Sequential and consolidated bioprocessing of biogenic municipal solid waste: A strategic pairing of thermophilic anaerobe and mesophilic microaerobe for ethanol production

https://doi.org/10.1016/j.biortech.2020.123260Get rights and content

Highlights

  • Sequential bioprocessing of municipal solid waste led to 0.26 g ethanol/g waste.

  • Positive microbial pairing of thermophilic anaerobe with mesophilic microaerobe.

  • Exogenous xylanase supplementation augmented ethanol titre by 1.53-folds.

  • Hexoses and pentoses are fermented to ethanol achieving 73.16% theoretical maxima.

Abstract

Feedstock availability and its pretreatment, high process economics and insufficient ethanol (HEt) titres necessitated the bioprocesses that are sustainable. The advanced consolidated bioprocessing (CBPSeq) strategy presently considered for improved HEt production involves, sequential coupling of CBP thermophile, Clostridium thermocellum ATCC-27405 with mesophilic microaerobe, Pichia stipitis NCIM-3498. Biogenic municipal solid waste (BMSW) pretreated with 0.5% NaOH (CSPBMSW) served as the sole carbon source. CBPSeq (23.99 g/L) fared better than CBP standalone (18.10 g/L) wherein 1.32-folds improvement in HEt titre was recorded. Considering insufficient xylanase titre in cellulosome complex of C. thermocellum, CBPSeq was performed employing exogenous xylanases (CBPSeqE) to improve xylan digestibility and HEt yield. CBPSeqE-II biosystem at pH 5 showed maximum HEt titre of 36.90 g/L which corresponds to yield of 0.26 g HEt/ g CSPBMSW. This study substantiates efficacy of CBPSeqE-II biosystem in sustainable bioethanol production from BMSW in a single reactor without laborious steps.

Introduction

Biogenic municipal solid waste (BMSW) is a sustainable and a global feedstock available to combat the escalating world energy demand in today’s time. The organic fraction of municipal solid waste also referred as the biogenic stream accounts to >50% of the total solid waste generated (Venkata Mohan et al., 2016). BMSW is a potential resource for biofuels and platform chemicals production due to its energy rich composition. It is mainly composed of carbohydrates (starch, cellulose and hemicellulose), proteins and lipids apart from lignin, tannin, fibre and pectin. The individual percentage of these high heating value biomolecules in BMSW is influenced by the waste collection site, urbanization rate and socio-economic status. The effective management of BMSW is challenging with the existing waste management and treatment technologies due to its burgeoning quantity. The traditionally followed landfill approach for BMSW management causes decomposition of the biogenic stream that releases greenhouse gases (GHGs) (Venkata Mohan et al., 2020). On the contrary, utilization of BMSW for ethanol (HEt) generation can curb the GHG emissions and high waste disposal costs apart from the added benefits of waste treatment and value addition. Biofuel production from BMSW as per EU Renewable Energy Directive (RED) I policy is reported to reduce GHGs by 9% relative to gasoline whereas under the US Energy Independence and Security Act (EISA) and California Low Carbon Fuel Standards (LCFS) policies, GHG reduction can be as high as 700% (Meng and McKechnie, 2019).

