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Batch Fermentation
Batch fermentations are the most commonly used and simplest models to study the fermentation activity of colonic microbes. These batch fermentors are usually anaerobic sealed bottles with pure cultures, defined mixed cultures or faecal slurry, and are used to study the effects of added NSP on the microbes and their fermentation activity. They model a certain part of the gastrointestinal tract and the run-times in batch fermentations range from 2 to 24 hours. The accumulation of fermentation products (e.g. SCFA) and the depletion of nutrients can alter the conditions and microbiota balance in the fermentor, thus affecting the in vivo relevance in longer simulations. This can be avoided by using more complex simulation models with several vessels and fluid transitions.

In batch fermentation, substrate and microorganism are loaded into the fermenter batchwise, and this is the most popular and simple method for ethanol production. Batch fermentation has the advantages of low investment costs, simple control and operations, and easy-to-maintain complete sterilization. However, seed culture is needed for each new batch. Bioethanol from corn in the USA is almost entirely produced using batch fermentation. A higher initial sugar concentration is required in order to achieve more efficient ethanol production; however, a high sugar concentration will inhibit the growth and function of fermenting microorganisms due to excessive osmosis to result in a low fermentation yield with a prolonged fermentation period. The batchwise fermentation method will alleviate this drawback. Fermentation is started with a low initial sugar concentration to allow robust growth of the microorganisms at an early stage, and sugar is added periodically when it is consumed. Batch fermentation is what is described as a ‘closed system’, whereby the substrate and producing microorganism are added to the system at time zero and are not removed until the fermentation is complete. This represents the simplest and most commonly employed method of fermentation for the production of bacteriocins. Optimisation of bacteriocin production in batch fermentation is achieved by the manipulation of growth conditions (pH, temperature etc.) and medium composition. Batch fermentation is ideal for studying bacteriocin production in the laboratory or in small scale trials, but is not economically viable on a commercial scale. Fed-batch fermentation is a modified form of batch fermentation whereby growth limiting substrates are fed into the fermenter at a controlled rate. This allows tight control over the growth rate and can alleviate problems such as catabolite repression. By controlling the sucrose feeding rate in a fed-batch system, Lv et al. (2005) increased the maximum nisin titre from 2658 IU ml−1 in batch fermentation to 4185 IU ml−1. Controlled carbohydrate feeding facilitated a maximum growth rate without substrate inhibition having an impact upon the rate of bacteriocin production. A number of studies have demonstrated that bacteriocin production can be improved through the use of fed batch, rather than batch fermentations (Ekinci and Barefoot, 2006; Guerra et al., 2005; Lv et al., 2005; Paik and Glatz, 1997). While promising, these experiments were performed on a small scale and thus it is difficult to determine how scaling up of the process would impact upon bacteriocin yields. In one study, bacteriocin production by Propionibacterium thoenii in small and large scale fed-batch fermentations has been assessed. Paik and Glatz (1997) found that scaling up of the process led to a reduction in bacteriocin activity. Even with the reduced activity, the authors concluded that fed-batch fermentations have the potential to facilitate the production of high concentrations of bacteriocins by propionibacteria.

Growth is a result of consumption of nutrients. Batch fermentation has 3 phases. The initial lag phase is a time of no apparent growth but actual biochemical analyses show metabolic turnover, indicating that cells are in the process of adapting to the environmental conditions and that new growth will eventually begin. There is then a transient acceleration phase as the inoculum begins to grow, which is quickly followed by an exponential phase. In the exponential phase, microbial growth proceeds at the maximum possible rate for that organism with nutrients in excess, ideal environmental parameters and growth inhibitors absent. However, in batch cultivations exponential growth is of limited duration and as nutrient conditions change, growth rate decreases, entering the deceleration phase, to be followed by the stationary phase, when overall growth can no longer be obtained owing to nutrient exhaustion. The final phase of the cycle is the death phase when growth rate has ceased. Most biotechnological batch processes are stopped before this stage because of decreasing metabolism and cell lysis. Typical microbial cultures in the laboratory (in a flask) are batch cultures.

