Methods to maximize fermentation output

Abstract

Disclosed herein is a continuous cell cycling fermentation method to produce a variety of fermentation products, including organic acids (e.g., propionic acid or lactic acid) from a microbe.

Description

[0001] METHODS TO MAXIMIZE FERMENTATION OUTPUT

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No.

[0004] 63 / 666.982, filed on July 2, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] TECHNICAL FIELD

[0006] This disclosure generally relates to methods, microbial cell lines, and culture media for producing organic acids using an optimized continuous fermentation protocol as well as methods for increasing fermentation productivity. For example, this disclosure relates to a continuous method for the purpose of efficient large-scale production. This disclosure also relates to a continuous method for producing organic acids.

[0007] BACKGROUND

[0008] Fermentation is any process in which the activity' of microorganisms facilitates a favorable transformation in a foodstuff or beverage. In microorganisms, fermentation is the primary' means of generating adenosine triphosphate (ATP) through the anaerobic degradation of organic nutrients. Fermentation ranging from informal, general usages to more scientific definitions, including preservation methods for food via microorganisms, any large- scale microbial process occurring with or without air (also known as industrial fermentation), any process that produces alcoholic beverages or acidic dairy products, any energy-releasing metabolic process that takes place only under anaerobic conditions (someyvhat scientific), any metabolic process that releases energy from a sugar or other organic molecule, does not require oxygen or an electron transport system, and uses an organic molecule as the final electron acceptor (most scientific).

[0009] SUMMARY

[0010] The productivity of fermentations is often hindered due to accumulations of products, depletion of carbon sources, and overcrowding of microorganisms. In addition, many methods require seed trains before every batch or expensive and technically challenging cell recycling, continuous fermentation, or extraction methods. The present disclosure provides a simple method that nearly doubles the productivity of an organic acid of interest, is technically simple, successful in commercial scaling-up, and can be conducted in a basic batch fermenter tank without any additional expensive axillary parts.

[0011] Provided herein are continuous methods of producing an organic acid. The methods comprise (a) seeding a microbe previously identified as an organic acid producing microbe at an initial density in a volume of fresh culture medium comprising a fermentable carbon source in a fermentation vessel for an initial fermentation; (b) culturing the microbe in the culture medium from about 1 day to about 10 days, thereby producing a fermentate at a titer; (c) removing at least about 50% of the fermentate and about 50% to about 99.5% of the microbes; (d) replacing the fermentate removed from the fermentation vessel in step (c) with the same volume of fresh culture medium, wherein the remaining microbes are maintained at a selected proportion of total microbes in step (b) for a subsequent fermentation; and (e) repeating steps (b) to (d) at least one time; thereby producing one or more volumes of the fermentate comprising the organic acid. The organic acid comprises propionic acid, lactic acid, acetic acid, butyric acid, formic acid, citric acid, succinic acid, gluconic acid, malic acid, itaconic acid, pyruvic acid, oxalic acid, ascorbic acid, or combinations thereof. In some embodiments, the organic acid is propionic acid.

[0012] In some embodiments, the microbe is only seeded into the fermentation vessel once. In some embodiments, the microbe in step (a) is seeded at an initial density of about 0.5% to about 20% of the culture medium. In some embodiments, the selected proportion at which the remaining microbes maintain in step (d) is about 0.5% to about 50% of the culture medium in each of the subsequent fermentations.

[0013] In some embodiments, the microbe comprises bacteria or fungi. In some embodiments, the bacteria comprise Propionibacterium spp., Veilonella spp., Bacteroides spp., Lactobacillus spp., Bacillus spp., Clostridium spp., Butyrivibrio spp., Acetobacter spp., or Gluconobacter spp. In some embodiments, the bacteria comprise Propionibacterium spp.. preferably Propionibacterium acidipropionici (P. acidipropionici), o Lactobacillus spp. In some embodiments, the fungi comprise Aspergillus spp., Penicillium spp., Rhizopus spp., or Candida spp.

[0014] In some embodiments, the fermentable carbon source comprises glucose, sucrose, fructose, lactose, maltose, starch, molasses, cellulose, xylose, arabinose, glycerol, or ethanol. In some embodiments, the fermentate removed in step (c) is from about 50% to about 99.5%. In some embodiments, the volume of the fermentation is about IL to about 100,000 gallons. In some embodiments, the volume of the first and subsequent fermentations is about IL, about 10 gallons, about 1,000 gallons, about 10. 000 gallons, or 50.000 gallons. In some embodiments, steps (b) to (d) in step (e) are repeated from about 1 time to about 20 times. Also provided herein are methods of continuous cell cycling (CCC) fermentation. The methods comprise (a) culturing a microbe at an initial density in a volume of fresh culture medium comprising a fermentable carbon source in a fermentation vessel for an incubation period of from about 2 days to about 10 days, thereby producing a fermentate at a titer for an initial fermentation; (b) removing about 50% to about 99.5% of the fermentate and about 50% to about 99.5% of the microbes from the fermentation vessel; (c) replacing the fermentate removed from the fermentation vessel in step (b) with the same volume of fresh culture medium, wherein the remaining microbes are maintained at a selected proportion of total microbes in step (a) for a subsequent fermentation; (d) incubating the subsequent fermentation for an incubation period of from about 1 day to about 8 days; and (e) repeating steps (b) to (d) at least one time; thereby producing one or more volumes of fermentate at about the titer said in step (a) during shortened incubation periods. In some embodiments, the microbe cultured in fermentations comprises bacteria, and fungi. In some embodiments, the microbe cultured in fermentations comprises one or more ty pes of microbes. In some embodiments, the microbe is only seeded into the fermentation vessel once. In some embodiments, the shortened incubation period for the subsequent fermentation in step (d) comprises shortened lag phase.

[0015] In some embodiments, the fermentate is an extracellularly excreted metabolite. In some embodiments, the fermentate removed in step (b) is at least about 70%. In some embodiments, the fermentate removed in step (b) is about 75% to about 99.5%.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0018] DESCRIPTION OF DRAWINGS

[0019] FIGS. 1A-1C shows an exemplary method of continuous cell cycling (CCC) for promoting productivity of fermentation. FIG. 1A shows that initial batch fermentation was conducted until the carbon was nearly consumed or was consumed. FIG. IB shows that between 75% to 99.5% volume (vol) of the media was drained through the harvest line. FIG. 1C shows that freshly prepared media was added to the fermenter.

