Isolation of Paraclostridium CR4 from sugarcane bagasse and its evaluation in the bioconversion of lignocellulosic feedstock into hydrogen by monitoring cellulase gene expression

https://doi.org/10.1016/j.scitotenv.2020.136868Get rights and content

Highlights

  • Paraclostridium was identified as autochthonous bacteria from sugarcane bagasse.

  • Paraclostridium produced H2 from glucose, cellulose and sugarcane bagasse.

  • The substrate type regulated the formation of the final product (acetic/butyric).

  • There was cell growth during all stages of H2 production.

  • The cellulase family protein expression was regulated by soluble sugars.

Abstract

Bioconversion of sugarcane bagasse (SCB) into hydrogen (H2) and organic acids was evaluated using a biomolecular approach to monitor the quantity and expression of the cellulase (Cel) gene. Batch reactors at 37 °C were operated with Paraclostridium sp. (10% v/v) and different substrates (5 g/L): glucose, cellulose and SCB in natura and pre-heat treated and hydrothermally. H2 production from glucose was 162.4 mL via acetic acid (2.9 g/L) and 78.4 mL from cellulose via butyric acid (2.9 g/L). H2 production was higher in hydrothermally pretreated SCB reactors (92.0 mL), heat treated (62.5 mL), when compared to in natura SCB (51.4 mL). Butyric acid (5.8, 4.9 and 4.0 g/L) was the main acid observed in hydrothermally, thermally pretreated, and in natura SCB, respectively. In the reactors with cellulose and reactors with hydrothermally pretreated SCB, the Cel gene copy number 3 and 2 log were higher, respectively, during the stage of maximum H2 production rate, when compared to the initial stage. Differences in Cel gene expression were observed according to the concentration of soluble sugars in the reaction medium. That is, there was no gene expression at the initial phase of the experiment using SCB with 2.6 g/L of sugars and increase of 2.2 log in gene expression during the phases with soluble sugars of <1.4 g/L.

Introduction

Lignocellulosic material has been used as an alternative substrate for sustainable production of biofuels such as hydrogen (Ratti et al., 2015; Soares et al., 2017; Rabelo et al., 2018b), methane (Guo et al., 2014; Tantayotai et al., 2017) and ethanol (Kumar et al., 2008; Rabelo et al., 2011). Some of the main advantages of using these materials are their wide availability and high carbon content. In addition, the use of lignocellulosic material adds waste reduction to clean energy generation (Shrestha et al., 2017; Ahmad et al., 2018).

According to its origin, lignocellulosic material can be obtained from the processing of forest residues (mainly wood), agricultural residues (soybean, rice and sugarcane, among others) and urban and industrial residues (solid or liquid). Brazil is the largest producer of sugarcane in the world and 620.8 million tons of sugarcane were produced in the 2018/19 harvest, and later about 0.2 million tons of sugarcane bagasse (SCB) (UNICA, 2019). Although the traditional use of SCB is a substrate for boiler combustion within the plants themselves, there is surplus that can be made available for hydrogen production.

The effective degradation of lignocellulosic materials, requires synergistic stages of cellulolytic enzyme production, polysaccharide hydrolysis and sugar fermentation. In this process, the speed and yield of the end products are related to the initial hydrolysis (Saratale et al., 2008; Juturu and Wu, 2014). Hydrolysis is generally limited by the complex structure of materials, including cellulose crystallinity, particle size and the presence of associated materials such as hemicellulose and lignin (Binod et al., 2011; Galbe and Zacchi, 2012).

Cellulolytic anaerobic bacteria can be obtained from ruminal fluid (Deng et al., 2017), from invertebrates (Gupta et al., 2012) and mollusks (Muñoz et al., 2014), soil (Talia et al., 2012), decomposition of grass (Desvaux, 2005) and sugarcane bagasse (Ratti et al., 2015; Rabelo et al., 2018a). Ratti et al. (2015) and Rabelo et al. (2018a) identified autochthonous cellulolytic bacteria from sugarcane bagasse and similar to Clostridium, Tepidimicrobia, and Paenibacillus. Both authors used this source of autochthonous bacteria as an inoculum for the bioconversion of pre-treated sugarcane bagasse into hydrogen and organic acids.

Cellulolytic, anaerobic and gram-positive bacteria are included in the Firmicutes phylum and more particularly in the Clostridia class, Clostridiales order, most of them belonging to the Clostridiaceae family (Whitman, 2010). These bacteria have an enzymatic cellulolytic system consisting of at least 3 different types of cellulases: exoglucanases, endoglucanases and β-glycosidases, which synergistically provide complete cellulose hydrolysis (Juturu and Wu, 2014).

Zhang et al. (2015) obtained high hydrogen production yields with cellulose (772 mL), carboxymethylcellulose (646 mL) and corn stalk (1308 mL) using Clostridium sartagoforme, which was isolated from cow dung compost. Different bacterial cellulolytic strains have been isolated from self-fermentation of cellulosic material and used as inoculum in fermentative systems. Mazareli et al. (2019) isolated Bacillus sp. from banana residues and used this strain as inoculum for the fermentative production of hydrogen from pure and complex substrates. The authors reported hydrogen production of 36.3 and 59.6 mL using xylose and maltose, respectively, and when banana residues were used as substrate, hydrogen production was 106.5 mL.

