Microbe assisted depolymerization of lignin rich waste and its conversion to gaseous biofuel
Graphical abstract
Introduction
Increasing concern over depleting fossil fuel resources combined with the escalating focus towards scientific battle against the climate change as well as to meet the sustainability goals has led researchers towards biofuels development using biomass, especially agricultural residues, as feedstock (Khosravanipour et al., 2018; Liu et al., 2017; Shrestha et al., 2017). Though, many ways have been identified for the conversion of cellulose and hemicelluloses fractions of biomass into bioenergy/biofuels, still effective utilization of residual lignin remains a major challenge for the economic viability and commercial success of biomass based biofuel technologies (Lee, 1997; Gutiérrez et al., 2020; Diego-Díaz et al., 2021). Lignin makes about 15–30% of the biomass fractions by weight and 40% by energy. About 800 to 1000 million MTPA rice straw is produced globally in which about 80% is produced in Asia alone, while wheat straw has an annual global production of 529 million MTPA (Govumoni et al., 2013; Kapoor et al., 2016; Karimi et al., 2006; Vasiliki et al., 2017). Ethanol fermentation of rice straw produces about 0.55–0.58 MT residue/MT of straw and is rich in carbon and nutrient (Ishizaki and Hasumi, 2014). The intended development of 79 billion liters of second generation biofuels (lignocellulosic ethanol) per annum in the USA was estimated to generate about 62 million MT of lignin (Langholtz et al., 2014). A typical bioethanol plant based on corn stover with a production capacity of 2000 TPA, will produce about 70,000 TPA lignin rich residue (Davis et al., 2013), which shows the left over energy potential of biomass. The maize cob is one of the highest available lignocellulosic waste feedstock worldwide and it can be potential option for anaerobic digestion as such or along with other low nitrogen bearing lignocellulosic wastes (Surra et al., 2019). Though, the lignocellulosic ethanol production has been established long back at pilot scale, commercial success of these plants is hindered mainly due to leaving behind the energy rich lignin residue as waste. Integration of novel approaches that can valorize the lignin rich residues to the lignocellulosic ethanol plant will contribute significantly for the commercial success of these plants and also help in establishing circular biobased economy (Lee, 1997; Xu et al., 2019). This will also redefine the biorefinery based energy landscape through balancing between the energy demand and environmental catastrophe (Beckham et al., 2016). Among various approaches, anaerobic digestion is being considered as potential option to valorize the lignin rich residue that can significantly improve the economic viability of the biomass (agricultural residues) (Davis et al., 2013; de Gonzalo et al., 2016; Zhang et al., 2009).
Anaerobic digestion, also named as biomethanation, is a proven technology for waste management where the organic content of waste is converted to biogas through microbial action. Biomethanation/anaerobic digestion is carried out by synergistic association of a group of microbes in 4 different stages, viz., hydrolysis, acidogenesis, acetogenesis and methanogenesis (Fitzgerald, 2018; Kreuger et al., 2011; Yuan et al., 2019; Zhou et al., 2019). Anaerobic digestion has been studied well using wide range of waste/wastewater but reports on the biomethanation of lignin rich waste are limited (DeAngelis et al., 2011; Ko et al., 2009; Mulat et al., 2018). Depolymerization of recalcitrant, irregular, complex, and highly heterogeneous aromatic structured lignin is the major challenge for establishment of full scale process for anaerobic digestion of lignin waste (Pu et al., 2013; Ragauskas et al., 2014).