[0020] FIG. 2 shows that the method was conducted in IL-scale bioreactors with a proprietary propionic acid bacteria (PAB) strain, with the initial and eight consecutive fermentations being derived from a single initial inoculum. The titer of propionic acid is reported as g / L (y-axis). The initial standard fermentation is inoculated and labeled as such, with CCC runs demarcated w ith a “CF” and a digit corresponding to the number of cycles the inoculum has gone through in the title. The initial standard fermentation was inoculated at 10% (vol / vol, e.g., combining a microbe-containing inoculum that constitutes 10% of the total volume with fresh medium that constitues 90% of the total volumn); the subsequent CCC fermentations maintained a 25% inoculum (vol / vol, e.g., combining a microbecontaining fermentate retention that constitues 25% of the total volume from the previous fementation with fresh medium that constitutes 75% of the total volumn) from the previous fermentation.

[0021] FIG. 3 shows that the CCC method was repeated in IL-scale bioreactors with a PAB strain for the initial and two CCC consecutive fermentations. The titer of the propionic acid is reported as g / L (y-axis). The initial standard fermentation is inoculated and labeled as such, with CCC runs demarcated with a “CF” and a digit corresponding to the number of cycles the inoculum has gone through in the title. The initial standard fermentation was inoculated at 10%; the subsequent CCC fermentations maintained a 10% inoculum from the previous fermentation.

[0022] FIG. 4 shows that the CCC method was tested at the production scale of 50,000 gallons with a PAB strain for the initial and three CCC consecutive fermentations. The titer of the propionic acid is reported as g / L (y-axis). The initial standard fermentation is inoculated and labeled as such (50K_Initial), with CCC runs demarcated with a “50K_CF” and a digit corresponding to the number of cycles the inoculum has gone through in the title. The initial standard fermentation was inoculated at 5%; the subsequent CCC fermentations maintained a 10% inoculum from the previous fermentation.

[0023] FIGS. 5A-5C show large commercial-scale batch results comparing fermentation kinetics between standard-batch (“batch”) and CCC fermentations (“CF method”). Error bars are reported by one standard deviation of the mean. FIG. 5A shows that productivity is defined by (g / L of product) / (hours post-inoculation). FIG. 5B shows that product titer is defined as (g of product) / (per 1 L of fermentation). FIG. 5C shows that yield percent (%) is defined as (gram of product) / (gram of input carbon) x 100, representing the weight percent of input to product. The initial standard fermentation was inoculated at 5%; the subsequent CCC fermentations maintained a 10% inoculum from the previous fermentation.

[0024] FIG. 6 shows that the CCC method was conducted in IL-scale bioreactors with a lactic acid bacteria (LAB) strain for a total of three consecutive fermentations. The titer of lactic acid is reported as g / L (y-axis). The initial standard fermentation is inoculated and labeled as such, with CCC runs demarcated with a “LAB CF” and a digit corresponding to the number of cycles the inoculum has gone through in the title. The initial standard fermentation was inoculated at 10%; the subsequent CCC fermentations maintained a 10% inoculum from the previous fermentation.

[0025] DETAILED DESCRIPTION

[0026] The economic viability’ of industrial fermentations is highly dependent on the final yield and titer of the desired metabolite and productivity (i.e., the amount of product produced with respect to time) of its biosynthesis. Productivity can especially be affected if the desired product acts as a feedback inhibition response regulator to its own production. This is particularly the case when considering organic acid production. Traditional batch or fed-batch fermentations often fall victim to this caveat. Several techniques have been developed to circumvent the feedback inhibition problem: Continuous, extractive, cellrecycling, and high-cell density inoculums of fermentations(1-7).

[0027] Although several reports of said techniques have shown great promise at the laboratory scale, especially in the case of organic acid production, oftentimes, their commercial scale-up has proven impossible or economically unviable. The present disclosure provides methods, referred to herein as continuous cell cycling (CCC), that show groundbreaking lab and commercial-scale success in which productivity is doubled over traditional batch fermentation at both the lab- and commercial-scale. This increase in productivity was demonstrated at the commercial scale to exhibit no reduction in final product titer and yield with respect to standard batch fermentation. CCC requires no extra extractive system, centrifugation, or ceramic membranes as the previously mentioned methods require, which are very expensive at the commercial scale, and, depending on the product of interest, may thus render the technology financially unviable. In addition, the standard batch fermentation method requires a seed train for biomass generation every fermentation run. The methods of the present disclosure (e.g., CCC) have shown no major reduction in productivity’ after numerous sequential iterations of fermentations, thus negating the need for a seed train between batches. In addition, the reduction of seed train iterations has the potential to greatly reduce ingredient costs, personnel costs, and time and increase tank turnover and overall output. In addition, with the reduction of seed interactions and no need for additional auxiliary equipment that must maintain sterility, the methods disclosed herein can highly reduce contamination frequency.

[0028] The continuous methods of producing organic acids described in the present disclosure begin with initial fermentation, which necessitates a seed train or initial inoculum of a microbe into the culture medium in a fermentation vessel. Following this, the fermentation is allowed to progress until it reaches or approaches completion. A portion of at least about 50% (e.g., about 75% to about 99.5%) of the fermentate from the initial cycle is drained, and fresh media is added to achieve the desired concentration. This draining and replenishing step is then repeated for subsequent cycles. The microbe is only inoculated (or seeded) into the fermentation vessel once in the initial fermentation.

[0029] The methods of continuous cell cycling (CCC) fermentations described in the present disclosure exhibit reduced lag-phase of microbial growth and fermentate production during the incubation periods in the consecutive subsequent cycles. This results in increased productivity at commercial scale (e.g., 50,000 gallons), without any reduction in final product titer and yield compared to standard batch fermentation. This may increase tank turnover and overall output. Furthermore, the present methods can negate the need for a seed train for the subsequent fermentation cycles, potentially reducing ingredient costs, personnel costs, fermentation time, or contamination frequency. Additionally, the present methods eliminate the need for additional extractive systems, centrifugation, or ceramic membranes, making this technology more financially feasible.

[0030] As used in the specification and the appended claims, the singular forms “a,” '‘an’’ and “the” include plural referents unless the context clearly dictates otherwise.

[0031] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is ± 10% of the referenced value.

[0032] As used herein, the term “plurality” refers to a state of having a plural (e.g.. more than one) number of different types of things (e.g., a cell, a microbe, a subject, a system, or a fermentate).

[0033] Fermentation

[0034] Fermentation typically encompasses several stages during the incubation or fermentation period, including inoculation (seeding), lag phase, exponential or log phase, stationary phase, and decline or death phase.

