B IOPROCESS C ONDITIONS

5.3.2 B IOPROCESS C ONDITIONS

The bioprocess conditions during the production of microbial functional polysac- charides influence significantly microbial physiology, and thus the concentration and

structure of the final product. In several bioprocesses the composition, molecular weight, and chemical structure of polysaccharides can be altered by different process

conditions. Apart from the case of fruit bodies of (medicinal) mushrooms, which are cultivated on solid media (soil enriched with nutrients, sawdust, etc.) over a long period (up to months) under partly controlled conditions, all other processes for polysaccharide production take place in bioreactors (or fermentors) using a liquid medium, which offers easy and automated control of the process. Such processes can be batch (only one addition of carbon source at the beginning of the process), fed-batch (additional supply of carbon source at some point of the process), or continuous (continuous addition of process medium and simultaneous withdrawal of equal amount of medium and product), although the industrial fermentations for microbial polysaccharide production are usually either batch or fed-batch. Of course, the environmental parameters that influence the numerous polysaccharide processes do not have the same effects in all processes, since each individual microorganism has different responses to process conditions, such as medium composition, temper-

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ature, pH, oxygen, agitation, and aeration. The impact of process parameters depends on the relation between cell growth and product synthesis. Microbial polysaccharide synthesis may follow either growth-associated or nongrowth-associated kinetics; in the former, the polysaccharide is synthesized mainly during the exponential growth phase, whereas in the latter the polysaccharides are formed mainly toward or after the end of the growth phase (secondary metabolites). Polysaccharide syntheses with mixed kinetic response also exist. 108,109,124

For several bioactive polysaccharides the physiology of the producer microor- ganism has not been studied yet, and reports on their production process are scarce. Based on the available data, some general principles regarding the effect of process parameters will be cited below, including examples from some processes.

5.3.2.1 Composition of the Process Medium

The composition of the process medium or fermentation medium (or fluid) is a key factor that influences the progress of the process, and careful design of the medium is essential in process optimization. In well-defined media the carbon source is usually a monosaccharide (e.g., glucose or fructose) or a disaccharide (e.g., sucrose or lactose), but in some processes complex carbon sources such as molasses, hydro- lyzed starch, or dairy whey have been used, due to their low cost. 50,108,109,130–132 Also,

a nitrogen source is needed to enhance cell growth. Nitrogen may be supplied in organic (e.g., peptone, yeast extract, corn steep liquor, soy hydrolyzate) or inorganic (e.g., ammonium or nitrate salts) form. In addition, phosphorous and sulfur ions are often included in the process medium, contributing to aminoacid synthesis, ATP synthesis (phosphorous), the uptake of other nutrients, and sometimes acting as pH buffers. Apart from the above macronutrients (used in comparatively high concen- trations), micronutrients (in low concentrations) such as Mg, K, Na, Ca, or other metal ions are often added to the process medium, serving as enzyme co-factors or as components of the polysaccharide structure. The fermentation medium may also contain vitamins, aminoacids, organic acids, or other substances, which assist cell growth or stimulate biopolymer synthesis.

In scleroglucan production, a typical fermentation medium contains (per liter of water) glucose or sucrose (30.0 to 35.0 g), NaNO 3 (3.0 g), KH 2 PO 4 (1.3 g), KCl (0.5 g), MgSO 4 × 7H 2 O (0.5 g), FeSO 4 × 7H 2 O (0.05 g), ZnSO 4 × 7H 2 O (3.3 mg), and yeast extract (YE) (1.0 g). Citric acid (0.7 g/l) and thiamine × HCl (3.3 g/l) may also be added. 50,133 Under these conditions, scleroglucan concentrations of 8.5 to 10.0 g/l can be obtained. However, these values are considerably lower than the maximal concentrations reported for other polysaccharides, such as xanthan (27.0 g/l). 134 The increase in initial sugra concentration might make more carbon available to glucan biosynthesis, but, in some cases, growth inhibition might occur when the sugra content exceeds a certain level, having an adverse effect on polysaccharide synthesis. 135 Schizophyllan can normally reach concentrations similar to that of scleroglucan, but when citric acid (up to 5.0 g/l) was added to the fermentation medium, cell growth rate decreased and glucan production rate (g glucan/l/h) and glucan product yield (g glucan/g glucose) showed a 6-fold increase, 136 making the process more economic.

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The type and amount of nitrogen source may also affect biopolymer formation. Generally, high levels of nitrogen favor cell growth at the expense of scleroglucan synthesis, and more glucan is produced when ammonium is replaced by nitrate ions. 137 The inhibitory effect of ammonium on polysaccharide synthesis has also

been demonstrated in alginate-producing cultures of Azotobacter vinelandii. 99 A high initial carbon/nitrogen (C/N) ratio and N depletion (toward the end of the growth phase) stimulate the production of (nongrowth-associated) polysaccharides, such as xanthan 138 and curdlan. 139 Furthermore, the number of side pyruvate groups of xanthan was found to be higher when the total N content of the process medium increased, which in turn affected the rheological properties of the biopolymer; 140 viscosity of xanthan solutions rose when pyruvilation degree increased.

