Elsevier

Bioresource Technology

Volume 164, July 2014, Pages 100-105
Bioresource Technology

Isolation of Paenibacillus glucanolyticus from pulp mill sources with potential to deconstruct pulping waste

https://doi.org/10.1016/j.biortech.2014.04.093Get rights and content

Highlights

Abstract

Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. A microorganism was isolated from a black liquor sample collected from the Department of Forest Biomaterials at North Carolina State University. The organism was identified as Paenibacillus glucanolyticus using 16S rRNA sequence analysis and was shown to be capable of growth on black liquor as the sole carbon source based on minimal media growth studies. Minimal media growth curves demonstrated that this facultative anaerobic microorganism can degrade black liquor as well as cellulose, hemicellulose, and lignin. Gas chromatography–mass spectrometry was used to identify products generated by P. glucanolyticus when it was grown anaerobically on black liquor. Fermentation products which could be converted into high-value chemicals such as succinic, propanoic, lactic, and malonic acids were detected.

Introduction

The pulping of wood is a chemically intensive process that has been optimized over many years to create a system with minimal waste. However, some outputs of this process could be used to produce valuable product(s) (other than steam and heat). An example of an underutilized material is black liquor. It is generated from the kraft process which is responsible for 85% of world lignin production (Tejado et al., 2007). The kraft process (Fig. 1) uses temperature (160–200 °C), pressure (120 psig), and the chemicals contained in white liquor (sodium hydroxide and sodium sulfide) to dissolve lignin from wood fibers (Biermann, 1996, Brannvall, 2009). Black liquor contains lignin, organic acids, and polysaccharide degradation by-products. Currently, black liquor is incinerated in the recovery boiler to produce steam for thermal energy and to recover chemicals for reuse in the pulping process. However, the amount of black liquor created by pulping can exceed the amount of black liquor that the recovery boiler can effectively process. Disposing of the black liquor as effluent negatively affects aquatic flora and fauna and thus is not an option. Isolation of microorganisms within the pulp mill that use black liquor for growth would be beneficial in terms of increasing chemical recovery and generation of value-added product(s) that would improve the overall life cycle analysis of the pulping system. The US Department of Energy (US DOE) published a list of chemicals that can be produced from biomass and converted into high-value chemicals. Among the top 30 are succinic, propanoic, lactic, and malonic acids which could be potentially produced by microbial fermentation (Werpy and Peterson, 2004). However, the recalcitrant nature of the lignocellulose, the chemical derivatives which constitute the majority of available organic carbon within black liquor, and the basic pH of black liquor present challenges to microbial biodegradation of this material.

The degradation of black liquor may involve enzymes with the ability to degrade lignin, cellulose, and hemicellulose. Many microorganisms can degrade the polysaccharide components in black liquor, but not the lignin. Primarily, the complex and amorphous structure of lignin limits the biodegradation capacity of microorganisms. Lignin limits a microorganism’s access to the carbon sources contained in the plant cell wall which is cellulose or hemicellulose. Trametes elegans is a lignin-degrading white-rot fungus that has been extensively studied that uses cellulose as its carbon source (Lara et al., 2003). Fungal lignin degradation has been studied for over 30 years, nonetheless there is no commercial biocatalytic process of lignin depolymerization (Pawlak, 2009). Bugg et al. (2011) attribute this to the challenges associated with fungal protein expression and genetic manipulation. Despite the structural complexity of lignin, some bacteria are capable of lignin degradation such as Aneurinibacillus aneurinilyticus, Bacillus sp., Bacillus cereus C10-1, Critobacter sp., Nocardia, Novosphingobium sp. B-7, Paenibacillus sp., Pandoraea norimbergensis, Pseudomonas jessenii PS06, Pseudomonas putida, Rhodococcus jostii RHA1, Serratia marcescens, Sphingomonas paucimobilis mt-2. Streptomyces, and Viridosporus T7A (Bandounas et al., 2011, Bugg et al., 2011, Chandra et al., 2011, Chen et al., 2012, Raj et al., 2007).

Bacillus glucanolyticus was first isolated from environmental soil samples by Alexander and Priest (1989). This gram positive, rod-shaped, facultative anaerobic bacterium is characterized by its terminal spore formation, motile colonies, and ability to degrade a variety of β-glucans (Alexander and Priest, 1989). B. glucanolyticus was shown to be capable of hydrolyzing carboxymethyl cellulose (β, 1–4 linked glucose), curdlan (β, 1–3 linked glucose), pustulan (β, 1–6 linked glucose), and xylose (Alexander and Priest, 1989, Kanzawa et al., 1995). Bacillus glucanolyticus was renamed Paenibacillus glucanolyticus in 1997 by Shida et al. (1997), based on 16s rRNA gene similarity. There has been no subsequent work published on P. glucanolyticus. However, bacteria within the genus Paenibacillus have been found to degrade black liquor components like cellulose and xylan (Ko et al., 2007).

