Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21

Accepted Manuscript Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21 Nisha Singh, Munish Puri, Deepak K. Tuli, Ravi P. Gupta, Colin J. Barrow, Anshu S. Mathur PII:

S1075-9964(18)30071-4

DOI:

10.1016/j.anaerobe.2018.04.014

Reference:

YANAE 1877

To appear in:

Anaerobe

Received Date: 25 September 2017 Revised Date:

22 April 2018

Accepted Date: 24 April 2018

Please cite this article as: Singh N, Puri M, Tuli DK, Gupta RP, Barrow CJ, Mathur AS, Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21, Anaerobe (2018), doi: 10.1016/j.anaerobe.2018.04.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Manuscript for Anaerobe Journal

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Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-

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IOC-DC21

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Nisha Singha,b, Munish Puria, Deepak K Tulib, Ravi. P. Guptab, Colin J Barrowa, and Anshu S

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Mathurb*

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a

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Australia.

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Centre for Chemistry and Biotechnology, Waurn Ponds, Deakin University, Victoria 3217,

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b

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Oil Corporation Limited, Sector-13, Faridabad 121007, India

DBT-IOC Centre for Advance Bioenergy Research, Research & Development Centre, Indian

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Email addresses of all authors: [email protected], [email protected],

[email protected],

[email protected],

[email protected], [email protected]

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*Corresponding author

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Dr. A. S. Mathur

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DBT-IOC Centre for Advance Bioenergy Research, Research & Development Centre, Indian Oil

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Corporation Limited, Sector-13, Faridabad, India

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Tel: +91-129-2294583; Fax: +91-129-2286221

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Email address: [email protected]

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Abstract

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To overcome the challenges associated with combined bioprocessing of lignocellulosic biomass

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to biofuel, finding good organisms is essential. An ethanol producing bacteria DBT-IOC-DC21

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was isolated from a compost site via preliminary enrichment culture on a pure hemicellulosic

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substrate and identified as a Clostridium strain by 16S rRNA analysis. This strain presented

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broad substrate spectrum with ethanol, acetate, lactate, and hydrogen as the primary metabolic

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end products. The optimum conditions for ethanol production were found to be an initial pH of

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7.0, a temperature of 70 °C and an L-G ratio of 0.67. Strain presented preferential hemicellulose

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fermentation when compared to various substrates and maximum ethanol concentration of 26.61

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mM and 43.63 mM was produced from xylan and xylose, respectively. During the fermentation

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of varying concentration of xylan, a substantial amount of ethanol ranging from 25.27 mM to

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67.29 mM was produced. An increased ethanol concentration of 40.22 mM was produced from a

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mixture of cellulose and xylan, with a significant effect observed on metabolic flux distribution.

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The optimum conditions were used to produce ethanol from 28 g L-1 rice straw biomass (RSB)

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(equivalent to 5.7 g L-1 of the xylose equivalents) in which 19.48 mM ethanol production was

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achieved. Thus, Clostridium strain DBT-IOC-DC21 has the potential to perform direct microbial

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conversion of untreated RSB to ethanol at a yield comparative to xylan fermentation.

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Keywords: Thermoanaerobe, Bioethanol, Xylan fermentation, Rice straw biomass, Consolidated

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bioprocessing 2

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1. Introduction

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With the current state of the art, the processing of lignocellulosic biomass to bioethanol (also

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known as second generation bioethanol) will become an alternative replacement for currently

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used liquid transportation fuels, if produced in a cost-competitive manner [1]. Lignocellulosic

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biomass feedstocks (woody substrates, forestry residues, and agricultural waste etc.) is composed

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of various fermentable carbohydrates in the form of cellulose and hemicellulose [2]. In recent

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years, considerable research has focused on the bioconversion of crystalline cellulose to ethanol;

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less attention has been paid to the bioconversion of hemicellulose (the second most abundant

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polysaccharides of lignocellulose). Hemicellulose accounts for nearly 30% of the lignocellulosic

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biomass (usually separated from cellulose during various pretreatment processes) and could be a

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useable biomass product for the production of bioethanol by microbial processes [3].

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Furthermore, when considering the overall economics of a process, the effective conversion of

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the hemicellulosic fraction will be an obvious advantage to obtain viable ethanol production [4,

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5].