[0035] At the inoculation stage, the desired microorganisms are inoculated into the fermentation vessel, often from a pure culture or a seed culture. A seed culture is a small volume of microbial culture used to initiate a fermentation process. It typically consists of a specific strain or combination of strains of bacteria that are selected for their ability to produce desired products or perform specific metabolic activities. Once the seed culture reaches a certain cell density or activity level, it is transferred to the larger fermentation vessel to inoculate the fermentation medium and start the production process.

[0036] At the lag stage, microorganisms adjust to their new environment before actively growing and producing metabolites (or fermentates). During this phase, microbial cells undergo physiological changes to adapt to the available nutrients, temperature, pH, and other environmental conditions. The duration of the lag phase can vary depending on factors such as the specific microorganism, the composition of the fermentation medium, and the environmental conditions. In some embodiments, the duration of lag phase is shortened by replenishing a portion of the fermentate with the fresh culture medium and removing a portion of outgrow n microbes in a fermentation environment. In some embodiments, the fermentate removed is at least about 50% (by volume). In some embodiments, the fermentate removed is at least about 70% (by volume). In some embodiments, the fermentate removed is from about 75% (by volume) to about 99.5% (by volume).

[0037] Once the lag phase is complete, microbial growth and fermentation activity enter the exponential or log phase, characterized by rapid cell division and metabolite production. Furthermore, during the stationary phase, as nutrient availability decreases and waste products accumulate, microbial growth slows down, and the population reaches a plateau. Metabolite production continues, but at a reduced rate. Eventually, nutrient depletion, waste accumulation, and other unfavorable conditions lead to a decline in microbial activity. This may result in cell death, resulting in a reduction in biomass and metabolite production.

[0038] Fermentates are the mixture or solution produced by microorganisms during the fermentation process, which can include organic acids, chemicals, biofuels, enzymes, proteins, pharmaceuticals, and other microbial metabolites. Fermentates (e.g., propionic acid or lactic acid) can vary widely depending on the specific fermentation process (e.g., Propionibacterium acidipropionici, or Lactobacillus spp.), the microorganisms involved, and the starting materials used. In some embodiments, the fermentate is an extracellularly excreted metabolite. Industrial fermentation (also called commercial-scale fermentation) is any large-scale microbial process, whether aerobic or anaerobic, in which microbes (or microorganisms) are employed for the mass production of fermentates. The microbes that can be used for the large-scale cultivation include bacteria and fungi. The bacteria commonly used for large-scale fermentation include but not limited to Propionibacterium spp., Lactobacillus spp., Escherichia coli. Bacillus spp., Clostridium spp., Streptococcus spp., and Corynebacterium spp. The fungi used for large-scale fermentation include but not limited to Saccharomyces cerevisiae (Baker's yeast), Saccharomyces pastorianus (Brewer's yeast), Aspergillus spp., Penicillium spp., Rhizopus spp., and Trichoderma spp. In some embodiments, the microbe cultured in fermentations comprises one or more types of microbes.

[0039] Various scales of fermentations can be conducted depending on the production requirements, ranging from laboratory-scale research (e.g., from milliliters to a few liters) to large-scale commercial production. Large-scale usually includes volumes ranging from hundreds to thousands of liters (L) including but not limited to about 100 L, about 500 L, about 1,000 L, about 2,000 L, about 3.000 L, about 4,000 L, about 5,000 L, about 6,000 L. about 7,000 L. about 8,000 L, about 9.000 L, about 10,000 L. about 20.000 L, about 30,000 L (about 10,000 gallons), about 40,000 L, about 50,000 L, about 60,000 L, about 70,000 L, about 80,000 L, about 90,000 L, about 100,000 L, about 110,000 L, about 120,000 L, about 130,000 L, about 140,000 L, about 150,000 L, about 160,000 L, about 170.000 L, about 180,000 L. about 190,000 L (about 50,000 gallons), about 200,000 L. about 210.000 L, about 220,000 L, about 230,000 L, about 240,000 L, about 250,000 L, about 260,000 L, about 270,000 L, about 280,000 L, about 290,000 L, and about 300,000 L.

[0040] Several fermentation methods used in various industries for producing a wide range of products, including batch fermentation, fed-batch fermentation, and continuous fermentation. In standard batch fermentation, microorganisms are cultured in a closed vessel (fermenter) with a specific substrate and optimal conditions for growth and metabolism. The fermentation progresses until the substrate is depleted or the desired product concentration is achieved, without any subsequent additions or removals. Fed-batch fermentation is a modification of batch fermentation where additional nutrients or substrates are added to the fermentation vessel during the fermentation process to maintain optimal growth conditions for the microorganisms, leading to higher product concentrations and yields. Continuous fermentation involves the continuous addition of fresh substrate and removal of fermentation products from the fermentation vessel while maintaining steady-state conditions. This method allows for continuous production without the need for stopping and starting the process. Batch fermentation typically requires a seed train for biomass generation (or large- scale fermentation) every fermentation run. A seed train is a series of sequential inoculations or cultivation steps used to gradually scale up the microbial culture from a small initial inoculum to a larger fermentation volume. Known continuous fermentations at a large-scale may require one or more seed trains. The methods disclosed herein only requires a seed train once at the initial fermentation.

[0041] In continuous fermentation methods with cell recycling, microbial cells are continuously recycled back into the fermentation vessel after separation from the fermentation broth. Periodically or continuously, a portion of the fermentation broth is withdrawn from the bioreactor and subjected to cell separation techniques such as centrifugation, filtration, or membrane separation to separate the microbial cells from the liquid phase. Subsequently, the separated microbial cells are then recycled back into the bioreactor (fermentation vessel), either directly or after further processing (e.g., washing or concentration). However, cell separation requires additional equipment for centrifugation, filtration, or membrane separation, while repetitive cell recycling increases the risk of contamination in the continuous culture system. In the methods disclosed herein, the microorganisms contained in the removed portion of fermentate are not recycled back to the fermentation vessel.

[0042] The economic viability of industrial fermentations is highly dependent on the final yield and titer of the desired metabolite and productivity (e.g., the amount of product produced with respect to time) of its biosynthesis.

