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An effective microplate method (Biolog MT2) for screening native lignocellulosic-straw-degrading bacteria

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Abstract

Lignocellulosic wastes such as straw are attractive resources for biofuel production when subjected to biological treatment (hydrolysis). However, their complex lignocellulosic structure can hinder saccharification. The urgent need for microbial groups with high levels of straw saccharifying activities is therefore a key step in the bioconversion of lignocellulosic straw into fermentable monosaccharides. Existing traditional methods of qualitative and quantitative screening of lignocellulolytic microbial isolates are costly, time consuming and largely not environmentally friendly. In this study, a Biolog (MT2) microplate-based assay was evaluated for potential use as an alternative screening method. This was carried out using three commercially available substrates (cellulose, xylan and lignin) and four native lignocellulosic straws (wheat, rice, sugarcane, and pea ball-milled straws). Selected bacterial isolates from soil, compost and straws were screened quantitatively using both traditional crude enzyme and Biolog (MT2) microplate methods. Positive correlations (R 2 values up to 0.86) between Biolog and the traditional enzyme methodologies were observed with respect to these isolates and their lignocellulosic activities. Quantitative assays were less labor intensive and faster (3–7 days) in Biolog microplates than in traditional assays which lasted for 12–15 days. Ball-milled rice and sugarcane straws were bio-converted to monosaccharaides more readily than wheat and pea straws and the commercially available substrates (cellulose, xylan and lignin). Environmental scanning electron microscopy (ESEM) analysis of ball-milled rice and sugarcane straws suggested that this was due to their higher silica content. Overall, the Biolog (MT2) microplate system was shown to be an effective, time saving and inexpensive alternative method for the screening of both lignocellulose-degrading bacteria and different substrates for saccharification.

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Acknowledgments

The authors are grateful to RMIT University and the Egyptian Government for the provision of a PhD scholarship to Mohamed Taha. The authors also acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility (AMMRF) at the RMIT Microscopy & Microanalysis Facility, at RMIT University.

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Authors and Affiliations

  1. School of Applied Sciences, RMIT University, Bundoora, VIC, 3083, Australia

    Mohamed Taha, Krishna K. Kadali, Khalid AL-Hothaly, Andrew T. Smith, Andrew S. Ball & Eric M. Adetutu

  2. Department of Biochemistry, Faculty of Agriculture, Benha University, Moshtohor, 13736, Toukh, Egypt

    Mohamed Taha

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  1. Mohamed Taha
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  2. Krishna K. Kadali
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  3. Khalid AL-Hothaly
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  6. Eric M. Adetutu
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Correspondence to Mohamed Taha.

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Supplementary Table 1

Enzyme activities (cellulase, xylanase and strawase U mL−1) of the 30 different bacterial isolates using the traditional crude enzyme production assay method (DOCX 949 kb)

Supplementary Figure 1

Phylogenetic tree of selected bacterial isolates. Note: tree was constructed from 1,000–1,200 nucleotide positions. Distances were calculated with the maximum likelihood model using PhyML. Bootstrap values > 0.50 are shown. Asterisks refer to lowest enzyme activity (based on strawase assay) by the traditional assays and to the “negative isolates” by Biolog (MT2) microplates.(DOCX 18 kb)

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Taha, M., Kadali, K.K., AL-Hothaly, K. et al. An effective microplate method (Biolog MT2) for screening native lignocellulosic-straw-degrading bacteria. Ann Microbiol 65, 2053–2064 (2015). https://doi.org/10.1007/s13213-015-1044-y

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