Medium formulation for glucan production by S. cerevisiae should aim at max- imizing biomass, since the glucan is a component of the cell wall. The yeast forms ethanol (which can restrict growth) in high quantities, especially if a critical con- centration of carbon source is exceeded (at least 100 g/l of glucose or sucrose is usually used for ethanol production). A high C/N ratio or N limitation also stimulates ethanol synthesis and reduces not only cell growth but also the amount of glucans in the cell wall. 141,142 Thus, high initial N concentration or relatively low carbon concentration should be preferred for glucan production. Furthermore, significant variance in polysaccharide composition of the cell wall of S. cerevisiae with the nature of carbon source has been demonstrated. 142 Similarly, the production yield of polysaccharides by submerged cultures of Ganoderma lucidum was found to be influenced by medium composition. 143,144

Lactic acid bacteria (LAB) have different requirements in medium composition, depending on the individual species and the relation between growth and polysac- charide synthesis. It has been established that, in general, functional polysaccharides from mesophilic species (Lactococcus cremoris, Lactobacillus casei) are not growth associated, while thermophilic species (Lactobacillus bulgaricus, Lactobacillus hel- veticus ) produce growth-associated EPSs. 119,145 For example, the absence of N lim- itation (optimal balance between carbon and nitrogen) and the inclusion of vitamins in the process medium favor EPS synthesis from L. bulgaricus. 146 Lactose and glucose are often used as carbon sources for polysaccharide production from LAB, while fructose and mannose usually lead to low EPS yields. Milk, whey, or whey permeate (deproteinized) can also be used. 131 The choice of carbon source in LAB cultures may influence not only the quantity, but also the composition of the EPSs (e.g., ratio of sugar monomers in heteropolysaccharides). 147 Therefore, special care must be taken when functional polysaccharides are produced, since changes in chemical composition may affect their functionality. For this reason, the use of well- defined media is probably preferable. On the other hand, new bioactive LAB polysac- charides with novel composition might be designed in this way, i.e., by careful selection of the type of carbon source.

5.3.2.2 Temperature

Temperature influences the formation of bioactive polysaccharides in two ways: first, it affects cell growth, which in turn has an impact on biopolymer synthesis;

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second, it may affect the activity of key enzymes involved in polysaccharide biosynthesis. 108,109,116,124 For example, the control of temperature is crucial in scleroglucan synthesis. Glucan formation occurs in the range of 20 to 37°C; the optimal temperature for synthesis is 28°C, while the optimal temperature for cell growth is above 28°C. Additionally, below 28°C by-product (oxalic acid) formation takes place, reducing glucan production. 50,148 For most polysaccharide-producing fungi, yeast, and bacteria, the process temperature is around 30°C, which often represents a compromise between the optimal temperature for growth and that for

biopolymer synthesis. Levan from Zymomonas mobilis 88 and exopolysaccharides from Ganoderma lucidum 144 are also produced at 30°C. Although a temperature of 30°C is often used for xanthan production, it has been shown that in the range of 25 to 34°C, the xanthan MW is highest at 25°C. 149 Shu and Yang also noticed that process temperature can affect the link between xanthan and cell growth. 150 They found that at 24°C or lower, xanthan formation began toward the end of the exponential growth phase, whereas at 27°C or higher, it followed growth-associ- ated kinetics.

When batch cultures of S. cerevisiae were grown at 22, 30, and 37°C, the highest growth rate and cell wall content of the yeast occurred at 30°C. 142 However, more total β-glucans were contained in the cell wall of yeasts cultivated at 37°C. 142

The optimal temperature for functional EPS formation by LAB varies consid- erably between mesophiles and thermophiles. Low temperatures between 18 and 20°C enhance polysaccharide production by Lactococcus cremoris and Lactobacillus casei strains. These temperatures are suboptimal for growth, which is preferable for nongrowth-related kinetics. 131,145 Moreover, it has been noted that since there is antagonism for the isoprenoid lipid carriers between cell wall synthesis and EPS formation, the slow growth under low temperatures allows more lipid carriers to be used for polysaccharide production. 108 Conversely, high temperatures (37 to 42°C) are applied in EPS production by thermophiles (Lactobacillus bulgaricus and Lac- tobacillus helveticus ). 131,151

5.3.2.3 pH

The control of pH via automatic addition of alkali or acid in a bioreactor is essential for most polysaccharide processes (uncontrolled pH usually leads to low yields or even cell death). According to Kang and Cotrell, 152 polysaccharide production by fungi is generally optimal in the pH range of 4.0 to 5.5. However, many fungi are acidophilic and grow readily at lower pH values. Thus, Wang and McNeil 153 designed

a bi-staged process for scleroglucan production where growth was stimulated in the first stage at pH 3.5, and after a sufficient amount of cells was produced, pH was

shifted to 4.5 in the second stage to promote glucan formation. Under these condi- tions oxalic acid synthesis was also reduced (by 10%).