Several methods were reported for lignin depolymerization including thermo-chemical, hydrothermal (Belkheiri et al., 2018), acid hydrolysis (Liu et al., 2018), pyrolysis (Liu et al., 2008; Windt et al., 2009), hydrogenolysis (Huber et al., 2006), gasification (Huber et al., 2006), etc (Duan et al., 2016; Shi et al., 2013; Tamminen et al., 2010; Verma and Ekka, 2018). Each of these methods have their own limitations like cost and energy intensive nature, not environmentally benign and generating secondary waste which is again a major issue of disposal (Khan and Ahring, 2020; Mulat et al., 2018). Alternatively, biological methods based on fungi and enzymes were also attempted for lignin breakdown that are sustainable, eco-friendly as well as energy and cost effective in nature, and were extensively studied (Kong et al., 2017; Reiter et al., 2013; Vicuña et al., 1993; Wesenberg et al., 2003). But both the methods are lagging behind due to some limitations like requirement of reducing agents and recyclability in case of enzyme, slow reaction rates and difficulty in withstanding the extreme conditions in case of fungi. On contrary, the bacterial metabolic machinery was identified as robust and can withstand the extreme changes (pH variation, redox imbalance, concentrations of phenolics, etc.) in the media during breakdown of lignin residues and its metabolites due to the environmental adaptability and biochemical versatility of bacteria (Mulat et al., 2018). There are very limited studies in this direction of lignin breakdown and valorization. Understanding of bacterial metabolism for lignin breakdown followed by anaerobic digestion under controlled conditions can open potential applications beyond combustion of the lignin rich residue (Taherzadeh and Karimi, 2008). Some bacterial species that were reported for their efficiency of lignin depolymerization include, Streptomyces, Pseudomonas, Athrobacter, Acinetobacter, Rhodococcus etc., through extracellular enzymes (Beckham et al., 2016; Duan et al., 2016; Mulat et al., 2018; Susmel and Stefanon, 1993). Rhodococcus jostii RHA1 reported to act on β-O-4 linkages of lignin by secreting specific peroxidases (Sainsbury et al., 2015). R.jostii was reported for its robustness as it can withstand adverse conditions such as low nutrient, high cell density and has ability to tolerate toxic metabolites concentrations. Pseudomonas putida is another potential candidate for lignin degradation to low molecular weight chemicals (Lieder et al., 2015; Martínez-García et al., 2014). Other bacterial species like Bacillus sp. (Abd-Elsalam, 2009) Novosphingobium sp. (Chen et al., 2012), and Citrobacter freundii (Chandra and Bharagava, 2013), are also exhibited the lignin degradation abilities. However, use of enriched selective consortia for lignin depolymerization is need of the hour considering the valorization of lignin rich residue from bioethanol plant. The lignin rich streams obtained after ethanol production are heterogeneous and contain leftover enzymes, organic acids, fermentation intermediates, along with lignin based compounds which are resisting its direct use in many applications. A meritorious way for funnelling these lignin residues into target chemicals through microbial depolymerization for complete utilization of lignin is essential. This further provides an opportunity to completely utilize the biomass except its ash content for biofuel production. In this context, an attempt was made in this study with the following objectives:
- (i)
To evaluate the efficiency of selectively enriched microbial consortia for their ability to depolymerise lignin
- (ii)
Pretreatment of lignin rich residue with the enriched microbial consortia in comparison with hydrothermal and thermochemical methods was studied.
- (iii)
To establish the lignin depolymerization ability of selectively enriched consortia using commercial lignins, viz., alkali and de-alkali lignins.
- (iv)
To convert the depolymerized lignin rich residue into methane via anaerobic digestion
Section snippets
Substrate selection
Lignin rich residue (LRR) was obtained from operating bioreactor after ethanol fermentation of rice straw, through mild acid pretreatment followed by enzymatic hydrolysis. The LRR obtained as slurry and used after removing the residual alcohol content, if any, by heating on stirrer plate at 80 °C for 2 h. Alkali lignin (AL) and de-alkali lignin (DL) were procured from M/s TCI chemicals. All other chemicals used in this study were analytical grade and used as received.
Biocatalyst
Two different microbial
Characterization of substrates
The detailed characterization of each selected substrate was carried out prior to experimentation (Table 2). LRR showed high moisture (87.98%) and low TS (15.44%) content while AL and DL, showed significantly low moisture content (<15%) and high TS (80–90%). Hence, experiments with AL and DL were carried out at high dilution factors but on contrary, LRR based experiments conducted at 50% dilution only. Overall, the TS content for experiments was maintained about 4.0 ± 0.4 g/l. In addition to
Conclusions
Selectively enriched LDC depicted higher lignin depolymerization over corresponding hydro-thermal, acid and peroxide pretreatments, with commercial lignins as well as the lignin rich residue obtained from bioethanol plant. Peroxide treatment was more effective than hydro-thermal and acid pretreatment, while LDC dominated all other pretreatment methods in lignin depolymerization. All the substrates resulted in higher biogas yields after pretreatment with LDC along with significantly higher
Credit author statement
Ms. N.L. Radhika: Involved in conducting experiments, data generation & compilation, manuscript preparation and corrections. Prof. Sarita Sachdeva: Involved in manuscript corrections and finalization. Dr. Manoj Kumar: Involved in experimental planning, data interpretation, manuscript corrections and finalization.
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
NLR thank Indian Oil R&D for providing the lab space and facility to carryout the research.
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