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Hemicellulose is a category of hetero-polysaccharides containing a variety of glycosidic linkages

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composed of hexoses, pentoses, and occasionally methylpentoses and uronic acids; many

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hemicelluloses are also acetylated with organic acid residues such as ferulic acid or p-coumaric

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acid [4]. Thus, during the breakdown of hemicellulose, a plethora of sugars and other products

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are generated apart from the monomers and oligomers (xylose, glucose, xylooligosaccharides,

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arabinose, and galactose) [6]. Although hemicellulose hydrolysis is easier compared to that of

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crystalline cellulose, direct microbial fermentation of hemicellulose is limited [7-13]. The

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complete hydrolysis of hemicellulose requires cooperative action of a large variety of hydrolytic

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enzymes such as, endo-1,4- β -xylanase, acetyl esterase, α-glucuronidase, and β-xylosidase,

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having different modes of action [14]. In addition some accessory enzymes such as xylan

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esterases, ferulic and p-coumaric esterases, α-L-arabinofuranosidases, and a-4-O-methyl

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glucuronosidases etc. are also required. Very few bacteria, fungi, and yeast that can degrade

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hemicellulose to monosaccharides and yield ethanol as a fermentation product are known [15].

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Therefore, isolation and development of novel microbial strains capable of efficient ethanol

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production from hemicellulose are essential for the development of an industrial biomass

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conversion process.

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The cost-effective production of bioethanol from lignocellulosic feedstocks requires improved

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approaches of biomass processing. The conventional approach of biomass processing termed as

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separate hydrolysis and fermentation (SHF) has four main steps: biomass pretreatment,

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production of lignocellulolytic enzymes, enzymatic hydrolysis, and fermentation of the resulting

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hexose and pentose sugars to ethanol, makes this process undesirable from the economic point of

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view [2]. In the last decade, progressive research mounted to replace this conventional approach

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by more integrated bioprocessing approaches like simultaneous saccharification and

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fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF) that aim to

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circumvent main cost-increasing elements by combining one or more steps in a single reactor

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[16]. However, in all of these mentioned approaches, the economic burden offered by the

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exogenous addition of costly cellulolytic enzymes is still formidable. A consolidated

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bioprocessing (CBP) approach offers the potential to lower the cost by combining all the

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individual steps of biomass processing into a single step when combined with a proper CBP

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candidate microbe having specific traits [1, 16].

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In past few decades, anaerobes thriving under high-temperature conditions have gained special

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interest for bioethanol production via CBP due to several process benefits such as improved

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substrate solubility and higher conversion rates at a higher temperature, broad substrate

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spectrum, facilitated product recovery, and preclusion of contaminants etc. [1]. The major

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advantage of using thermoanaerobes for CBP is their natural ability to ferment a range of

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carbohydrates commonly found in lignocellulosic biomass and production of thermostable

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cellulases and xylanases [3]. Few thermoanaerobes such as Clostridium thermocellum and

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cellulolytic Caldicellulosiruptor species such as Caldicellulosiruptor bescii and

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Caldicellulosiruptor saccharolyticus were found to be extremely efficient hydrolyzer of

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crystalline cellulose [17-19]. However, substantial strain improvement efforts are underway to

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match the ethanol production level of these bacteria with conventional sugar fermenting

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ethanologens such as; Saccharomyces cerevisiae or Zymomonas mobilis [20]. In contrast to

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cellulose, fermentation of hemicellulose to bioethanol by thermophilic anaerobic bacteria has not

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been fully explored and is a subject of active research. Among wild-type thermophilic anaerobic

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bacteria previously isolated different members of the genera; Caldicellulosiruptor, Clostridium,

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Thermoanaerobacter, and Thermoanaerobacterium have been shown to utilize xylan, and only

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few can perform xylan fermentation to ethanol, albeit at very slow rates and lower yield [1, 7, 8,

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10, 11, 20].

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The aim of present study was to isolate and characterize a potential thermoanaerobe, which can

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produce bioethanol directly from the hemicellulosic fraction of lignocellulosic biomass. A

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composting facility in Northern India was chosen for isolating such bacteria using selective

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enrichment approach. Xylan is the most abundant hemicellulosic polysaccharides in plant cell

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wall. In this study, branched and substituted xylan derived from beechwood (Sigma Aldrich,

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India) was used as a pure hemicellulosic substrate, which upon degradation produces xylose and

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arabinose (Sigma information). Rice straw biomass (RSB) is one of the most important

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feedstocks available in India for second-generation bioethanol production with a potential to

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produce 2.1 billion liters of ethanol annually [21]. Here, the single step conversion of the high

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and low concentration of untreated rice straw biomass (UTRSB) was conducted to highlight the

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ability of the new isolate to grow and produce ethanol from real substrates via consolidated

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bioprocessing. So far, only a few studies have been conducted with thermophilic anaerobic

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ethanol producers on untreated lignocellulosic biomass [22-24].