[0043] The final yield in industrial fermentation is the amount of desired product obtained after the fermentation process is complete. It indicates the efficiency of the fermentation process and the effectiveness of the microorganism in converting the substrate into the desired product. The final yield is typically expressed in terms of the quantity of the product obtained per unit volume of fermentation broth or per unit weight of substrate used. Achieving a high final yield is important for maximizing production efficiency and reducing production costs in industrial fermentation processes. Yield is often expressed as yield percentage (yield %), calculated as the ratio of the weight of the product (in grams) to the weight of the input carbon source (in grams), multiplied by 100 ((g of product) / (g of input carbon source) x 100). This value represents the percentage of input carbon that is converted into the desired product.

[0044] The titer of the desired metabolite in industrial fermentation is the concentration or amount of the target compound produced by the microorganisms during the fermentation process. It indicates the productivity of the fermentation process and is usually measured at the end of fermentation. The titer is typically expressed in terms of mass concentration (e.g., grams per liter) or volumetric concentration (e.g., moles per liter) of the desired metabolite in the fermentation broth. Achieving a high titer is essential for maximizing the yield of the desired product and optimizing the overall efficiency of the fermentation process. In some embodiments, the titer is calculated as the amount of product (in grams) produced per liter of fermentation volume ((g of product) / (IL of fermentation volume)).

[0045] Productivity in the context of industrial fermentation is the efficiency with which microorganisms produce the desired metabolites or products during the fermentation process. It is a measure of the rate at which the target compound is synthesized or generated per unit of time and resources, typically expressed as grams per liter per hour (g / L / h) or moles per liter per hour (mol / L / h). Productivity is influenced by various factors such as the choice of microorganism, fermentation conditions (e.g., temperature, pH, agitation, aeration), substrate availability, and genetic engineering techniques. High productivity is desirable in industrial fermentation as it directly impacts the efficiency and economics of the production process. In some embodiments, productivity is calculated as the amount of product (in grams) produced per liter of fermentation volume per hour post-inoculation ((g / L of product) / (hours postinoculation)).

[0046] The economic viability of industrial fermentation is highly dependent on the final yield and titer of the desired metabolite and productivity (e.g., the amount of product produced with respect to time) of its biosynthesis. Productivity can especially be affected if the desired product acts as a feedback inhibition response regulator to its own production. Traditional batch or fed-batch fermentations often fall victim to this caveat. In some embodiments, productivity can be affected by depleting carbon source. In some embodiments, productivity can be affected by high cell density of the microbial cells. The methods described herein exhibit shortening fermentation periods while maintaining fermentate titer and yield, thereby increasing the productivity compared to other methods.

[0047] Organic acids

[0048] Organic acids are a class of organic compounds characterized by the presence of one or more carboxyl functional groups (COOH) attached to a carbon atom in the molecule. They are carbon-containing compounds having acidic properties. Organic acid production in industrial fermentation involves the controlled growth of specific microbes under optimized conditions to yield high concentrations of organic acids as the desired end-product. Examples of organic acids commonly produced through industrial fermentation include propionic acid, lactic acid, acetic acid, butyric acid, formic acid, citric acid, succinic acid, gluconic acid, malic acid, itaconic acid, pyruvic acid, oxalic acid, and ascorbic acid. Organic acids can be, e.g., used in the production of biodegradable polymers. They also have other important industrial applications, including as food and feed additives, mainly as preservatives.

[0049] Microbial production of organic acids by fermentation has been known and used for centuries. For example, Aspergillus niger and Yarrowia lipolytica have been used to produce citric acid; Lactobacillus has been used to produce lactic acid; Clostridium has been used to produce acetic acid; Aspergillus niger and Gluconobacter have been used to produce gluconic acid.

[0050] Propionic acid (PA) is a carboxylic acid that has gained significant commercial value and can be produced by microbial fermentation of sugars from several carbon sources (see, e.g., Ahmadi et al., 2017). The examples of fermentable carbon sources include but not limited to glucose, sucrose, fructose, lactose, maltose, starch, molasses, com syrup, cellulose, xylose, arabinose, glycerol, ethanol, acetic acid, lactic acid, and citric acid. Propionic acid has historically been produced commercially using a '■petrochemical” process because it was deemed more economically feasible. This has changed as the cost of crude oil and petrochemicals have increased over the years, as has consumer demand for a natural source of propionic acid for use in foods, resulting in increased interest in production methods using microbial fermentation. A number of culture systems such as batch, fed-batch, and continuous fermentation have been used.

[0051] Propionibacterium is the microorganism most often used in the production of PA (as well as vitamin B12 and Swiss cheese). Propionibacterium is a gram-positive, non-motile, non-spore forming, rod-shaped, anaerobic genus of bacteria that includes the species P. freudenreichii , P. acidifaciens , P. cyclohexanicum. P. auslrahense. P. acidipropionici, P. jensenii. P. thoenii, P. microaerophilum, P. olivae. P. damnosum, P. propionicum, P. acnes, P. avidum, P. granulosum, P. humerusii, and P. lymphophilum. For industrial PA production, the most commonly used strain is P. acidipropionici. (A proposal has been made to reclassify the species within the genus Propionibacterium into three novel genera: Acidipropionibacterium, Cull bacterium. and Pseudopropionib act er ium (Scholz & Kilian 2016). However, Propionibacterium acidipropionci and Acidipropionibacterium acidipropionici are still used somewhat interchangeably.) The optimal pH and temperature for Propionibacterium cell growth are about 6.0-7.0 and about 30-37°C, respectively (Ahmadi et al. 2017). Cell growth is inhibited in pH less than about 5.0, although fermenters started at neutral pH can reach pH 4.4 (Rehberger and Glatz 1998). Ahmadi et al. provides an overview of PA production on several carbon sources by various species of Propionibacterium as reported in the literature (Ahmadi et al. 2017) and is incorporated herein by reference.

[0052] PA can also be produced by other anaerobic bacteria, such as certain species of Anaerovibrio, Bacteroides, Clostridium. Fusobacterium, Megasphaera, Propionispira, Selenomonas. and Veillonella.

[0053] Lactic acid (LA) is a hydroxy acid, containing a hydroxyl group (OH) and a carboxyl group (COOH) on adjacent carbon atoms in its molecular structure. Lactic acid exists in two optical isomers: L-lactic acid and D-lactic acid, with L-lactic acid being the naturally occurring form, lactic acid is produced through the microbial fermentation of sugars, particularly glucose, by certain bacteria known as lactic acid bacteria. This fermentation process, called lactic acid fermentation, involves the conversion of glucose to lactic acid in the absence of oxygen. Lactic acid fermentation is commonly used in the production of fermented dairy products such as yogurt, cheese, and kefir, as well as in the fermentation of vegetables to produce sauerkraut and kimchi.