In a similar mode, a bi-staged process for Ganoderma lucidum exopolysaccha- rides with pH shift from 3.0 to 6.0 resulted in a remarkable increase in EPS concentration, namely, 20.1 g/l EPS was obtained, compared to 14.0 g/l when the

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pH was controlled at 6.0, 6.5 g/l when the pH was adjusted at 3.0, and 4.1 g/l when the pH was uncontrolled. 143

Low environmental pH also promotes cell wall synthesis in S. cereviciae and results in increased resistance to (1 →3)-β-glucanase action. 154 Decreased sensitivity of S. cerevisiae cell wall to zymolyase at low pH was also noted by Aguilar-Uscanga and François, 142 who found that the yeast grown at pH 5.0 had the highest growth rate and cell wall content (with high mannan concentration in the cell wall), whereas the proportion of total β-glucans and β-1,6-glucans in the cell wall was higher at a pH of 4.0. 142

For many bacterial polysaccharides, a neutral pH is preferable. 107 Thus, xan- than is produced at pH 7.0, 107,149 while for alginate synthesis a pH of 7.0 to 7.2 is normally used. 155 In most cases, the optimal pH for EPS synthesis by lactic acid bacteria is close to 6.0. 131 Nevertheless, it has been suggested that higher pH values may be beneficial to polysaccharide production by Lactobacillus bulgaricus, because they cause prolongation of the exponential growth phase, during which much of the EPS is formed (growth-associated kinetics). 153 Also, the control of pH results in greater EPS yields, compared to uncontrolled pH conditions where the medium is progressively acidified. In fact, the impact of pH adjustment seems to be greater than that of supplementation with nutrients. 131,146,156 This could pose

a limitation in the industrial exploitation of polysaccharide-producing LAB and their addition in functional fermented dairy products, since relatively low EPSs must be expected under uncontrolled pH conditions.

5.3.2.4 Agitation, Aeration, and Dissolved Oxygen

These additional fermentation parameters determine the efficiency of mass and oxygen transfer and uptake, as well as the rate of metabolism and metabolites release.

They are interconnected to some extent with one another, and are particularly important for all polysaccharide processes, because the viscous fluid that is usually

developed after some point in the process makes mixing of the fermentation fluid problematic. Usually, a high agitation (stirring) rate (above 250 rpm) is essential in

microbial polysaccharide production. In xanthan production, biopolymer synthesis was a function of stirring rate in an agitation range of 200 to 800 rpm, as a result of improved nutrient uptake by the microorganism. 157 In the scleroglucan process, despite the importance of agitation in effective mixing, it was revealed that the glucan is sensitive to shear-induced depolymerization under vigorous agitation (reduction in MW). 158

The effect of aeration and oxygen on polysaccharide formation is sometimes controversial as well. Several studies on polysaccharide synthesis support the idea that oxygen enhances biopolymer formation and production rate. For instance, this has been observed for xanthan 157 and curdlan. 159 On the other hand, it has been proposed for scleroglucan and schizophyllan that low dissolved oxygen (DO) in the bioreactor, expressed as a percentage of air saturation with oxygen, and especially DO limitation could enhance their biosynthesis, in contrast to cell growth. 158 This might happen either via the induction of oxygen-sensitive biosyn- thetic enzymes or via the direction of carbon flux primarily toward glucan syn-

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thesis, under growth-limiting conditions. Additionally, high DO is believed to be responsible for radical-induced degradation of xanthan 160 and scleroglucan 54 under oxidative stress conditions. During exopolysaccharide production by Ganoderma lucidum, the biopolymer concentration and production rate rose as the agitation rate increased in the range of 100 to 400 rpm. 161 Also, comparing a DO of 10% (of air saturation) with a DO of 25% showed that at low DO cell growth was restricted, due to oxygen limitation occurring in mycelia aggregates in the fermentor, but EPS production by G. lucidum was elevated. 162 Restriction (but not cessation) of growth of S. cerevisiae occurred at a DO of 0% (absence of aeration), compared to a process with the DO above 50%. In the second process, cell growth rate and cell wall content were higher, whereas mannan and glucan concentrations of the cell walls did not differ signifi- cantly between the two processes. 142 The alginate process exhibits an increase in biopolymer formation with increased agitation rate (from 300 to 700 rpm) and under relatively high aeration. 155 Lastly, polysaccharide production by lactic acid bacteria usually takes place under low agitation (e.g., 100 rpm), since EPS content is rarely above 1 g/l and the low viscosity allows efficient mixing. Minimal or no aeration is applied, because most strains are microaerophilic or can grow anaerobically. 131