Batch culture systems provide a number of advantages:

1 Reduced risk of contamination or cell mutation as the growth period is short.

2 Lower capital investment when compared to continuous processes for the same bioreactor volume.

3 More flexibility with varying product/biological systems.

4 Higher raw material conversion levels, resulting from a controlled growth period.

The disadvantages include:

1 Lower productivity levels due to time for filling, heating, sterilization, cooling, emptying and cleaning the reactor.

2 Increased focus on instrumentation due to frequent sterilization.

3 Greater expense incurred in preparing several subsultures for inoculation.

4 Higher costs for labour and/or process control for this non-stationary procedure.

5 Larger industrial hygiene risks due to potential contact with pathogenic microorganisms.

Fed Batch fermentation
Fed-batch fermentation is alternatively known as semi-batch culture where nutrient or substrates are supplied to the bioreactor according to the required amount. Thus, fed-substrate concentration can be controlled easily (Yamane & Shimizu, 2005). Earlier, in N-acetyl-d-glucosamine production Serratia marcescens QM B1466 was used to express chitinase in fed-batch fermentation (Kim, Creagh, & Haynes, 1998). The culture was grown in 10 L bioreactor for 7 days by using chitinaceous wastes (crab/shrimp chitin) in the media. The pH was set at 8.5 and temperature at 30°C with 3 h feeding time (Kim et al., 1998). Recently, Rao, Inman, Holmes, and Lalitha (2013) also reported the use of fed-batch fermentation for chitinase production in a mixed culture of Vibrio harveyi and Vibrio alginolyticus. In this study, a 10 L bioreactor was used with daily addition of 2% colloidal chitin (w/v) at 30°C, with 20% dissolved oxygen and 150 rpm agitation. With fed-batch fermentation up to threefold chitinase activity can be achieved. Thus, fed-batch fermentation is known to be effective process for chitinase production.

The fed batch method is characterized by the addition of small concentrations at the beginning of the fermentation and these substances continue to be added in small doses during the fermentation process. Despite the apparent similarity between the fed batch reactor model and the continuous culture model, they are very different. Whereas the chemostat process (continuous culture) for biomass accumulation is composed of a growth and removal process, the fed batch procedure is composed of a growth and dilution process. The concept of steady state cannot be easily applied to a fed batch reactor. It is significantly more difficult to maintain a specific growth rate in a fed batch system than in continuous culture. As cells are not removed during the fermentation, fed batch cultures are well suited for the production of compounds produced during very slow or zero growth. Unlike a continuous culture, the feed does not need to contain all the nutrients needed to sustain growth. The feed may contain only a nitrogen source or a metabolic precursor. Contamination and/or mutation will not have the same dramatic effect on a fed batch fermenter. A fed batch fermenter can be operated in a variety of ways, e.g., the reactor can be operated in the following sequence: Batch => Fed batch = > Batch. The feed can also be manipulated to maximize product formation. During fermentation, the feed composition and feed flow rate can be adjusted to match the physiological state of the cells. Fed batch reactors can maintain low nutrient and substrate concentrations and are thus well suited for producing product or cells when the substrate is inhibitory by allowing the maintenance of low levels of substrate so that cells are not inhibited. They are very useful for the production of vinegar and amylase. Fed batch fermentations are also useful when the product or biomass yield is highest at low substrate concentrations as in the case of mammalian cell systems for recombinant protein, baker’s yeast products and antibiotic production. Another suitable application is when the product formation is dependent on a specific nutrient composition, e.g., specific carbon to nitrogen ratio.

Advantages of fed batch systems:

1 Higher yield, resulting from a well-defined cultivation period during which no cells are added or removed.

2 Increased opportunity for optimizing environmental conditions of the microorganisms in regard to the phase of growth or production and age of the culture.