The harvest of embodied energy in BMSW is impeded by its high recalcitrance arising mainly from lignin which suggests an effective pretreatment is vital to recover maximum energy. Different pretreatment techniques grouped under physical, chemical, thermal, microbial and enzymatic treatment methods and their combinations thereof are widely being practised for depolymerizing lignin matrix and loosening of integrated carbohydrate reserve (Althuri et al., 2017, Kumar et al., 2019). Mechanical and radiation driven methods are categorized under physical pretreatment strategies. The methods that are operative at commercial scale involve ball milling and microwave irradiation while ultrasound treatment, gamma ray irradiation, electron beam irradiation and pulsed electric energy are still being explored at laboratory stage. These majorly cause the reduction in particle size and increase in surface area apart from reducing the cellulose crystallinity. Chemical based approaches include treatment using acid, alkali, organic solvents, deep eutectic solvents, ozone, H2O2, ionic liquids, sodium benzoate and metal salts. Acid based pretreatment is generally carried out using HCl, H2SO4 and H3PO4 as catalysts that mainly trigger the hydrolysis of hemicellulose portion to sugar monomers consequently increase biomass porosity. Alkali pretreatment using NaOH, Ca(OH)2 and NH3 on the other hand result in depolymerization of lignin and cleave the linkages (ester, aryl–ether, C–C bond) amidst lignin and carbohydrate fraction. Further, microbial and enzymatic pretreatment approaches have gained increased attention due to lower inhibitor formation and high sugar yields, however, enzyme cost and longer microbial reaction time are continuing to be restraining factors. Thus, the pretreatment method adopted significantly impacts the overall cost and reactor configuration of waste biorefinery (Kumar et al., 2020). The cost of pretreatment accounts to 18–20% of the total incurred cost for bioethanol production. The lignin recalcitrance of BMSW was dealt in this study using alkali pretreatment.

Further, the rate limiting step of bioethanol production process is the enzyme mediated hydrolysis of the carbohydrate pool present in waste to reducing sugars which is commonly referred as saccharification process. The high cost of commercially available hydrolysing enzymes is the major limiting factor that is found to negatively influence the minimum ethanol selling price (MeSP). Besides, the concluding step focussed on the fermentation of reducing sugars to HEt is also a key determining factor of MeSP (Althuri et al., 2016 & 2017). Thus the probe for low-cost technologies is still continuing to enable production of bioethanol at comparable cost as commercial ethanol. In line with this, recently single pot bioprocessing (SPB) approach otherwise known as consolidated bioprocessing (CBP) has emerged wherein, the three challenging steps of HEt production are carried out in a single reactor under same conditions. This can strategically subdue the capital expenditure (CAPEX) and operating expenditure (OPEX) of the bioprocess along with minimizing associated labour and process time (Althuri and Venkata Mohan, 2019).

CBP can be driven either by HEt producing aerobic strains supplemented with exogenous hydrolysing enzymes (Althuri and Venkata Mohan, 2019) or by anaerobic strains that are both cellulolytic and ethanologenic in nature (Singh et al., 2019). The latter approach is considered as a true CBP method and the microbe as a model CBP candidate. The cellulose degrading trait of anaerobic bacteria, namely C. thermocellum considered in this study, is owing to their ability to produce an efficient multi-enzyme system termed ‘cellulosome complex’ specialized in crystalline cellulose depolymerisation to glucose which in some cases is comparable to the degree of hydrolysis mediated by commercial enzymes (Lynd et al., 2002). C. thermocellum, an anaerobic Gram positive thermophile, is advantageous for HEt generation through CBP due to its sustainability and functionality at higher temperature that is generally considered optimum for the action of carbohydrate hydrolysing enzymes. Moreover, these cellulolytic thermophiles evade contamination and coalesce with the well-established process streams without major modification in the unit operation (Akinosho et al., 2014). The growth of C. thermocellum is detected on several carbon sources such as glucose, cellobiose, carboxymethyl cellulose, Avicel (Sato et al., 1993) and microcrystalline cellulose (Singh et al., 2018). Most of the HEt research done on Clostridium thermocellum as a model CBP strain is limited to commercial cellulose and its variant forms as the substrate but a detailed study on bioconversion of recalcitrant BMSW is not reported. Thus this study examines the amenability of BMSW as a carbon source for C. thermocellum ATCC-27405 growth and HEt production. Further, considering the lower HEt tolerance of C. thermocellum (Herrero and Gomez, 1980) attempt was made to develop a CBP strain with improved ethanol tolerance. Moreover, this strain was assessed for composition of its hydrolytic enzyme complex and HEt production ability on a variety of carbon sources.

