Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA

Abstract

The present study investigated the cultivable mesophilic (37 degrees C) and thermophilic (60 degrees C) cellulose-degrading bacterial diversity in a weathered soil-like sample collected from the deep subsurface (1.5 km depth) of the Homestake gold mine in Lead, South Dakota, USA. Chemical characterization of the sample by X-ray fluorescence spectroscopy revealed a high amount of toxic heavy metals such as Cu, Cr, Pb, Ni, and Zn. Molecular community structures were determined by phylogenetic analysis of 16S rRNA gene sequences retrieved from enrichment cultures growing in presence of microcrystalline cellulose as the sole source of carbon. All phylotypes retrieved from enrichment cultures were affiliated to Firmicutes. Cellulose-degrading mesophilic and thermophilic pure cultures belonging to the genera Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and selected cultures were studied for enzyme activities. For a mesophilic isolate (DUSELG12), the optimum pH and temperature for carboxymethyl cellulase (CMCase) were 5.5 and 55 degrees C, while for a thermophilic isolate (DUSELR7) they were 5.0 and 75 degrees C, respectively. Furthermore, DUSELG12 retained about 40% CMCase activity after incubation at 60 degrees C for 8 h. Most remarkably, thermophilic isolate, DUSELR7 retained 26% CMCase activity at 60 degrees C up to a period of 300 h. Overall, the present work revealed the presence of different cellulose-degrading bacterial lineages in the unique deep subsurface environment of the mine. The results also have strong implications for biological conversion of cellulosic agricultural and forestry wastes to commodity chemicals including sugars.

Enhanced Cellulase Production by a Novel Thermophilic Bacillus licheniformis 2D55: Characterization and Application in Lignocellulosic Saccharification

“…The basal medium used in this study contained the following: 1.0 g/L KH2PO4, 1.145 g/L K2HPO4, 0.5 g/L MgSO4.7H2O, 5.0 g/L NH4SO4, 0.05 g/L CaCl2.2H2O, and 1 mL Nitsch's trace element solution (2.2 g MnSO4, 0.5 g ZnSO4, 0.5 g H3BO3, 0.016 g CuSO4, 0.025 g Na2MoO4, and 0.046 g CoCl2) (Rastogi et al 2009). The pH of the medium was corrected to pH 7.…”

“…Previous report showed that thermostable cellulase from B. licheniformis JK7 (Seo et al 2013), retained only about 25% of its original activity when incubated at 80 °C for 1 h. Another Bacillus sp. isolated from deep surface of homestake gold mine exhibited a thermostable cellulase (Rastogi et al 2009); however, the enzyme completely lost its activity over 1 h incubation at 90 and 100 °C.…”

Genetic engineering and enzyme research in lignocellulosic ethanol production

“…Several cellulose-degrading Firmicutes including, Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and isolates were found with optimum temperature for carboxymethyl cellulase (CMCase) of up to 75°C (pH 5.0). One particular isolate retained 26% CMCase activity at 60°C up to a period of 300 h (Rastogi et al, 2009). These thermostable enzymes and robust thermophilic fermentative microbes could facilitate the development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process used to convert lignocellulosic biomass into biofuels.…”

Isolation of a Thermostable Alkaline Cellulase-producing Bacterium Strain from a Garbage Dump

“…Nucleotide sequence of 16S rDNA was sequenced and identified through the online BLAST program. Multiple alignment was carried out with the CLUSTALX and a neighbor-joining phylogenetic tree was reconstructed using the MEGA program version 4.0 (Rastogi et al, 2009).…”