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In addition, comparative fermentation performances of the strain on various carbohydrates

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commonly found in the lignocellulose and their mixture were explored. We also compared the

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substrate utilization spectrum of the newly isolated strain with previously described xylan

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fermenting thermophilic anaerobes, in order to obtain a deeper insight into their contrasting

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characteristics. Process parameters such as pH, temperature, an initial concentration of xylan and

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liquid to headspace ratio were optimized to achieve best ethanol titers. This study suggests the

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importance of both RSB as a substrate and fermenting strains as a potential CBP candidate for

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their future applicability for bioethanol production.

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2. Materials and methods

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2.1. Chemicals

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Powdered cellulosic substrate; Avicel PH-101 (Cat. no. 11365) and the hemicellulosic substrate;

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Xylan from beechwood (Cat. no. X4252) were procured from Sigma Aldrich (Bengaluru, KA,

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India). All other chemicals used in this study were purchased from Himedia Laboratories, India.

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All gases used were high purity and purchased from Inox Air Products Limited, India.

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2.2. Sampling site

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Samples were collected from a composting facility at Lucknow (Uttar Pradesh), North India. The

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compost material was rich in food waste, animal waste, manure and dead plant material. Samples

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collected from the middle of the compost heap (with temperature ranging from 45-60 °C) in

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Anaero Bag system (LE007, Himedia Laboratories, India) followed by simultaneously placing a

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cut opened Anaerobic Gas Pack (LE002A, Himedia Laboratories, India) and Anaerobic indicator

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tablet (LE065, Himedia Laboratories, India). The Anaero Bag was sealed immediately with the

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clamp provided to ensure anaerobic conditions. Samples were transported to laboratory and

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processed immediately.

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2.3. Growth medium and inoculum preparation

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The composition of chemically defined anaerobic medium M (pH 7.0) used throughout this study

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was in accordance to [25] and prepared under strict anaerobic conditions inside a Coy anaerobic

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chamber (USA) having headspace of N2: CO2: H2 = 90:5:5, as previously described [26]. All the

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insoluble carbon sources including complex polysaccharides and RSB were supplemented to 50

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mL M medium prior to sterilization (121 ºC for 15 min), at a concentration specified in the text,

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unless otherwise specified. Concentrated stocks of vitamins and sugar solutions were sterilized

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by filtration using 0.22 µm filters and added to the pre-autoclaved anaerobic bottles. An

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anaerobic sterile stock solution containing L-cysteine HCl as reducing agent was used for the

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preparation of pre-reduced medium and added to the autoclaved medium just before inoculation.

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The final pH of the medium was adjusted using the sterile anaerobic stock solutions of 1N HCl

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and 1N NaOH.

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Inoculum for each experiment set was prepared by sub-culturing frozen glycerol stock of single

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colony of isolate grown on M media (50 mL, pH 7.0) containing 5 g L-1 xylose and incubated at

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70 °C for 24 h without shaking. A log-phase culture (OD600 ~ 0.8-1.0) obtained after three such

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repeated transfers were used as seed at 10 % (v/v) inoculum size. All experiments were

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performed in triplicates and duplicate controls unless otherwise noted.

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2.4. Isolation and identification of hemicellulose-fermenting thermophilic anaerobic

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bacteria

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Standard anaerobic culture techniques were used for enrichment and subsequent studies.

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Compost samples (10 %, w/v) were inoculated into serum bottles (125 mL, Wheaton)

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containing 50 mL M medium (pH 7.0). The medium was supplemented with 5 g L-1 xylan

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as the sole carbon source to selectively enrich hemicellulose-fermenting thermophilic

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anaerobic bacteria. Enrichment cultures were incubated at 70 °C without shaking till

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positive growth was evidenced in the form of pH drop and gas production. Single

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colonies from the positive enrichment cultures were purified using Hungate roll tube

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technique of culture purification, in accordance with the procedure published earlier [26].

