Strain improvement of Acremonium cellulolyticus for cellulase production by mutation Xu Fang, Shinichi Yano, ⁎ Hiroyuki Inoue, and Shigeki Sawayama Biomass Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2-2-2 Hiro-suehiro, Kure 737-0197, Japan Received 17 July 2008; accepted 10 November 2008 In the search for an efficient producer of cellulolytic enzymes, Acremonium cellulolyticus strain C-1 was subjected to mutagenesis using UV-irradiation and N-methyl-N′nitro-N-nitrosoguanidine (NTG) and strain CF-2612 was isolated. Strain CF-2612 exhibited higher filter paperase (FPase) activities (17.8 U/ml) than the parent strain C-1 (12.3 U/ml). Soluble protein production and β-glucosidase activity from strain CF-2612 were also significantly improved. FPase activity, cellulase productivity and yield of CF-2612 using batch culture with 5% Solka Floc in a 2-l jar fermentor at 30 °C reached 18.0 U/ml, 150.0 FPU/l/h and 360.0 FPU/g carbohydrate, respectively; when fed-batch culture was used with Solka Floc, these values reached 34.6 U/ml, 240.3 FPU/l/h and 346.0 FPU/g carbohydrate, respectively. It was observed that more hydrolyzed glucose was released from pretreated eucalyptus with the enzyme of strain CF-2612, compared with that of the commercial cellulase GC-220. This result was attributed to the higher ratio of β-glucosidase/FPase activity of strain CF-2612. Three distinguishable phases including the periods of primary or second mycelial growth and mycelial fragmentation were proposed in batch culture by A. cellulolyticus. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Acremonium cellulolyticus; Bioethanol; Mutagenesis; Cellulase; Batch culture; Fed-batch culture] Bioethanol is the most promising renewable and carbon neutral alternative liquid fuel (1). Consumption of bioethanol has been rapidly increasing as the world strives to decrease its dependence on fossil fuels as well as reducing greenhouse gases (GHGs) (2). At present, bioethanol is made from crops such as sugarcane or corn. Searchinger et al. (3) found that while crop-based ethanol increases GHGs through emissions from land-use change, the value of using waste products for ethanol production was highlighted. The cellulosic biomass is an abundant, renewable and underutilized global carbon source (4). Moreover, it is concluded that bioethanol produced from cellulosic biomass has the potential to reduce GHGs emissions by up to 86% (5). Thus, technology to produce bioethanol from cellulosic biomass should be developed. To produce bioethanol from cellulosic biomass, it must first be hydrolyzed to fermentable sugars using cellulolytic enzymes or acids. Enzymatic hydrolysis is environment-friendly and can achieve high sugar yields for ethanol fermentation, making it amenable to industrial production. However, the high cost of cellulases remains the most significant barrier to the economical production of bioethanol from cellulosic biomass. Cellulase is an enzyme complex found in some fungi and bacteria (6–11). Filamentous fungi, typically Trichoderma reesei, are the preferred source of industrial cellulase preparations because of their excellent capacity for extracellular protein production. It is well known that T. reesei produces the CBH and EG components of cellu- lase enzyme complex in large quantities. However, the amount of β-glucosidase secreted by T. reesei is insufficient (12). Acremonium cellulolyticus, isolated in 1982 from soil in north- eastern Japan, is a cellulose-degrading filamentous fungus (13). A. cellulolyticus wild strain Y-94 secretes a large amount of cellulo- lytic enzyme and the ratio of β-glucosidase/filter paperase (FPase) activity in its enzyme system is higher than that of T. reesei QM9414 (13). Furthermore, mutant strains TN, C-1 have been isolated from strain Y-94 with random mutagenesis in these successive generations (Yamanobe, T. et al., Japanese patent,1998-63197, 1998; 2003-135052, 2003). A. cellulolyticus has been exploited in Japan as a cellulolytic enzyme-producing industrial microorganism. In the development of a more economical production of cellulolytic enzymes, an efficient approach is the isolation of cellulase hyper- producing mutants. In this study, A. cellulolyticus strain C-1 was treated by random mutagenesis and then strain CF-2612 was isolated. Moreover, strain CF-2612 was investigated for cellulase production using batch and fed-batch culture. The morphology of the mycelium in different phases was observed microscopically and three representa- tive phases in batch culture was proposed. The hydrolysis capability between the cellulolytic enzyme from A. cellulolyticus and the commercial enzyme from T. reesei was compared. MATERIALS AND METHODS Fungal strain and mutagenesis The parental strain A. cellulolyticus C-1 (FERM P-18508) was obtained from Tsukishima Kikai Co. Ltd. (TSK; Tokyo). This strain was inoculated on potato dextrose agar (PDA; Difco, Sparks, MD, USA) slants, incubated for Journal of Bioscience and Bioengineering VOL. 107 No. 3, 256 – 261, 2009 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 823 72 1935; fax: +81 823 73 3284. E-mail address: s-yano@aist.go.jp (S. Yano). 1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2008.11.022