Friday, March 30, 2018

Fermentative Production of Gellan Gum

Fermentative Production of Gellan Gum 

The growth media suitable for the production of different exopolysaccharides by microorganisms vary widely,
and this probably reflects the differing role of each exopolysaccharide in nature.
It is instructive to consider the effect on polymer biosynthesis rates, yields and composition of varying growth media during fermentative production of these exopolysaccharides.

Factors affecting gellan gum production

Media components

The media used for production of gellan gum are simple media containing carbon source, nitrogen source
and inorganic salts. The exact quantity of carbon utilization depends in part upon the other ingredients of the
medium. A copious secretion of exopolysaccharide is usually most noticeable when the bacteria are supplied with an abundant carbon source and minimal nitrogen. Sometimes complex medium ingredients supplying vitamins can also enhance the cell growth and production. Effects of various medium components on gellan gum production are as follows. 

Effect of carbon source on gellan gum production

Carbon source is the most important component of the media used for the production of exopolysaccharides because it directly affects the production yields, compositions, structures, and properties of bacterial exopolysaccharide. According to Kang et al. , carbohydrates such as glucose, fructose, maltose, sucrose and mannitol can be used either alone or in combination as carbon source. 

The amount of carbon source usually varies between 2–4 % by mass. Kang et al.  and
Lobas et al.used glucose as carbon source for production of gellan gum with approximate yields of 8–10
g/L. Ashtaputre and Shah studied sucrose as carbon source for gellan gum production using Sphingomonas paucimobilis GS1 and obtained yield of 6.6 g/L of gellan gum. Fialho et al. compared gellan gum production by using glucose, lactose and sweet cheese whey as carbon source and yields obtained were 14.5, 10.2 and
7.9 g/L, respectively. Nampoothiri et al. and Bajaj et al. compared soluble starch, glucose, lactose, maltose and sucrose as carbon source for gellan gum production and found soluble starch to be the best carbon
source for gellan gum production with yields of 24–28 g/L.
Banik et al.  developed a molasses-based medium for the production of gellan by Sphingomonas paucimobilis ATCC 31461. They applied Plackett–Burman design criterion to study the effect of various nutrient
supplements on gellan production using molasses. 

Among  the 20 variables tested, molasses, tryptone, casamino acid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. Molasses 112.5 g/L, tryptone 1 g/L, casamino acid 1 g/L, disodium hydrogen orthophosphate 1 g/L and manganese chloride 0.947 g/L produced maximum (13.81 g/L) gellan gum.

Effect of nitrogen source on gellan gum production

Following carbon source, nitrogen is the most important medium component for gellan gum production.
In general, the type and concentration of nitrogen source in the medium influenced the flow of carbon to either
biomass or product formation. Abundant secretion of the exopolysaccharide is usually most noticeable when
bacteria are supplied with abundant carbon source and minimal nitrogen.

The choice of the nitrogen source has strong effect on gellan broth characteristics. Dreveton et al. reported that organic nitrogen accelerates cell growth and biosynthesis of gellan gum. Hence broth with organic nitrogen is more viscous as compared to the broth without organic nitrogen, and therefore requires proper impeller system to provide enough oxygen transfer during gellan gum production.

Organic nitrogen sources like corn steep liquor  and inorganic nitrogen sources like ammonium nitrate and potassium nitrate  have been tried for gellan gum production. Hyuck et al.  compared bactopeptone and soybean pomace (an agroindustrial by-product) for gellan gum production from Sphingomonas paucimobilis NK 2000 and achieved maximum yield of 3.27 and 7.33 g/L, respectively. Nampoothiri et al.  compared various organic and inorganic nitrogen sources for gellan gum production from Sphingomonas paucimobilis ATCC 31461, and reported maximum gellan gum production of 32.1 g/L with tryptone. Bajaj et al. 
studied the effect of different nitrogen sources on gellan gum production. Among the various nitrogen sources
used, yeast extract supported the maximum gellan gum production.

Effect of the addition of precursors

Addition of precursor molecules is of considerable importance in the polysaccharide synthesis in terms of
metabolic driving force. In case of polysaccharides, higher intracellular levels of nucleotide phosphate sugars
under nitrogen-limited conditions reportedly enhance metabolite flux of exopolysaccharide synthesis.
Many researchers have described the pathway for the synthesis of the repeating tetrasaccharide unit of
gellan gum by Sphingomonas paucimobilis. It is assumed that gellan synthesis requires activated precursors before the repeating unit is assembled. These gellan precursors were detected by enzyme assays, and they were found to be nucleotide phosphate sugars . The repeating unit of gellan gum is a tetrasaccharide composed of the glucose, rhamnose and glucuronic acid.
The sugar nucleotides providing the activated precursors for synthesis of this tetrasaccharide are assumed to
be respectively UDP-glucose, TDP-rhamnose and UDP-glucuronic acid. Bajaj et al. studied the effect of the
addition of guanosine-5’-monophospate (GMP), uridine -5’-diphospate (UDP), adenosine-5’-diphospate (ADP), cytidine-5’-monophospate (CMP), and adenosine-5’-triphospate (ATP) on gellan gum production, and observed that ADP at 1 mM to give maximum gellan gum (32.15 g/L) production.

