Expressing Vitreoscilla Hemoglobin in Statically Cultured Acetobacter xylinum with Reduced O 2 Tension Maximizes Bacterial Cellulose Pellicle Production Magdiel Inggrid Setyawati, Liang-Jung Chien, and Cheng-Kang Lee* Department of Chemical Engineering, National Taiwan University of Science and Technology Taipei, 106, Taiwan *Corresponding author: cklee@mail.ntust.edu.tw Vitreoscilla hemoglobin was constitutively expressed in Acetobacter xylinum to enhance bacterial cellulose (BC) production. The enhancement was much pronounced in static culture than in shaken culture. Reducing O 2 tension in gaseous phase of the culture by tightly sealing the culture tube could also enhance BC production by 70 %. O 2 tension reduced from 21 % to 15 % in the sealed and static culture of VHb-expressing A. xylinum after 7 days cultivation, while 7.36 g/l of BC with yield of 0.44 were obtained. BC pellicle production was successfully scaled-up in a sealed 4 l disposable zip lock plastic bag with BC yield of 0.35 and concentration of 6.31 g/l. Keywords: Bacterial cellulose; Acetobacter xylinum; Vitreoscilla hemoglobin; reduced O 2 tension. 1. Introduction Bacterial cellulose (BC) produced by Acetobacter xylinum in a static culture condition is known to form a thick, leatherlike white pellicle at the air-liquid interface of the culture. The pellicle is characterized by a 3-D structure consisting of an ultrafine network of cellulose nanofibers (3-8 nm) that are highly uniaxially oriented (Czaja et al., 2004). This unique nanomorphology results in a very high water-holding capacity, great elasticity, high wet strength, and conformability that are different from those of plant cellulose (Ross et al., 1991). The unique physical and mechanical properties of BC as well as its purity enable its successful applications in fields of biomedical materials (Fontana et al., 1990; Svensson et al., 2005; Czaja et al., 2006) for wound-healing dressing and scaffold of tissue engineering. A. xylinum is a Gram-negative, rod-shaped, and obligate aerobic bacterium, and it is a generally accepted hypothesis that cellulose synthesis and secretion by the highly aerobic A. xylinum help the cells to float and reach the oxygen rich surface (Schramm and Hestrin, 1954). BC could also be produced in the form of spherical pellets in submerged culture; however, the yield as compared to static surface culture diminished significantly. The spontaneous selection of cellulose-non-productive variants and shear stress damage the cellulose synthesis in the 1