Fermentation process of Gluconobacter xylinus for biosynthesis of BC 26

Gluconobacter xylinus grows on various culture substrates. Under laboratory conditions, Gluconobacter xylinus and other cellulose-producing strains were cultured under static and agitated conditions and up-scaled to semi-industrial size bioreactors^[78]. Static culture con-sists in inoculating aseptically a broth medium with a viable bacteria population and letting it grow statically for a certain cultivation time at a predefined temperature between 28^◦ to 37^◦ for thermotolerant Gluconobacter xylinus strain^[79]. In this configuration, the bacteria produce a BC film at the air-liquid interface^[80], where the thickness of BC film increases as a function of culture duration from a few micrometers to few centimetres^[21,81], as exemplified in Figure 3.5a. On the other hand, agitated culture consist in the inoculation of a culture medium where the subsequent cultivation is carried out under a constant and controlled ag-itation, using an orbital shaker. The agitated culture condition enhances liquid-gas transfer leading to rapid growth of the bacteria population to the detriment of BC production. BC synthesised under agitated condition forms round balls in the broth instead of a well-organised film as in static culture^[64], as exemplified in Figure 3.5b. Both aforementioned types of cul-ture have a relatively low yield of BC since they are carried out in flasks with a restricted surface area. To improve BC production, cellulose-producing bacteria were cultured in an

3.1 Introduction

instrumented bioreactor, as seen in Figure 3.6. However fermentation in bioreactor resulted in low yield of BC, although, oxygen and mass transfer were dramatically improved by mixing and agitation. When a Gluconobacter xylinus population was subjected to stress imposed by agitation in a bioreactor, it gave rise spontaneously to non-cellulose-producing mutants^[64,72]. The mutants phenotypes throve from the excess of oxygen present in the culture medium.

Moreover, the mutation undertaken was stable as long the culture medium remained agitated.

Interestingly, if those mutant phenotypes were re-established into a static culture, they once again reverted to cellulose-producing bacteria. Furthermore, the BC accumulated during fer-mentation in the bioreactor increases the medium viscosity, thereby lowering the effectiveness of both mixing and aeration due to decrease in homogeneity of the culture medium^[65].

Traditional culture medium, such as Hestrin and Schramm (H&S) medium^[20], are com-posed of different carbon sources (glucose, fructose, sucrose, mannitol, arabitol), complex nitrogen (yeast extract), vitamin source (polypeptone) and other additives, those of which are costly and economically unsustainable at an industrial scale^[82,83,84]. The cost of culture medium generally accounts for at least 30% of the total cost of fermentation. Therefore, cost-effective culture medium were investigated, such as, corn steep liquor (CSL) and molasses medium, by-products of corn wet-milling and the sugar industry, respectively^[78,85].

Despite the fact that bacteria population of Gluconobacter xylinus cultured in agitated condition tended to mutate, thus significantly reducing BC production, the economical gain occurring from this fermentation process led to the development of strains of Gluconobacter xylinus more tolerant to stressing conditions and shearing inherent to agitated fermentation process, as discussed in Section 3.1.3. Meanwhile, bioreactor configurations were developed to tackle the requirements posed by culture of Gluconobacter xylinus strain in agitated condi-tions, such as an enhanced oxygen aeration through the culture medium for bacteria growth, reduction of shear forces to avoid mutation, as well as reasonable energy consumption^[82]. Several configurations of bioreactors were reported in recent literature, such as a stirred tank bioreactor fitted with various type of impellers^[86], a low-shear stress air-lifted bioreactor (steering-free), a spherical type bubble column bioreactor, a rotary disk bioreactor and a plastic composite support (PCS) biofilm reactor[78,82,87,88,89].

3.1.6 Properties of bacterial cellulose

BC is characterised by a high degree of crystallinity between 70% to 90%^[23,90]. As much as 70% of its crystalline structure is composed of metastable cellulose type Iα (triclinic

con-27

a) b)

Figure 3.5: Shape of biosynthesised BC by Gluconobacter xylinus BPR2001 after 3 d of culture at 30^◦C under; a) static Erlenmeyer flask; b) agitated Erlenmeyer flask.

formation), compared to 30% for plant cellulose^[65]. Furthermore, the weight-average degree of polymerisation (DP w) of nitrated BC samples, which were nitrated in a solution of ni-¯ tric acid/diphosphorous pentoxide was determined using a gel permeation chromatography (GPC) for static and agitated culture, where theDP w were 14400 and 10900, respectively¯ ^[23]. In addition BC is insoluble in common organic solvents owing to its molecular configuration constituted of parallel and alignment arrangement of β(1 → 4) glycosidic AGU chain, so called cellulose type I^[72]. The structural characteristics and features of BC nanofibrils are dependent on the bacterial strain producing it, the type of culture and composition of broth medium.

The dimension of BC nanofibrils varies from 1 to 25 nm in width, which corresponds to stacking of 10 to 250 AGU chains, thus forming highly regular intra- and interchain hydrogen bonds. The length of BC nanofibrils varies between 1 to 9 µm, which is equivalent to 2 000 to 18 000 AGU^[72,91]. BC ribbons formed by aggregation of individual BC nanofibrils are approximately 100 nm wide and 3 to 8 nm thick^[25]. Yamanaka et al.^[92] reported the use of antibiotics and other chemical reagents added to the culture medium to change the morphology of bacteria cells of Gluconobacter xylinus, which in time synthesised wider BC ribbons having thus modified both the mode of aggregation and structural features of their biosynthesised BC nanofibrils. Single BC nanofibrils exhibits outstanding mechanical per-formance. Guhados et al.^[93] calculated a Young’s modulus of 78 GPa using atomic force microscopy (AFM) where the calculation was based on a clamped-beam model. However,

3.1 Introduction

Tanaka et al.^[94]reported for elementary of cellulose I_β a Young’s modulus between 124 and 155 GPa, which was determined by molecular simulation.

The properties of BC depend on whether the BC tested was a swollen-gel (i.e., pellicle) or a dried-film or else individual nanofibrils. Purified and subsequently dehydrated BC gel formed during static culture exhibits interesting in-plane mechanical properties. In fact, a Young’s modulus of 30 GPa was measured for a BC film, which was heat-press dried after removal of cell debris by subsequent treatments with 0.5% NaClO and 5% NaOH solution, respectively^[95]. Such outstanding performance arose from the extensive hydrogen bonding of BC nanofibrils and the inherent high degree of crystallinity of BC. However the swollen-gel possesses an out-of-plane elastic response when squeezed^[24,25]. BC gel has an excellent H₂O holding capacity of nearly 100 times its own weight. The H₂O retention of BC gel was improved by addition of a small concentration of water-soluble polymers (WSP) to the H&S culture medium, such WSP included were carboxymethyl cellulose (CMC), methyl cellulose (MC), cyclodextrin, cationic starch or polyethylene glycol (PEG)^[96,97].