3 Nearly stationary operation, important with slightly mutating microorganisms and those at risk for contamination.

Disadvantages include:

1 Lower productivity levels due to time for filling, heating, sterilization, cooling, emptying and cleaning the reactor.

2 Higher costs in labour and/or dynamic process control for the process.

Continuous Fermentation
Continuous fermentation is a microbial process with a constant flow of culture medium through the reactor (Fig. 31.1). The main difference compared with an animal cell perfusion process is that no device prevents the biomass from staying in the culture vessel in a continuous fermentation.1 The volume in a continuous fermentation is usually constant in industrial applications, but it can fluctuate in specific processes such as waste water treatment. The concept of continuous fermentation processes is closely linked to the chemostat, where one nutrient is growth limiting and used to determine the growth rate. However, there are several other, less common ways by which a continuous fermentation can be controlled: through constant pH (pH-auxostat), constant optical density (turbidostat), and constant substrate. Continuous fermentation starts as a batch process. At a certain point, for example, when the culture reaches the exponential growth phase, or when the culture becomes substrate limited, a feed with fresh growth medium is started, and an equal volume of culture broth is removed. Continuous fermentation is a superior tool in research, but the number of industrial applications is limited. Reasons for this include an increased risk for contamination, risk for genetic drift in the culture, and difficulties to control the process.

In continuous fermentation an open system is set up. Sterile nutrient solution is added to the bioreactor continuously and an equivalent amount of converted nutrient solution with microorganisms is simultaneously taken out of the system (Fig. 2.3). In a homogenously mixed bioreactor, we can have a chemostat or a turbidostat. In the chemostat, in the steady state adjusting the concentration of one substrate controls cell growth. In the turbidostat, cell growth is kept constant by using turbidity to monitor the biomass concentration and the rate of feed of nutrient solution is appropriately adjusted. In the chemostat, constant chemical environment is maintained, while in a turbidostat constant cell concentration is maintained. In a chemostat the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium is allowed to enter the growth chamber at a rate that limits the growth of the bacteria. The rate of addition of fresh medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient. Thus the chemostat relieves the insufficiency of nutrients, the accumulation of toxic substances and the accumulation of excess cells in the culture which are the parameters that initiate the stationary phase of the growth cycle.

There are several major advantages of using continuous cultures as opposed to batch cultures:

1 Continuous reactions offer increased opportunities for system investigation and analysis. As the variables remain unchanged, a benchmark can be determined for the process results, and then the effects of even minor changes to physical or chemical variables can be evaluated. By changing the growth-limiting nutrient, changes in cell composition and metabolic activity can be tracked. The constancy of the continuous process also provides a more accurate picture of kinetic constants, maintenance energy and true growth yields.

2 Continuous culture provides a higher degree of control than a batch culture. Growth rates can be regulated and maintained for extended periods. By varying the dilution rate, biomass concentration can be controlled. Secondary metabolite production can be sustained simultaneously along with growth. In steady state continuous culture, mixed cultures can be maintained using chemostat cultures – unlike in a batch process where one organism usually outgrows another.

3 Bioreactors operated as chemostats can be used to enhance selectivity for thermophiles, osmotolerant strains or mutant organisms with high growth rates. Also the medium composition can be optimized for biomass and product formation using a pulse- and shift- method that injects nutrients directly into the chemostat. As changes are observed, the nutrient is added to the medium supply reservoir and a new steady state is established.

4 Because of the steady state of continuous culture, the results are not only more reliable but also more consistent leading to a better quality product.

5 It also results in higher productivity per unit volume, as time consuming tasks, such as cleaning and sterilization are unnecessary.

6 The ability to automate the process makes it more cost-efficient and less sensitive to the impact of human error.

Disadvantages include:

1 The control of the production of some non-growth related products is not easy. For this reason, the continuous process often requires feed-batch culturing and a continuous nutrient supply.

2 Wall growth and cell aggregation can also cause wash-out or prevent optimum steady-state growth.

3 The original product strain could be lost over time if a faster growing one overtakes it.

4 The viscosity and heterogenous nature of the mixture can also make it difficult to maintain filamentous organisms.

5 Long growth periods not only increase the risk of contamination but also dictate that the bioreactor must be extremely reliable and consistent, incurring a potentially larger initial expenditure in higher quality equipment.