[0054] Lactobacillus is the microorganism most often used in the production of LA. Lactobacillus is a genus of Gram-positive, rod-shaped bacteria belonging to the family Lactobacillaceae. Some common species that produce lactic acid include Lactobacillus acidophilus, Lactobacillus plantarum. Lactobacillus casei. Lactobacillus rhamnosus, and Lactobacillus bulgaricus.

[0055] However, productivity of organic acids can be hindered by feedback inhibition responses, in which the organic acid being produced inhibits its own production. Traditional batch or fed-batch fermentations often show organic acid feedback inhibition responses, resulting in decreased final yield and titer. Although several reports of the technologies have shown great promise at the laboratory scale, especially in the case of organic acid production, oftentimes, their commercial scale-up has proven impossible or economically unviable. There is a clear need for improved methods of fermentation of organic acids that are less dependent on petrochemical-dependent processes and that overcome challenges posed by feedback inhibition.

[0056] Culture medium

[0057] The composition of microbial culture medium for organic acid production in industrial fermentation typically includes carbon source, nitrogen source, phosphorus, sulfur, micronutrients, buffer agents, antifoaming agents, oxygen and aeration. pH control agents, inducers, and substrate analogs. Any suitable microbial culture medium known in the art can be used in the methods described herein. The exact composition and concentrations of these components can vary depending on the specific microorganism, fermentation conditions, and desired organic acid product. Additionally, other additives or supplements may be included to optimize fermentation performance and product yield. Common fermentable carbon sources include but are not limited to glucose, sucrose, fructose, lactose, maltose, starch, molasses, cellulose, xylose, arabinose, glycerol, and ethanol.

[0058] As described herein, the composition of culture medium for culturing propionic acid or lactic acid producing bacteria for propionic acid production typically includes fermentable carbon source, nitrogen source, and pH control agents.

[0059] Continuous cell cycling (CCC) fermentation

[0060] Continuous cell cycling (CCC) fermentation begins with an initial fermentation, which requires an initial seed train or inoculum of a microbe into the culture medium in a fermentation vessel to produce a fermentate (e.g., organic acid). Following this, a portion of about 50% to about 99.5% (e.g., about 75% to about 99.5%) of the fermentate (e.g., organic acid) is drained and replaced with fresh culture medium. This draining and replenishing step is iterated for subsequent fermentation cycles. Notably, the microbes are only inoculated (or seeded) into the culture medium once in the initial fermentation. The microbes contained in the removed portion of fermentate are not reintroduced into the fermentation vessel. The microbial cells from the initial fermentation undergo amplify and partial passage into the subsequent fermentation cycles (“CF”) with partly refreshed culture medium, resulting in shortened fermentation periods while maintaining desired titer and yields of the fermentate (e.g., organic acid). Consequently, CCC fermentation increases fermentation productivity compared to tradition methods. In some embodiments, the fermentation period of the subsequent fermentation cycles (“CF”) is shortened compared to the fermentation period of the initial fermentation. In some embodiments, the fermentation period of the subsequent fermentation cycles (“CF”) is shortened compared to the fermentation period of traditional batch fermentation. In some embodiments, the shortened fermentation period is the lag phase.

[0061] The disclosed methods of continuous cell cycling (CCC) fermentation include (a) culturing a microbe at an initial density in a volume of fresh culture medium comprising a fermentable carbon source in a fermentation vessel for an incubation period of from about 3 days to about 10 days, thereby producing a fermentate (e.g., organic acid) at a titer for an initial fermentation; (b) removing about 50% to about 99.5% of the fermentate (e.g.. organic acid) and about 50% to about 99.5%of the microbes from the fermentation vessel; (c) replacing the fermentate (e.g., organic acid) removed from the fermentation vessel in step (b) with the same volume of fresh culture medium, wherein the remaining microbes are maintained at a selected density7for a subsequent fermentation; (d) incubating the subsequent fermentation for an incubation period of from about 1 day to about 8 days; and (e) repeating steps (b) to (d) at least one time; thereby producing one or more volumes of fermentate (e.g.. organic acid) at about the titer said in step (a) during shortened incubation periods.

[0062] The microbe cultured in CCC fermentation can be bacteria and fungi. One or more types of microbes can be cultured in one fermentation vessel to produce one or more fermentates. The fermentates are extracellularly excreted metabolites (e.g., organic acids).

[0063] As describe herein, the microbe is inoculated only once at the initial (first) standard fermentation throughout multiple continuous fermentation cycles. One fermentation cycle or fermentation period is the duration when the fermentation proceeds until all carbon in the medium is consumed, or until a desired level of product is obtained. For example, the initial standard fermentation can proceed for about 2 to about 10 days, e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 days, or any range therebetween with those endpoints. The subsequent fermentation cycles (“CF”) can proceed for about 2 to 10 days, or for about 1 to 8 days, e.g.. about 1, about 2. about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days, or any range therebetween with those endpoints.

[0064] As described herein, the number of microbial cells (e.g., bacteria, fungi, or viruses) in a sample that are viable can be estimated by colony -forming unit (CFU). The standard measure for CFU is the number of culturable microbial cells (e.g., bacteria, fungi, or viruses) present per 1 mL of culture (CFU / mL) determined by serial dilution and spread plating techniques. For each timepoint, a 1:2, 1:5, 1: 10, 1 :20, 1 :50, or other appropriate dilution series of the batch culture is performed. As described in the present disclosure, the microbes inoculated at the initial standard fermentation were about 1 x 102CFU / mL to about 1 x 1010CFU / mL (e.g., about I x lO3CFU / mL to about l* 109CFU / mL, about I xlO4CFU / mL to about I xlO8CFU / mL, about I xlO5CFU / mL to about IxlO7CFU / mL, about I xlO2CFU / mL, about I xlO3CFU / mL, about I xlO4CFU / mL, about I xlO5CFU / mL, about IzI O6CFU / mL, about I xlO7CFU / mL, about I xlO8CFU / mL, about I xlO9CFU / mL, or about Ix lO10CFU / mL).

[0065] As described herein, the microbe can be inoculated at about 0.5% to about 20% at the initial standard fermentation. For example, the microbe can be inoculated at about 0.5%. about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%. about 18%, about 19%, about 20%, or any range therebetween with those endpoints, at the initial standard fermentation. In the disclosed methods of CCC fermentation, the microbe is inoculated at about 5% or about 10% at the initial standard fermentation. Unless the context clearly dictates otherwise, the percentage (%) at which the inoculum is inoculated (e.g., 5% or 10%) is a proportion made up of inoculum volume to total volume (vol / vol). For example, a 10% inoculum (the microbe is inoculated at 10%) is 10% vol / vol, meaning combining a microbe-containing inoculum that constitutes 10% of the total volume with fresh medium that constitutes 90% of the total volume).