Media used for production of gellan gum usually contain complex medium ingredients that supply vitamins and amino acids to enhance cell growth and gellan production . Amino acids have been used by some researchers as nitrogen source or as stimulator for improving gellan gum production. Studies carried out by Bajaj et al. demonstrated that tryptophan at 0.05 % concentration gave maximum (39.5 g/L) yield of gellan gum.


pH plays a very important role in production of gellan by Sphingomonas paucimobilis, as it significantly influences both cell growth and product formation. In general, optimal pH value for bacterial exopolysaccharide production is somewhat higher than that of the fungal glucan production . The pH value usually recommended for gellan production ranges from 6.5 to 7. More acidic or more alkaline environment reduces the cell growth, and consequently gellan production.

Agitation rate

Dreveton et al. studied the effect of agitation rate on gellan gum production. Fermentations were carried out in a 14-liter vessel with an initial working volume of 10 L. Culture temperature was maintained at 30
°C and pH of the broth was regulated at pH=6.5.

An agitation of 250 rpm using a helical ribbon impeller is adequate for the mixing of gellan gum broth.
Lower levels of agitation were insufficient for homogenous conditions and the broth exhibited gelling characteristics. On the other hand, the same authors observed high stirring rates (600 to 800 rpm) with Rushton turbines to lead to cavitations in impeller zone suggesting that high shear thinning properties of the broth result in
formation of stagnant zone. Consequently, the medium became heterogeneous with increasing agitation rate.
This is a major drawback as it causes limitations in heat and mass transfer, and substrate exhaustion could occur in stagnant zones.

Giavasis et al. investigated the effects of agitation and aeration on the synthesis and molecular mass
of the gellan gum in batch fermentor cultures of the bacterium Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced the growth of S. paucimobilis.

Although gellan formation occurred mainly parallel with cell growth, the increase in cells able to synthesize gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth
was stimulated at the expense of biopolymer synthesis. Maximal gellan gum concentration can be obtained at
the agitation of 500 rpm at 1 and 2 vvm aeration (12.3–12.4 g/L gellan). At low agitation rates (250 rpm), an increase in aeration from 1 to 2 vvm enhances gellan synthesis.

Banik and Santhiagu studied the effect of agitation rate on cell growth and gellan gum production.
Growth of Sphingomonas paucimobilis increased up to 5.4g dry cells/L with an agitation rate of up to 700 rpm.
Specific growth rate was high at 700 rpm (0.38 h–1) and was comparatively low at 1000 rpm (0.29 h–1). This was
contrary to the report given by Giavasis et al. (31), in which the authors reported higher cell growth at 1000
rpm. Gellan production increased up to 500 rpm (14g/L) due to increased mass and oxygen transfer and decreased at 700 rpm (13 g/L) because of stimulation of cell growth.

Dissolved oxygen and oxygen transfer capacity

Rho et al. suggested O2 to be vital for gellan synthesis, as depletion in oxygen concentration decreased
the growth, and hence gellan gum production. The best gas dispersion conditions of the turbine systems were
accomplished by high gellan production. In contrast to the above, Rau et al. observed an improvement of
exopolysaccharide production when cultures of Sclerotium glucanicum were grown under limited oxygen supply. Clearly the high oxygenation rate that promotes optimal gellan synthesis is in distinct contrast with the low
or limiting oxygen levels which contribute to high concentrations of fungal glucans. One explanation for these
observations may be that in the case of glucans, exopolysaccharide synthesis follows the growth phase; whereas with gellan, biopolymer is produced at a higher rate during the growth phase.

Banik and Santhiagu studied the effect of dissolved oxygen tension (DOT) on cell growth and gellan
gum production, and found that DOT levels above 20 % have no effect on cell growth; gellan gum yield, however, increased to 23 g/L with increase in DOT level to 100 %. DOT level acts as a driving force for increasing
oxygen uptake rate by the cells, which resulted in higher gellan production. Higher DOT levels reportedly improve the viscosity and molecular mass of the polymer with change in acetate and glycerate content of the polymer .


Most of the fermentations involving gellan gum production are carried out at 30 °C. However, it is
reported that gellan yield reaches its peak at 20 °C, remains quite high at 25 °C, and significantly decreases
above 30 °C.

Influence of fermentation hydrodynamics on the physicochemical properties of gellan gum

Dreveton et al.  revealed that the degree of esterification, the average molecular mass and the intrinsic
viscosity of the gellan polymer depend on the fermentor hydrodynamics. Comparing several helical ribbon impellers with Rushton turbine impellers, they found that degree of esterification with acetate and glycerate was
higher for products produced by process using HR250 and HR125 impellers, both of which are characterized
by oxygen limitation. Hence, it was assumed that acetate and glycerate substitutes are related to oxygen limitation or the physiological state of the cells.

Using a range of impellers and dissolved oxygen regimes, Dreveton et al. noted that gellan molecular
mass is related to the degree of homogeneity in the fermentor. Under the most homogeneous conditions, the
average molecular mass of gellan gum doubled compared to heterogeneous conditions. They also noted that
the least viscous broth had the lowest molecular mass biopolymer, and intrinsic viscosity of gellan gum broth
seemed to be a function of molecular mass of gellan gum. However, oxygen limitation did not seem to influence the molecular mass of gellan gum.

Recently, Wang et al.have proposed kinetic model for understanding, controlling, and optimizing the
fermentation process for gellan gum production. Fermentation was carried out by Sphingomonas paucimobilis ATCC31461. Logistic and Luedeking-Piret models were confirmed to provide a good description of gellan gum fermentation. Analysis of kinetics in batch fermentation process demonstrated that gellan gum production is largely growth associated. Based on the model prediction, fedbatch fermentation for gellan gum production was carried out. Higher gellan gum production and higher conversion efficiency were obtained at the same total substrate concentration.