Improvement of cellulase production in cultures of Acremonium cellulolyticus using pretreated waste milk pack with cellulase targeting for biorefinery
Introduction
Cellulase production is the most important step in the commercial production of ethanol and other chemicals from renewable cellulosic materials. To date, many potential cellulase producers have been isolated (Atanasova et al., 2010) and used to produce cellulase (Fujii et al., 2009) in submerged culture processes. However, cellulase producers require the relatively expensive cellulose powder as the sole carbon source, and this hinders the industrial application of cellulase in bioconversion processes. To overcome this problem, the use of waste agro-biomass as a carbon source for the production of cellulase has been suggested due to its lower cost (Krishna, 1999, de Lima et al., 2005, Sukumaran et al., 2009). Agricultural residues present in abundance, such as corn stover (Yao et al., 2010), wheat straw (Emtiazi and Nahvi, 2000), rice straw (Sun et al., 2008), bagasse (Adsul et al., 2004), and lignocellulose (Hendriks and Zeeman, 2009), have been used for cellulase production. These raw materials are cheaper, but pretreatment is generally required to improve their utilizability as carbon sources for the cellulase producer, which increases the costs considerably. The cellulolytic biomass has to be hydrolyzed to reducing sugars by cellulolytic enzymes or acids. Cost-efficient acids can be used as hydrolysis agents, but these are not environmentally friendly because the process requires high temperatures and the disposal of acid wastes is a problem. Thus, improving the process to produce cost-efficient cellulolytic enzymes from microorganisms is an important objective in the biorefining of cellulosic biomass.
Waste paper is a cellulolytic biomass that has been targeted for recycling. In Japan, approximately 30.6 million tons of paper is produced and consumed each year. The importance given to recycling has resulted in increased public awareness such that in 2008, 75.1% of the annual paper production was collected and 61.8% was reused (http://www.jpa.gr.jp/states/used-paper/index.html). When paper materials are recycled, they are usually converted into lower grade paper products, for example, office paper is converted to magazine paper and cardboard is converted to sanitary products. As the paper is recycled further, the length of the cellulose fibers decrease. This shortening of cellulose fibers reduces the paper quality; therefore, the maximum ratio of paper-to-paper recycling is considered to be 65%. Thirty-five percent of all paper is not fit for recycling and is disposed of as paper sludge, which is incinerated or landfilled without reuse.
One of the aggressive utilizations of waste paper and paper sludge is to use it as an alternative carbon source for cellulase production. Waste paper is a good carbon source for cellulase production by microorganisms because waste paper is composed of delignified biomass. Toyama et al. (2002) tried to isolate cellulase hyperproducers of Trichoderma reesei for the utilization of waste paper. They used 1% (w/v) paper powder medium for the 1st screening and found that the hydrolytic activity of the selected strain was 3.5-fold higher than that of the original strain. Waste paper hydrolysate was also reported to induce cellulase in cultures of T. reesei (Chen and Wayman, 1991). Ju and Afolabi (1999) showed that the enzymatic hydrolysate of wastepaper induced cellulase activity in a continuous culture of T. reesei, and they attributed this to the higher concentrations of oligomer-inducing intermediates in the wastepaper hydrolysate. Similarly, paper sludge is a good carbon source for cellulase production. Paper mill sludge was used for cellulase production with mixed cultures of T. reesei and Aspergillus niger (Maheshwari et al., 1994). However, cellulase production from paper sludge was low compared to that obtained when pure cellulose was used as the carbon source. The reason for this was that paper sludge contains impurities such as clay and several kinds of metal ions. To improve the cellulase production rate, the paper sludge was pretreated with ammonium hydroxide or hydrogen peroxide. This resulted in the production of 2.4 FPU/ml of cellulase from the T. reesei culture, which was half of the value obtained when steam-exploded wood was used as the substrate (Shin et al., 2000). Prasetyo et al. (2011) optimized culture condition in the culture of Acremonium cellulolyticus using untreated waste paper sludge as the carbon source, and achieved 9.31 FPU/ml of cellulase activity at the flask scale and 10.96 FPU/ml in a 3-L fermentor. However, cellulase production from paper sludge was only approximately 60% of that obtained when pure cellulose was used as the carbon source (Ikeda et al., 2007).