[0066] As describe herein, the subsequent CCC fermentations maintain microbes at about 0.5% to about 50% at each subsequent fermentation cycle (“CF”). For example, each "CF" maintains microbes at 0.5%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5%, about 25%, about 27.5%, about 30%, about 32.5%, about 35%, about 37.5%, about 40%, about 42.5%, about 45%, about 47.5%, about 50%. or any range there between with those endpoints. In the disclosed methods of CCC fermentation, the subsequent CCC fermentations maintain microbes at about 10% or about 25%. Unless the context clearly dictates otherwise, the percentage (%, e.g., 10% or 25%), at which microbes remain in the culture vessel from the previous fermentations, is a proportion made up of remaining fermentate volume to total volume (vol / vol). For example, a 25% inoculum for the subsequent “CF” is 25% vol / vol, meaning combining a microbecontaining fermentate retention that constitues 25% of the total volume with fresh medium that constitues 75% the total volumn)

[0067] At each subsequent fermentation cycle (“CF”), a volume of fermentation medium that includes the product (e.g., organic acid), containing a portion of the microbes, is removed for further processing of the product. The volume that is removed can be at least 50% and up to 99.5%. For example, the volume of the fermentate (e.g., organic acid) that is removed can be at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99.5%, or any range therebetween with those endpoints. In some embodiments, the volume of the fermentate (e.g., organic acid) that is removed can be about 75% to about 99.5%. The density of the remaining microbes in the refreshed culture medium is about 5% to about 50% from the previous fermentation, e.g., about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%. or any range therebetween with those endpoints, in each of the subsequent fermentations. Preferably, the microbes remaining in the refreshed culture medium is about 10% to about 25% from the previous fermentation. As described herein, subsequent CCC fermentation cycles can be repeated from about 1 to about 20 times, e.g.. about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 times. In some embodiment, subsequent CCC fermentation cycles can be repeated from about 8 to about 15 times. In some embodiment, the microbes can maintain genetic stability’ across multiple consecutive fermentation cycles.

[0068] The disclosed CCC fermentation methods can be used for industrial scale fermentation. Thus, the volume of the initial and subsequent fermentations can be at least about 1,000 gallons to about 300,000 gallons, e.g., about 1,000 gallons, about 10, 000 gallons, about 30, 000 gallons, about 50,000 gallons, about 75,000 gallons, about 100, 000 gallons, about 200. 000 gallons, about 300, 000 gallons, or any range therebetween with those endpoints.

[0069] In the disclosed methods described herein, a seed train is only required once for the initial fermentation for continuous large-scale fermentations. This reduction in seed train iterations has the potential to significantly reduce ingredient and personnel costs, as well as save time, while boosting tank turnover and overall output. Moreover, by minimizing seed interactions and eliminating the need for extra auxiliary equipment to maintain sterility7, it is reasonable to anticipate a substantial reduction in contamination frequency with the present methods described herein.

[0070] As disclosed herein, the microorganisms contained in the removed portion of fermentate are not recycled back to the fermentation vessel. Unlike previously mentioned methods, the disclosed methods do not require extra extractive system, centrifugation, or ceramic membranes. These technologies can be prohibitively expensive at commercial scale and may render the overall technology financially unfeasible, particularly depending on the specific product of interest.

[0071] Methods for producing organic acid

[0072] Several techniques have previously been developed to address feedback inhibition of fermented organics and include continuous fermentation, extractive fermentation, cellrecycling fermentation, and high-cell density inoculations. See, for example, Colomban et al., Biotechnol Bioeng. 1993;42(9): 1091-8; de Assis et al., Food Bioproc Tech. 2022;15(4):734- 49; Dishisha et al., Bioresour Technol. 2012;118:553-62; Liu et al., Bioresource Technology. 2016;216:856-61; Miyano et al.. Biochem Eng J. 2000;6(3):207 Stowers et d ., J Ind Microbiol Biotechnol. 2014;41(5):837-52; and Wang et al., Biotechnol Bioeng. 2015; 112(3):502-l l. However, commercial scale-up of these fermentation methods to reach large fermentation batches (e.g., at least 10,000 gallons fermentations batches) has proven difficult or economically unviable. One reason is that the commercial scale-up process often requires a seed train, or repeated cultures of microbes in successively larger volumes to obtain enough of the microbes to successfully initiate fermentation in large fermentation batches, and sometimes between each sequential fermentation batch. Each time another culture is inoculated in a seed train increases the likelihood of contamination and increases the costs of using proper sterile technique.

[0073] The present methods can be used for producing organic acids, e.g., propionic acid or lactic acid. The method disclosed herein include (a) seeding a microbe previously identified as an organic acid producing microbe at an initial density in a volume of culture medium comprising a fermentable carbon source in a fermentation vessel for an initial fermentation; (b) culturing the microbe in the culture medium for from about 2 to about 10 days, thereby producing a fermentate comprising an organic acid at a titer; (c) removing about 50% to about 99.5% of the fermentate comprising an organic acid and a plurality of the microbes; (d) replacing the fermentate removed from the fermentation vessel in step (c) with the same volume of fresh culture medium, wherein the remaining microbes are maintained at a selected density7for the subsequent fermentation; and (e) repeating step (b) to (d) at least one time; thereby producing one or more volumes of the fermentate comprising an organic acid.

[0074] The organic acids (e.g., propionic acid or lactic acid) produced by disclosed methods include but not limited to propionic acid, lactic acid, acetic acid, butyric acid, formic acid, citric acid, succinic acid, or combinations thereof. In some embodiments, the organic acid produced by disclosed methods is propionic acid. In some embodiments, the organic acid produced by disclosed methods is lactic acid.

