. In other words, the ATP production rate during catabolism must equal the ATP consumption rate during anabolism. When a substrate containing cellulose or starch is supplied, cellulose or starch is hydrolyzed to glucose by cellulosomes or amylases, respectively (1). When glucose is subsequently catabolized, VFAs are produced and hydrogen ions (H⁺) are released from them (2), causing the pH of the culture solution to decrease. Once cellulose and starch are completely digested, amino acids are catabolized to maintain ATP production (4). During amino acid catabolism, ammonium ions (NH₄⁺) are released through deamination, in which amino acids are converted to pyruvate. This is why the pH of the culture solution increases when the carbohydrate-containing substrate (cellulose and starch) has been fully digested. We refer to this pH change as “Catabolic pH shift,” which occurs when catabolism transitions from (2) to (4). catabolic pH-shifts were observed at the end of substrate digestion. Therefore, the catabolic pH-shift of RMS indicates that the carbohydrates contained in the substrate have been completely digested. Ruminal bacterial cells are synthesized by anabolism ((3) and (5)) using ATP generated through catabolism. When the substrate does not contain protein, such as cellulose and starch, ammonium-containing artificial saliva serves as a nitrogen source (3). Ruminal bacteria also constitutively supply amino acids through their autolysis (6).

13/03/2026

3-3. Feedstuffs are digested by flocs and biofilms of ruminal bacteria.
Representative images of flocs (A, B) and biofilms (C, D) of ruminal bacteria formed during cultivation using RMS. Microscopic observation of the flocs that enfolded some Avicel particles. The biofilms were formed on the surface of rice straw.

05/03/2026

2-6. Typical pH pattern observed during substrate digestion and digestion time of substrate
When 20 g of rice straw pulverized to less than 100 µm was added to the reactor, the pH of the culture solution immediately decreased due to VFA production (Fig. A). The pH was controlled so that it did not fall below 6.50 by the automatic addition of artificial saliva. After 36.3 h, the pH increased above 6.50 because the rice straw had been completely digested and VFA production had ceased (Fig. A). The digestion time of the rice straw was 36.3 h, and 1670 ml of effluent was collected. The amounts of acetic acid, propionic acid, and butyric acid in the effluent were 59.4, 17.2, and 3.4 mmol, respectively. Therefore, the total amount of VFAs produced from 20 g of rice straw was 79.9 mmol, and the total VFA yield was 4.00 mmol/g-substrate. Twenty grams of pulverized alfalfa hay was also evaluated for its nutritional value in a similar manner (Fig. B). The digestion time and the total amount of VFAs produced from 20 g of alfalfa hay were 14.6 h and 29 mmol, respectively, and the total VFA yield was 1.45 mmol/g-substrate.

23/02/2026

3-9. Specific growth rate of ruminal bacteria
The specific growth rate (µ) is the proportional rate at which a microbial population increases in biomass per unit time and is formally expressed as the first order kinetic coefficient in the differential growth equation:
dX/dt=μX
where X is the biomass concentration (g dry cell/L), and the unit of µ is h⁻¹. It quantifies how rapidly cells grow during the exponential phase and is influenced by environmental and nutritional conditions. The medium exchange rate is defined by the dilution rate D (h⁻¹), which describes the relationship between the medium inflow rate F (L/h) and the culture volume V (L) as follows:
D=F/V
When continuous cultivation reaches steady state conditions, the specific growth rate becomes equal to the dilution rate of the bioreactor, and a constant biomass concentration is maintained. Therefore, the specific growth rate in continuous cultivation can be calculated as:
µ=D
If an average flow rate calculated from the digestion time (h) and the effluent volume (L) is used as the flow rate, the specific growth rates of ruminal bacteria cultivated using corn starch, Avicel, rice straw, and alfalfa hay (hay cubes) as substrates were estimated as shown in Table 3 9 and Fig. 3 9. When starch is used as the substrate, the growth rate of amylolytic bacteria becomes the rate determining step. Similarly, when cellulose is used as the substrate, the growth rate of cellulolytic bacteria becomes the rate determining step. Therefore, the specific growth rates of ruminal bacteria cultivated using Avicel, rice straw, or alfalfa hay were almost the same because the main carbohydrate in these substrates is cellulose.

30/01/2026

6-6-8. SCFAs have a potential therapeutic effect on Inflammatory Bowel Disease.
Inflammatory bowel disease (IBD) is a chronic condition characterized by recurrent intestinal inflammation. Its etiopathogenesis involves a series of events that disrupt the mucosal barrier, alter the healthy balance of the intestinal microbiota, and abnormally stimulate intestinal immune responses. Short-chain fatty acids (SCFAs), particularly butyrate, play a critical role in regulating intestinal inflammation [1] and may serve as a therapeutic strategy to increase the levels of SCFA-producing bacteria with potential anti-inflammatory effects when administered orally to patients with various colonic diseases. Although preliminary findings are promising, further research is required to confirm the therapeutic role of SCFAs in IBD and to establish clinically applicable treatment protocols [2, 3].

References:
1. Neurogastroenterology & Motility. 2020; 32: e13914. (doi: 10.1111/nmo.13914)
2. Zhang et al. Cell Communication and Signaling 2022, 20:64 (doi: 10.1186/s12964-022-00869-5)
3. Int. J. Mol. Sci. 2024, 25, 10879. (doi: 10.3390/ijms252010879)