In Japan, about 251,000 tons of paper packs were used as containers for food, beverages, and milk in 2008, which accounted for 0.7% of the total paper consumption (http://www.yokankyo.jp/cat02.html). Paper packs are made from conifers and coated with polyethylene film, and 70% of these are used as milk containers. Due to their good safety, light weight, easy handling, and renewability, the usage of paper pack has increased every year by approximately 1%. In Japan, the recycling ratio of paper packs has increased every year and was reportedly 32% in 2008 (http://www.yokankyo.jp/cat02.html). Eighty-five percent of the recovered paper packs was reused as sanitary products, despite the fact that this material was composed of high-quality cellulose. One way to reuse paper packs is to produce cellulase for the saccharification of cellulosic biomass to reducing sugars. These sugars can then be converted (for example, by fermentation) to value-added bioproducts such as ethanol or other chemicals (Scott et al., 1994, Wayman et al., 1993).
Filamentous fungi, typically T. reesei, have been used for industrial cellulase production due to their ability to produce extracellular protein in high amounts. Unfortunately, the amount of β-glucosidase secreted by T. reesei is insufficient for an efficient saccharification (Sternberg et al., 1977). A. cellulolyticus, which was isolated in 1987 (Yamanobe et al., 1987) and subsequently engineered to enhance its performance, produces both cellulase and β-glucosidase in addition to carbomethyl cellulose-hydrolyzing enzyme (CMCase) and small amounts of xylanase, β-1,3-glucanase, and amylase. This microorganism has been used as an alternative cellulase producer (Ikeda et al., 2007, Park et al., 2002, Prasetyo et al., 2010). The A. cellulolyticus strain uses Solka Floc (SF), which is composed of 100% cellulose and contains 70–80% crystalline cellulose and 20–30% amorphous cellulose, as the carbon source for cellulase production. However, SF is an expensive carbon source, and this is an obstacle in the industrial production of cellulase from A. cellulolyticus.
There are no specific reports on cellulase production from MP cellulose. Waste MP is very clean, pure, and delignified cellulose, but it has been reused only as a sanitary product. In this study, we developed a method for utilizing MP cellulose as a carbon source for producing cellulase from A. cellulolyticus, which is a value-added bioproduct. MP was first pretreated with cellulase and then used as the carbon source for an A. cellulolyticus culture. This pretreated MP cellulose improved significantly the production of cellulase, which was similar to the cellulose activity level obtained with SF as the carbon source. The physicochemical and morphological changes of cellulase-pretreated MP were investigated.
Section snippets
Cellulose materials used
Ten types of cellulose materials (cellulose type I) from different manufacturers were used in this study to investigate cellulase production (Table 1). Waste MP was collected from local supermarkets in Shizuoka city (Japan). MP is made from conifers and coated with polyethylene film. SF (CAS #9004-34-6; International Fiber Corp., New York, USA) was used as the carbon source for the preculture of A. cellulolyticus. SF is a fine white powder that is used as an industrial filtration material. It
Effect of the degree of polymerization (DP) and crystallinity of cellulose on cellulase production
The DP and CI of 10 different types of cellulose were measured before use. The DP varied in the range 70–850, while the CI varied in the range 0.59–0.91 (Fig. 1A and B). Cellulase production was examined in flask cultures. The cellulase activity increased up to a DP of 400 but decreased when the DP was higher than 400 (Fig. 1A). In the case of CI, the cellulase activity was high when cellulose with low CI was used, but the activity was low when the CI was higher than 0.75 (Fig. 1B). This
Conclusions
Cellulase production is a key step in biorefining in the production of second-generation bioethanol as an alternative fuel (Santos et al., 2010). When A. cellulolyticus was cultured in pretreated MP cellulose with 3 FPU/g MP for 12 h, the cellulase activity significantly increased to 16 FPU/ml in a 3-L fermentor. This was 25-fold higher than the activity achieved with untreated MP cellulose. This result would be useful to bioconvert MP cellulose to the high-value product cellulase because
Acknowledgements
We thank Mr. Hiroyuki Fukazawa of Shizuoka Industrial Research Institute for measuring the length and width of the pretreated MP cellulose fibers. We also thank Dr. Vipin Kumar Deo of Shizuoka University for manuscript editing. This study was supported by a Comprehensive Support Programs for Creation of Regional Innovation in Japan Science and Technology Agency.
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