The aims of these experiments were to (1) isolate a microorganism from the pulping waste black liquor, (2) identify the microorganism isolated, (3) characterize its growth on black liquor and black liquor constituents, and (4) identify fermentation products produced when it is grown on black liquor.

Section snippets

Chemicals

NaClO2 was purchased from Sigma Aldrich (MO) and CaCl2 from VWR (PA). NaOH was obtained from EMD Millipore (MA). Switchgrass was sourced from Cherry Research Farm (Goldsboro, NC). Filter paper (size 1) was purchased from Whatman (UK). Ampicillin, glucose, and isopropyl-β-d-thiogalactopyranoside (IPTG) were supplied by Fisher (MA). X-gal was purchased from Qiagen (CA) and xylose from Acros organics (NJ).

Solutions

1,4-Dioxane (ACS grade), ethanol (microbiology grade), and H2SO4 (ACS grade) were purchased

P. glucanolyticus optimal growth conditions

P. glucanolyticus was previously isolated and described by Alexander and Priest (1989) as a facultative anaerobe characterized by long thin cells that form flat, smooth, opaque and motile colonies which produces terminal spores. In the current study, the P. glucanolyticus strain isolated from black liquor was evaluated for growth in response to pH (Fig. 2A) and temperature (Fig. 2B) and was found to have an optimal growth pH of 9.0 and a temperature of 37 °C, respectively, when it was grown

Conclusions

This study characterized the growth of P. glucanolyticus isolated from the pulping by-product black liquor. P. glucanolyticus grows optimally at pH 9 and 37 °C and can use black liquor as a carbon and fixed nitrogen source. P. glucanolyticus can also use glucose, xylose, cellulose, hemicellulose, and lignin as sole carbon sources under aerobic and anaerobic conditions. Finally, P. glucanolyticus can metabolize black liquor to produce value-added products including ethanol, succinic, propanoic,

Acknowledgements

The authors thank Jim McMurray for providing black liquor, Dr. Dhana Savithri for her help with the HPLC analysis, Dr. Ali Ayoub for his help with the switchgrass extraction, and Dr. David Tilotta and August Meng for their help with the GC–MS analysis.

Funding source: National Needs Fellowship (NNF) sponsored by USDA-CSREES.

References (25)

  • B. Alexander et al.

    Bacillus glucanolyticus, a new species that degrades a variety of B-glucans

    Int. J. Syst. Bacteriol.

    (1989)
  • L. Bandounas et al.

    Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

    BMC Biotechnol.

    (2011)
  • Cited by (37)

    • Recent progress and challenges in biological degradation and biotechnological valorization of lignin as an emerging source of bioenergy: A state-of-the-art review

      2022, Renewable and Sustainable Energy Reviews
      Citation Excerpt :

      In addition to Paenibacillus sp. ITRC S6, P. glucanolyticus SLM1, which was isolated from pulp mill waste (black liquor), was shown to degrade lignin under both aerobic and anaerobic conditions [87,88]. The most diverse distribution of lignin-degrading bacteria at the genus level has been reported for the phylum Proteobacteria, of which Pseudomonas is one of the potential genera for lignin catabolism.

    • Application of ligninolytic bacteria to the enhancement of lignocellulose breakdown and methane production from oil palm empty fruit bunches (OPEFB)

      2022, Bioresource Technology Reports
      Citation Excerpt :

      The highest value of TSP was obtained from sample inoculated by Paenibacillus sp which is 39% higher than a non-treated sample (Fig. 2a). Paenibacillus sp was confirmed to be able to hydrolyze cellulose and hemicellulose for growth and was able to grow on lignin media as the sole carbon both in aerobic and anaerobic conditions (Mathews et al., 2014). Other studies showed that C. testosteroni and Agrobacterium sp were also able to release TSP about 74.6% and 22.7% respectively from pine wood (Rashid et al., 2017).

    • Paper and pulp mill wastewater: Characterization, microbial-mediated degradation, and challenges

      2022, Nanotechnology in Paper and Wood Engineering: Fundamentals, Challenges and Applications
    • Enhanced bioremediation of pulp effluents through improved enzymatic treatment strategies: A greener approach

      2021, Renewable and Sustainable Energy Reviews
      Citation Excerpt :

      Besides, LiPs have also played a significant role in the degradation of organic chemicals in the presence of veratryl alcohol (VA) mediator and hydrogen peroxidase co-factor [43]. Therefore, these enzymes catalyze the degradation of phenolic chemicals (vanillyl alcohol and guaiacol) and non-phenolic compounds (1-(3,4-diethoxyphenyl-1,3-dihydroxy, 4-methoxy-phenyl) in the presence of hydrogen peroxidase [44] and possess high potential for degradation of pulp and paper mill effluents. Manganese peroxidase (MnP) is an oxidoreductase enzyme secreted by several fungi and bacteria.

    View all citing articles on Scopus
    View full text