When combined, qPCR and RT-qPCR analyses are useful tools for the specific detection and quantification and analysis of transcriptional cellulolytic activity (Dollhofer et al., 2016). These analyses have been widely applied in both environmental samples (Béra-maillet et al., 2009; Singh et al., 2014; Dollhofer et al., 2016) and in fermentative reactors (Lu and Lee, 2015; Salimi and Mahadevan, 2013) for monitoring bacterial activity (Béra-maillet et al., 2009; Lu and Lee, 2015; Salimi and Mahadevan, 2013) and cellulolytic fungi (Dollhofer et al., 2016).

Béra-maillet et al. (2009) evaluated expression of cellulolytic enzymes of the bacterium Fibrobacter succinogenes from sheep rumen microbiota by RT-qPCR analysis. The authors compared cellulase expression in sheep containing F. succinogenes as the only cellulolytic bacterium with the complex microbiota of a conventional sheep. The level of gene transcription in the monoculture was about two logs higher than that measured in the conventional animal, since F. succinogenes in monoculture does not compete with other species for the degradation of vegetal fibers.

The qPCR analysis was used by Dollhofer et al. (2016) for the quantification of anaerobic fungi in environmental samples. According to the results of these authors, the quantification of anaerobic fungi (in number of copies/mL) in biogas digesters was lower (1.78 × 108) than the amount reported in rumen fluid (1.69 × 1010) and in cattle feces (1.88 × 109 to 6 × 109), since it does not represent their natural habitat. Based on the RT-qPCR analyses, there was no transcriptional cellulolytic fungal activity in biogas digesters. Bacterial cellulolytic activity in biogas digesters was evaluated by Wei et al. (2015) and, contrary to fungal activity, it was shown that bacterial endoglucanases were significant in these environments.

In the present study, the cellulolytic bacterium Paraclostridium sp. CR4 was isolated from self-fermentation of sugarcane bagasse. The bacterium was used as inoculum in fermentative reactors for the hydrolysis and fermentation of cellulose and also sugarcane bagasse for hydrogen production. Quantification and expression of the cellulase family protein gene were monitored by real-time polymerase chain analysis (qPCR) and reverse transcription followed by qPCR (RT-qPCR). These analyses were applied to samples of fermentative reactors with cellulose and sugarcane bagasse as substrates and Paraclostridium sp. CR4. The building stages of specific primers were necessary for this bacterial species, as well as for the study of reference genes to normalize gene expression.

Section snippets

Strain isolation

Sugarcane bagasse (SCB) used as a source of autochthonous bacteria was supplied by Usina São Martinho (Pradópolis, SP, Brazil).

SCB self-fermentation assays were performed using 500 mL Duran® flasks with 250 mL autoclaved PCS medium (1.0 g/L yeast extract, 5.0 g/L peptone, 2.0 g/L CaCO3, and 5.0 g/L NaCl), and sugarcane bagasse (7.0 g/L). The reactors were subjected to N2 atmosphere (100%) for 15 min, closed with butyl cap and plastic screw, and incubated at 37 °C for 10 days.

Samples of the

CR4 identification and characterization

The isolated strain CR4 of SCB auto-fermentation grew on agar surface and formed circular, smooth and cream color colony. According to microscopic examination, the strain was rod-shaped, gram-positive, 1.2 × 3.3 μm, and forming terminal endospores. The occurrences of cells were single, in pairs or in chains (Fig. A1, Appendices document).

The nucleotide sequence of the isolated strain CR4 was similar (100%) to Paraclostridium genus. Fig. 1 shows the genetic distance dendrogram, the bootstrap

Conclusion

Cellulolytic and fermentative bacteria similar to Paraclostridium sp. CR4 were isolated from sugarcane bagasse (SCB) and used as inoculum for the fermentative production of H2 from glucose (162.4 mL), cellulose (78.4 mL) and SCB. The pretreatment applied to SCB favored hydrolysis and fermentation, as 92.0, 62.5 and 51.4 mL of H2 were obtained from hydrothermally pretreated SCB, heat pretreated SCB and in natura SCB, respectively. Metabolic route change was observed according to the substrate,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Process no. 150.446/2018-7) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Process no. 2015/06246-7and 2018/26470-7).

References (81)

  • X.M. Guo et al.

    Hydrogen production from agricultural waste by dark fermentation: a review

    Int. J. Hydrog. Energy

    (2010)
  • Y.C. Guo et al.

    Co-producing hydrogen and methane from higher-concentration of corn stalk by combining hydrogen fermentation and anaerobic digestion

    Int. J. Hydrog. Energy

    (2014)
  • M. Gupta et al.

    Co-fermentation of glucose, starch, and cellulose for mesophilic biohydrogen production

    Int. J. Hydrog. Energy

    (2014)
  • V. Juturu et al.

    Microbial cellulases: engineering, production and applications

    Renew. Sust. Energ. Rev.

