Contents lists available at ScienceDirect New BIOTECHNOLOGY journal homepage: www.elsevier.com/locate/nbt Response surface statistical optimization of bacterial nanocellulose fermentation in static culture using a low-cost medium Ana Cristina Rodrigues a , Ana Isabel Fontão a,1 , Aires Coelho a,1 , Marta Leal a , Francisco A.G. Soares da Silva a,1 , Yizao Wan b , Fernando Dourado a, ⁎ , Miguel Gama a a CEB- Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal b School of Materials Science and Engineering, East China Jiaotong University, Nanchang, 330013, China ARTICLE INFO Keywords: BNC production optimization Low-cost substrates Response surface methodology-central composite design Culture medium depth Surface area ABSTRACT This work aimed at the optimization of bacterial nanocellulose (BNC) production by static culture, using Komagataeibacter xylinus BPR 2001 (K. xylinus). Response surface methodology - central composite design was used to evaluate the effect of inexpensive and widely available nutrient sources, namely molasses, ethanol, corn steep liquor (CSL) and ammonium sulphate, on BNC production yield. The optimized parameters for maximum BNC production were % (m/v): molasses 5.38, CSL 1.91, ammonium sulphate 0.63, disodium phosphate 0.270, citric acid 0.115 and ethanol 1.38% (v/v). The experimental and predicted maximum BNC production yields were 7.5 ± 0.54 g/L and 6.64 ± 0.079 g/L, respectively and the experimental and predicted maximum BNC productivity were 0.829 ± 0.046 g/L/day and 0.734 ± 0.079 g/L/day, after 9 days of static culture fermen- tation, at 30 °C. The effect of surface area and culture medium depth on production yield and productivity were also studied. BNC dry mass production increased linearly with surface area, medium depth and fermentation time. So long as nutrients were still available in the culture media, BNC mass productivity was constant. The results show that a high BNC production yield can be obtained by static culture of K. xylinus BPR 2001 using a low-cost medium. These are promising conditions for the static industrial scale BNC production, since as com- pared to agitated bioreactors, higher productivities may be reached, while avoiding high capital and operating costs. Introduction Bacterial nanocellulose (BNC) is an exopolysaccharide produced by Komagataeibacter xylinus (formerly Gluconacetobacter xylinus), a Gram negative and strictly aerobic bacterium of the Acetobacteraceae family [1–6]. BNC shows several unique physicochemical and mechanical properties, including high purity, high crystallinity, high degree of polymerization [7], an ultrafine fiber network, high water holding and absorbing abilities [8], high tensile strength in the wet state [9], and the possibility to be shaped into 3D structures during synthesis. It is biocompatible and biofunctional [10]. Due to these properties, the biopolymer has been studied in several applications, including tissue regeneration, drug delivery systems, vascular grafts, in vitro and in vivo scaffolds for tissue engineering, electronic paper displays and in food applications [11–17]. These properties and applications have generated a growing interest in the development of new strategies aimed at large- scale BNC production. Several fermentation technologies have been attempted, such as agitated, air-lift, membrane and horizontal bior- eactors, using different fermentation media and overproducing mutant strains. Stirred tank reactors can prevent the heterogeneity of the cul- ture broth, at the expense of a high energy cost for generation of me- chanical power. Airlift reactors typically require only one sixth of the energy power used in stirred tank reactors. Nonetheless, the agitation power of an airlift reactor is limited, resulting in low fluidity of the culture broth, especially at high cellulose concentrations. In addition, both agitation and aeration systems have been reported to result in the development of cellulose-negative mutants (non-cellulose producers, Cel - )[18–20]. In the case of membrane bioreactors, the major draw- backs include high operating costs and difficulty in collecting the cel- lulose from the reactors following fermentation [9,18–23]. https://doi.org/10.1016/j.nbt.2018.12.002 Received 5 June 2018; Received in revised form 5 December 2018; Accepted 5 December 2018 Abbreviations: BNC, bacterial nanocellulose; K. xylinus, Komagataeibacter xylinus; CSL, corn steep liquor; RSM, Response Surface Methodology; CCD, Central Composite Design; HS medium, Hestrin-Schramm culture medium; S, surface fermentation area; L, culture medium depth; V, culture medium volume ⁎ Corresponding author. E-mail address: fdourado@deb.uminho.pt (F. Dourado). 1 These authors contributed equally to this work. New BIOTECHNOLOGY 49 (2019) 19–27 Available online 06 December 2018 1871-6784/ © 2018 Elsevier B.V. All rights reserved.