[0075] As disclosed herein, the organic acids (e.g., propionic acid or lactic acid) producing microbes include bacteria or fungi. Organic acids producing bacteria include but not limited to Propionibacterium spp., Veilonella spp., Bacteroides spp., Lactobacillus spp., Bacillus spp., Clostridium spp., Butyrivibrio spp., Acetobacter spp., or Gluconobacter spp. In some embodiments, the bacteria are propionic acid bacteria (PAB). which include but not limited to Propionibacterium jreudenreichii, Propionibacterium acidipropionici, Propionibacterium jensenii. Propionibacterium thoenii, Propionibacterium shermanii, and Propionibacterium cyclohexanicum. In some embodiments, the PAB is P. acidipropionici (ATCC 25562). In some embodiments, the PAB is a strain adapted from / ’, acidipropionici (ATCC 25562). PAB strains adapted from P. acidipropionici (ATCC 25562) can include any of those disclosed in U.S. Patent No. 10,808,266, the disclosure of which as it relates to the PAB strains is incorporated herein by reference. In some embodiments, a PAB strain suitable for use herein is deposited under the name NFS-2018 on Jul. 10, 2019, in the American Type Culture Collection (10801 University Blvd. Manassas, Va. 20110-2209) and assigned Accession Number ATCC PTA-125895. In some embodiments, the PAB strain NFS-2018 is also known as "‘Strain 3-1” (see, e.g., U.S. Patent No. 10,808,266).

[0076] In some embodiments, the bacteria are lactic acid bacteria (LAB), which include but not limited to Lactobacillus spp. Organic acids producing fungi include but not limited to Aspergillus spp., Penicillium spp., Rhizopus spp., or Candida spp.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0078] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0079] EXAMPLES

[0080] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0081] Materials and Methods

[0082] The following materials and methods were used in the Examples below.

[0083] Experimental design

[0084] Continuous cell cycling (CCC) requires only a traditional batch fermenter with no additional axillary equipment (FIGS. 1A-1C).

[0085] 1. First, a batch fermentation requiring a seed train or initial inoculum is necessary.

[0086] 2. Allow the fermentation to run to or near completion (i.e., all carbon source is consumed).

[0087] 3. Drain 75% to 99.5% of the fermentate of the initial batch and replenish with fresh media te the desired concentration. 4. When the subsequent batch is finished, step 3 is repeated.

[0088] Methods

[0089] In current disclosure, IL lab-scale fermentations were conducted with a proprietary propionic acid bacteria (PAB) strain, P. acidipropionici (acid adapted from ATCC 25562, e.g., NFS-2018), to develop the CCC method. An organic acid served as the product of interest. Several recipes and process conditions were tested. Consecutive CCC fermentations were executed for eight additional cycles to assess the concept's robustness. At the end of each cycle, cells were collected, and frozen stocks were created (25% glycerol) and stored at - 80°C. Cells generated at the 6th (CF6) and 8th (CF8) consecutive CCC cycles were selected for resequencing to determine the genetic stability of the parent strain. Whole genomic DNA was extracted, purified, and sequenced using Illumina MiSeq system. The sequencing depth was > 150 x with respect to the complete genome of the parent strain to ensure the accuracy of any reported genetic drift. Percent average nucleotide identity (ANI) of assembled CF6 and CF8 genomes was used to determine genetic drift from the parent strain. In addition, commercial-scale (50,000 G) fermentations were conducted with the same PAB strain and organic acid product of interest as used in the runs. Input ingredients were identical for the batch and CCC fermentations at this scale. The organic acid profile was determined using a reversed-phase HPLC method. A two-tailed unpaired student’s T-test was used to determine significant differences (p-value < 0.05) in final productivity, carbon-to-product yield, and final titer were compared between the standard batch and subsequent CCC fermentations of commercial runs. Finally, IL lab-scale fermentations were conducted with a lactic acid bacteria (LAB) strain to demonstrate that the disclosed method could be used with genetically distinct bacteria and with a different product produced by fermentation.

[0090] Example 1. Propionic acid (PA) production through continuous cell cycling (CCC) fermentations with a PAB strain at the IL scale

[0091] CCC fermentations were run and optimized at the IL scale. A single inoculum of a parent strain, P. acidipropionici (ATCC 25562), was added at 10% in the initial standard fermentations. When the fermentations were completed (i.e., the carbon source was near depletion), 75% of the fermentate was drained and replaced with fresh media; this was done for a total of eight cycles. The initial fermentation and the consecutive eight fermentations were derived from a single initial inoculum (FIG. 2). Differences in final titer and productivity were due to media and process changes for optimization for scale-up to 50,000 gallons. As shown in FIG. 2, the results of consecutive fermentations show that the CCC method may be applied up to at least nine total fermentations without any obvious reduction in final titer or yield of propionic acid.

[0092] Cells derived from the 6th(CF6) and 8thCCC (CF8) runs were isolated and sequenced for assessment of genetic drift and compared to a high-quality and complete genome of the parent strain. Both CF6 and CF8 assemblies exhibit 100% nucleotide identity and 100% genome coverage with regard to the parent strain, indicating that all genomes were virtually identical. These results showed that CCC had caused no measurable genetic drift (at least with our organism and process controls), and the PAB strain was genetically stable.

[0093] Example 2. PA production through CCC fermentations with a PAB strain following process optimization at the IL scale

[0094] The CCC method was repeated at IL size from the initial frozen PAB stock, and two cycles were performed to increase confidence in the robustness of the CCC method within the disclosed system. As expected, highly similar yields and titers were observed in the initial regular fermentation as in the two subsequent CCC runs, with a drastic increase in productivity observed in the CCC runs due to process optimization (e.g., fine tuning parameters such as inoculum retainment, temperature, agitation and pH) (FIG. 3).

[0095] Example 3. PA production through CCC fermentations with a PAB strain at the scale of 50, 000 gallons or large commercial scale

[0096] After optimization of CCC fermentations at the IL scale, the method was successfully scaled up and run in production-size vessels for a total of four consecutive fermentations without an additional seed inoculum (50,000 gallons, FIG. 4). Data from several commercialscale batch and CCC fermentation iterations were compiled and analyzed (FIGS. 5A-5C). CCC fermentations were able to be completed in four days, while batch fermentation runs needed eight days. There were no significant differences between the two processes when considering product yield with respect to carbon input and in final product titer (p-value > 0.05). However, striking differences exhibited in final productivity (g / L / total hrs. of fermentation), where CCC significantly outperformed the batch method (p-value < 0.001).

[0097] Example 4. CCC fermentations with a lactic acid bacteria (LAB) strain at the IL scale

[0098] A Lacticaseibacillus rhamnosus strain was used to test if the CCC method could be applied to bacteria from other genera and a different final product of interest. The results showed that after three consecutive fermentations with an LAB, no reduction in final titer of lactic acid was observed, and all three runs finished (e.g., consumed all carbon sources determined by high performance liquid chromatography using a YSI analyzer) within 24 hrs.

