Elsevier

Bioresource Technology

Volume 101, Issue 22, November 2010, Pages 8798-8806
Bioresource Technology

Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains

https://doi.org/10.1016/j.biortech.2010.06.001Get rights and content

Abstract

The composition of thermophilic (60 °C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 °C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had Km values of 3.11 and 1.08 mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 °C for 1 day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.

Introduction

Lignocellulosic biomass (all plants and plant-derived materials) is the Earth’s most abundant and renewable organic material with great potential for production of bioenergy and commodity chemicals. In April 2005, a joint study conducted by the United States Department of Energy and United States Department of Agriculture reported the availability in the US of nearly 1.3 billion dry tons biomass per year with a biofuels potential that could meet about one-third of the country’s demand for transportation fuels (Somerville, 2006). Recently, much attention has been paid to the thermophilic bioprocessing of cellulosic biomass to biofuels which, due to the use of elevated temperatures, offers several potential advantages such as (i) improved hydrolysis of cellulosic substrates, (ii) higher mass-transfer rates leading to better substrate solubility, (iii) lowered risk of potential contamination, and (iv) increased flexibility with respect to process design thus improving the overall economics of the process. However, the cellulose recalcitrance to biodegradation poses several major bottlenecks in the thermophilic digestion of biomass with one being the lack of robust cellulases that can function efficiently at high temperatures and at a broad range of pH. Thus, thermophilic cellulose-degrading bacteria and their enzymes have great potential in the development of viable technologies for the production of alternative fuels from agricultural, forestry, and municipal cellulosic wastes.

Thermophilic cellulose-degrading bacteria have been isolated from various environments such as compost systems (Lu et al., 2005, Mayendea et al., 2006, Ng et al., 2009), soils (Abdel-Fattah et al., 2007, Lee et al., 2008), wastewaters (Tai et al., 2004), and from the deep subsurface of the former Homestake gold mine in Lead, SD, now known as the NSF Deep Underground Science and Engineering Laboratory (DUSEL) (Rastogi et al., 2009a). The deep biosphere of gold mines harbors harsh environments similar to those in industrial processes for microorganisms, both natural and anthropogenic, including extreme temperature, pH, pressure, low-oxygen concentration, and inhibitors including toxic metals. In our previous study (Rastogi et al., 2009a), we reported the isolation and characterization of a thermophilic strain, DUSELR13 (Bacillus sp.), that was capable of growing at 60 °C using various carbon sources including cellulose. The objectives of the present study are to: (1) characterize the thermophilic cellulases secreted by DUSELR13; and (2) characterize the major thermophilic cellulose-degrading bacterial lineages in a compost facility. Additionally, the thermophilic cellulases secreted by a selected compost strain WSUCF1 (Geobacillus sp.) were characterized, compared to those of DUSELR13, and their potential applications in thermophilic bioprocessing are discussed.

Section snippets

Composting facility and sample collection

The Compost Facility started its operations in October 1994 at Washington State University (WSU), Pullman, WA and has been processing about 12,000 tons of campus-generated organic waste (e.g., shredded wood waste, animal manure, yard waste, food waste), annually. The composting facility consists of standard open air windrows. For the isolation of thermophilic cellulose-degrading bacteria (CDB), compost samples were aseptically obtained from the core (40 cm from the top where the temperature was 70

Bacterial communities in mixed cellulose-degrading enrichment culture

Bacterial communities play important role in the production of composts and thermogenic composting processes have been shown to support a variety of thermophilic and mesophilic bacteria (Blanc et al., 1999, Adams and Frostick, 2009). Bacterial diversity of mixed cellulose-enrichment culture was examined to get a broad picture of major bacterial communities that are involved in cellulose-degradation during composting. A total of 37 clone sequences were included in phylogenetic analysis that

Conclusions

CMCases secreted by a Bacillus sp. and Geobacillus sp. isolated from compost and deep gold mine environments, respectively had high thermo-tolerance. We believe that our findings on the production of cellulose-degrading enzymes under unoptimized medium and culture conditions, the remarkable thermostability of the CMCases and their broad temperature and pH activity range, and utilization of microcrystalline cellulose, make a contribution to our current knowledge on extremophiles and their

Acknowledgements

This research was funded by the Nelson Research Award Funds and South Dakota Board of Regents Competitive Research Grant (Award No. SDBOR/SDSMT 2010-09-05). The active support of the Center for Bioprocessing Research and Development and Department of Chemical and Biological Engineering at the South Dakota School of Mines and Technology is gratefully acknowledged. We would also like to thank the anonymous reviewers whose critiques were instrumental in improving the quality of our manuscript.

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    Present address: Department of Plant Pathology, University of California, Davis 95616, USA.

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