The by-products usually detected during CBP along with the mainstream HEt include short chain carboxylic acids such as acetic acid (HAc), lactic acid (HLa) and formic acid (HFo). These organic acids can have either inhibitory or stimulatory effect (He et al., 2009) on HEt production ability of the CBP bacterium. To decipher the possible influence of the major by-product of fermentation i.e., acetic acid on C. thermocellum HEt titre, the effect of exogenous sodium acetate at varying concentrations was studied along with deducing the ambient reaction conditions for maximum bioethanol production from BMSW through CBP.

The incompetence of C. thermocellum to utilize pentose (C5) sugars is the major limiting factor that impedes its industrial utility for HEt production (Singh et al., 2017). On the other hand, for commercial viability of BMSW derived HEt, C5 sugar utilization along with the mainstream glucose (C6) moieties is indispensable. The total sugar fermentation significantly enhances HEt yield and directly improves the economics of the process (Althuri et al., 2016). Sequential CBP (CBPSeq) is attempted in this study as an alternative strategy to enhance HEt titre wherein the residual pentose (C5) rich stream obtained after C. thermocellum mediated CBP was inoculated with C5 fermenting yeast, Pichia stipitis NCIM-3498 for additional HEt production. Also, the effect of exogenous addition of hydrolytic enzyme cocktail (mainly dominated by xylanases) on the CBPSeq biosystems (referred as CBPSeqE-I-III) was evaluated to comprehend the suitable process for maximum HEt production from BMSW. Thus the novelty of this study lies in the sequential CBP of BMSW using ethanol tolerant cellulolytic and ethanologenic anaerobes, coupled sequentially with mesophilic microaerobes, in presence of exogenous xylanases, for maximum carbohydrate hydrolysis to C6 and C5 sugars and subsequent fermentation to HEt. The prominence of the CBPSeqE process is that it can be conducted in a single reactor without the need for solid–liquid separation thereby can reduce capital and operational costs. This CBPSeqE strategy can significantly improve HEt production from BMSW when compared to CBP standalone in a sustainable and eco-friendly manner.

Section snippets

Carbon source

BMSW pulverized sample with particle size ≤0.2 mm was prepared as per our previous report (Althuri and Venkata Mohan, 2019). The test sample consisted of powdered food and vegetable residues, yard trimmings, waste newspaper, cardboard waste, textile and wood chips in definite proportions. The total carbohydrate content was 65.95% (w/w) while total lignin content was found to be 22.22% (w/w).

Anaerobic inoculum

Clostridium thermocellum ATCC-27405 was procured from American Type Culture Collection (ATCC), USA and

Effect of NaOH concentration on acid soluble lignin (ASL) and acid insoluble lignin (AIL) of BMSW

Alkali pretreatment using NaOH at mild concentration is a potential method for cleaving the linkages amidst lignin and the carbohydrate portions of organic waste mediated by salvation and saponification reactions. This results in increased porosity of biomass, cellulose swelling and reduced cellulose crystallinity. The rationale for evaluating different NaOH concentrations is to arrive at a mild concentration sufficient to depolymerize lignin. Moreover, relative to acid and hydrothermal

CRediT authorship contribution statement

Avanthi Althuri: Conceptualization, Methodology, Data curation, Writing - original draft, Investigation, Validation, Funding acquisition. S. Venkata Mohan: Visualization, Supervision, Writing - review & editing, Resources, Project administration, Funding acquisition.

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

AA sincerely thanks Council of Scientific & Industrial Research (CSIR), India for financial assistance in the form of CSIR-Nehru Science Postdoctoral Fellowship (HRDG/CSIR-Nehru PDF/LS/EMR-I/04/2018). Work is supported by Mission Mode project (Waste to Wealth-MLP 0042) funded by CSIR, India under Ecology, Environment & Ocean Sciences & Water (E3OW) theme. Authors acknowledge the Director, CSIR-IICT for kind support in carrying out this work (IICT/Pubs./2020/032).

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