[0099] (FIG. 6)

[0100] REFERENCES

[0101] 1. Colomban A. Roger L. Boyaval P. Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling. Biotechnol Bioeng. 1993;42(9):1091-8.

[0102] 2. de Assis DA, Machado C, Matte C, Ayub MAZ. High Cell Density Culture of Dairy Propionibacterium sp. and Acidipropionibacterium sp.: A Review for Food Industry- Applications. Food Bioproc Tech. 2022;15(4):734-49.

[0103] 3. Dishisha T, Alvarez MT, Hatti-Kaul R. Batch- and continuous propionic acid production from glycerol using free and immobilized cells of Propionibacterium acidipropionici. Bioresour Technol. 2012;118:553-62.

[0104] 4. Liu Z, Ge Y, Xu J, Gao C, Ma C, Xu P. Efficient production of propionic acid through high density culture with recycling cells of Propionibacterium acidipropionici. Bioresource Technology. 2016;216:856-61.

[0105] 5. Miyano K, Ye K, Shimizu K. Improvement of vitamin B(12) fermentation by reducing the inhibitory metabolites by cell recycle system and a mixed culture. Biochem Eng J. 2000:6(3):207-14.

[0106] 6. Stowers CC, Cox BM, Rodriguez BA. Development of an industrializable fermentation process for propionic acid production. J Ind Microbiol Biotechnol. 2014;41(5):837-52.

[0107] 7. Wang Z, Jin Y, Yang ST. High cell density propionic acid fermentation with an acid tolerant strain of Propionibacterium acidipropionici. Biotechnol Bioeng.

[0108] 2015; 112(3):502-l 1.

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Claims

WHAT IS CLAIMED IS:

1. A continuous method of producing an organic acid, the method comprising:(a) seeding a microbe previously identified as an organic acid producing microbe at an initial density in a volume of fresh culture medium comprising a fermentable carbon source in a fermentation vessel for an initial fermentation;(b) culturing the microbe in the culture medium from about 1 day to about 10 days, thereby producing a fermentate at a titer;(c) removing about 50% to about 99.5% of the fermentate and about 50% to about 99.5% of the microbes from the fermentation vessel;(d) replacing the fermentate removed from the fermentation vessel in step (c) with the same volume of fresh culture medium, wherein the remaining microbes are maintained at a selected proportion of total microbes in step (b) for a subsequent fermentation; and(e) repeating steps (b) to (d) at least one time; thereby producing one or more volumes of the fermentate comprising the organic acid.

2. The method of claim 1, wherein the organic acid comprises propionic acid, lactic acid, acetic acid, butyric acid, formic acid, citric acid, succinic acid, gluconic acid, malic acid, itaconic acid, pyruvic acid, oxalic acid, ascorbic acid, or combinations thereof.

3. The method of claim 1 or 2, wherein the organic acid is propionic acid.

4. The method of any one of claims 1-3, wherein the microbe is only seeded into the fermentation vessel once.

5. The method of any one of claims 1-4, wherein the microbe in step (a) is seeded at an initial density of about 0.5% to about 20% of the culture medium.

6. The method of any one of claims 1-5, wherein the selected proportion at which the remaining microbes maintain in step (d) is about 0.5% to about 50% of the culture medium in each of the subsequent fermentations.

7. The method of any one of claims 1-6, wherein the microbe comprises bacteria or fungi.

8. The method of any one of claims 1-7, wherein the bacteria comprise Propionibacterium spp., Veilonella spp., Bacteroides spp.. Lactobacillus spp.. Bacillus spp., Clostridium spp., Butyrivibrio spp., Acetobacter spp., or Gluconobacter spp.

9. The method of one of claims 1-8, wherein the bacteria comprise Propionibacterium spp., preferably Propionibacterium acidipropionici (P. acidipropionici) , or Lactobacillus spp.

10. The method of any one of claims 1-9, wherein the fungi comprise Aspergillus spp., Penicillium spp., Rhizopus spp., or Candida spp.

11. The method of any one of claims 1-10, wherein the fermentable carbon source comprises glucose, sucrose, fructose, lactose, maltose, starch, molasses, cellulose, xylose, arabinose, glycerol, or ethanol.

12. The method of any one of claims 1-11, wherein the fermentate removed in step (c) comprises removing about 75% to about 99.5% of the fermentate from the fermentation vessel.

13. The method of any one of claims 1-12, wherein the volume of the fermentation is about IL to about 100,000 gallons.

14. The method of any one of claims 1-13, wherein the volume of the first and subsequent fermentations is about IL, about 1 gallon, about 10 gallons, about 1,000 gallons, about 10,000 gallons, or about 50,000 gallons.

15. The method of any one of claims 1-14, wherein steps (b) to (d) in step (e) are repeated from about 1 time to about 20 times.

16. A method of continuous cell cycling (CCC) fermentation, the method comprising: (a) culturing a microbe at an initial density in a volume of fresh culture medium comprising a fermentable carbon source in a fermentation vessel for an incubation period of from about 2 days to about 10 days, thereby producing a fermentate at atiter for an initial fermentation;(b) removing about 50% to about 99.5% of the fermentate and about 50% to about 99.5% of the microbes from the fermentation vessel;(c) replacing the fermentate removed from the fermentation vessel in step (b) with the same volume of fresh culture medium, wherein the remaining microbes are maintained at a selected proportion of total microbes in step (a) for a subsequent fermentation;(d) incubating the subsequent fermentation for an incubation period of from about 1 day to about 8 days; and(e) repeating steps (b) to (d) at least one time; thereby producing one or more volumes of fermentate at about the titer said in step (a) during shortened incubation periods.

17. The method of claim 16, wherein the microbe cultured in fermentations comprises bacteria and fungi.

18. The method of claim 1 or 17, wherein the microbe cultured in fermentations comprises one or more types of microbes.

19. The method of any one of claims 16-18. wherein the microbe is only seeded into the fermentation vessel once.

20. The method of any one of claims 16-19, wherein the shortened incubation period for the subsequent fermentation in step (d) comprises shortened lag phase.

21. The method of any one of claims 16-20 wherein the fermentate is an extracellularly excreted metabolite.

22. The method of any one of claims 16-21. wherein the fermentate removed in step (b) comprises removing at least about 70% of the fermentate from the fermentation vessel.

23. The method of any one of claims 16-22, wherein the fermentate removed in step (b) comprises removing about 75% to about 99.5% of the fermentate from the fermentation vessel.

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