    (2014)
  • W.C. Kuo et al.

    Bioaugmentation strategies to improve cellulolytic and hydrogen producing characteristics in CSTR intermittent fed with vegetable kitchen waste and napiergrass

    Energy Procedia

    (2012)
  • J. Liu et al.

    Evaluation of Clostridium ljungdahlii DSM 13528 reference genes in gene expression studies by qRT-PCR

    J. Biosci. Bioeng.

    (2013)
  • H. Lu et al.

    Effects of cellulose concentrations on the syntrophic interactions between Clostridium cellulovorans 743B and Rhodopseudomonas palustris CGA009 in coculture fermentation for biohydrogen production

    Int. J. Hydrog. Energy

    (2015)
  • H. Luo et al.

    Recent advances and strategies in process and strain engineering for the production of butyric acid by microbial fermentation

    Bioresour. Technol.

    (2018)
  • R.C.S. Mazareli et al.

    Bacillus sp. isolated from banana waste and analysis of metabolic pathways in acidogenic systems in hydrogen production

    J. Environ. Manag.

    (2019)
  • Y. Mu et al.

    Determining optimum conditions for hydrogen production from glucose by an anaerobic culture using response surface methodology (RSM)

    Int. J. Hydrog. Energy

    (2009)
  • H.J. Oh et al.

    Enhanced butyric acid production using mixed biomass of brown algae and rice straw by Clostridium tyrobutyricum ATCC25755

    Bioresour. Technol.

    (2019)
  • L. Ozkan et al.

    Effects of pretreatment methods on solubilization of beet-pulp and bio-hydrogen production yield

    Int. J. Hydrog. Energy

    (2011)
  • C.M. Pan et al.

    Statistical optimization of process parameters on biohydrogen production from glucose by Clostridium sp. Fanp2

    Bioresour. Technol.

    (2008)
  • E. Petitdemange et al.

    Effect of carbon sources on cellulase production by Clostridium cellulolyticum

    Biomass Bioenergy

    (1992)
  • R.S. Prakasham et al.

    Fermentative biohydrogen production by mixed anaerobic consortia: impact of glucose to xylose ratio

    Int. J. Hydrog. Energy

    (2009)
  • A. Pugazhendhi et al.

    Process performance of biohydrogen production using glucose at various HRTs and assessment of microbial dynamics variation via q-PCR

    Int. J. Hydrog. Energy

    (2017)
  • M. Quemeneur et al.

    Changes in hydrogenase genetic diversity and proteomic patterns in mixed-culture dark fermentation of mono- , di- and

    Int. J. Hydrog. Energy

    (2011)
  • S.C. Rabelo et al.

    Production of bioethanol, methane and heat from sugarcane bagasse in a biorefinery concept

    Bioresour. Technol.

    (2011)
  • C.A.B.S. Rabelo et al.

    Optimization of hydrogen and organic acids productions with autochthonous and allochthonous bacteria from sugarcane bagasse in batch reactors

    J. Environ. Manag.

    (2018)
  • U. Ramachandran et al.

    Hydrogen production and end-product synthesis patterns by Clostridium termitidis strain CT1112 in batch fermentation cultures with cellobiose or a-cellulose

    Int. J. Hydrog. Energy

    (2008)
  • R.P. Ratti et al.

    Thermophilic hydrogen production from sugarcane bagasse pretreated by steam explosion and alkaline delignification

    Int. J. Hydrog. Energy

    (2015)
  • N. Ren et al.

    Dark fermentation of xylose and glucose mix using isolated Thermoanaerobacterium thermosaccharolyticum W16

    Int. J. Hydrog. Energy

    (2008)
  • S. Shrestha et al.

    Biological strategies for enhanced hydrolysis of lignocellulosic biomass during anaerobic digestion: current status and future perspectives

    Bioresour. Technol.

    (2017)
  • K.M. Singh et al.

    Study of rumen metagenome community using qPCR under different diets

    Meta Gene

    (2014)
  • P. Talia et al.

    Biodiversity characterization of cellulolytic bacteria present on native Chaco soil by comparison of ribosomal RNA genes

    Res. Microbiol.

    (2012)
  • P. Tantayotai et al.

    Effect of cellulase-producing microbial consortium on biogas production from lignocellulosic biomass

    Energy Procedia

    (2017)
  • M. Wang et al.

    Monitoring dark hydrogen fermentation performance of indigenous Clostridium butyricum by hydrogenase gene expression using RT-PCR and qPCR 33

    (2008)
  • G. Yang et al.

    Enhancement of biohydrogen production from grass by ferrous ion and variation of microbial community

    Fuel

    (2018)
  • G. Yang et al.

    Microbial community diversity during fermentative hydrogen production inoculating various pretreated cultures

    Int. J. Hydrog. Energy

    (2019)
  • J.N. Zhang et al.

    Direct degradation of cellulosic biomass to bio-hydrogen from a newly isolated strain Clostridium sartagoforme FZ11

    Bioresour. Technol.

    (2015)
  • Cited by (19)

    View all citing articles on Scopus
    View full text