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Briefly, the stable enrichment culture responsible for maximum ethanol production was

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transferred from medium containing 5 g L-1 xylan to roll-tubes with solid medium

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containing 5 g L-1 xylose. Colonies in the roll-tubes were picked and transferred back to

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liquid medium with xylan and incubated till growth observed. The same procedure was

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repeated several times till single colony forms were predominant.

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One of the bacterial strains designated as DBT-IOC-DC21 purified in this study,

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presented maximum ethanol production during xylan fermentation and selected for

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further studies. The culture was checked for purity by microscopy and 16S rRNA gene

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sequencing. The pure isolate was preserved under 30 % deoxygenated glycerol at -80 ºC

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and revived before each experiment.

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The genomic DNA of the strain DBT-IOC-DC21 was isolated using DNeasy blood and

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tissue kit (Qiagen India Pvt. Ltd), following the manufacturer’s instructions. The 16S

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rRNA was amplified by polymerase chain reaction, in accordance with the conditions

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described previously [26] and sequenced by Institute of Microbial Technology,

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Chandigarh, India. The obtained gene sequence was analyzed for evolutionary history and

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a phylogenetic tree was constructed, as described previously [26] and references therein.

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2.5. Chemotaxonomic characterization of the new isolate

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The Gram reaction of exponentially growing cells was performed using a Gram staining kit

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(Himedia Laboratories, India). Growth optimization for pH and temperature conditions were

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conducted in triplicates, as described previously [26] except 5 g L-1 xylose was used as the sole

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carbon source. The substrate utilization spectrum for strain DBT-IOC-DC21 was tested using

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various soluble (cellobiose, glucose, xylose, arabinose, mannose, galactose, fructose, maltose,

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lactose, carboxymethyl cellulose sodium salt, and sucrose) and insoluble (starch, Whatman no. 1

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filter paper, xylan, microcrystalline cellulose; Avicel PH-101, washed native and dilute acid pre-

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treated RSB) carbon sources supplemented to Hungate culture tubes (16 × 125 mm, Bellco glass,

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India) containing 10 mL M medium and incubated under respective optimum conditions for 48-

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96 h. Cultures that exhibited gas production, decrease in pH and increase in optical density at

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600 nm (OD600) compared with the control culture were considered positive for substrate

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utilization. The controls were the uninoculated culture containing same carbon source.

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2.6. Effect of initial ethanol concentration

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The effect of ethanol on cell growth and ethanol production from xylose and xylan (5 g L-1) was

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tested in triplicates with 50 mL M medium containing ethanol at a concentration ranging from 0

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to 5% (v/v) in 0.5% increments and 5 mL (10 % v/v) of the strain DBT-IOC-DC21 suspension at

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exponential phase (OD600 ~ 0.8-1.0). Inoculated bottles were incubated under the optimized

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growth conditions for 48 h (xylose) and 96 h (xylan).

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2.7. Effect of initial xylan concentration

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Influence of different initial concentration of xylan on cell growth and metabolic end products

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was also investigated in triplicates by this strain in 50 mL M medium containing 5, 10, 15, 20, 25

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and 30 g L-1 xylan and incubated under optimum conditions for 96 h. After incubation,

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fermentation products produced by strain DBT-IOC-DC21 were determined, as described in

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analytical methods.

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2.8. Ethanol production from xylan at varying liquid-to-headspace ratio

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The impact of liquid-to-headspace ratio on xylan fermentation was investigated in 125 mL serum

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bottles containing varying working volumes of M medium (pH 7.0), ranging from 10.0 mL to

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90.0 mL. Each experiment set was performed in triplicates and inoculated with an exponentially

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growing culture of the strain DBT-IOC-DC21 followed by incubation at 70 ºC without shaking.

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Uninoculated control with carbon source was included as negative control. Samples were

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collected after 96 h for the determination of growth, final pH, residual sugars and soluble

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metabolic end products. The optimized liquid-to-headspace ratio determined was thereafter used

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in all experiments performed.

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2.9. Ethanol production from simple sugars, amorphous cellulose, cellulose, xylan, a

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mixture of cellulose and xylan

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Batch experiments were carried out in triplicates to test the fermentation performance of

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Clostridium strain DBT-IOC-DC21 using different carbon sources such as monosaccharides

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(glucose, xylose, cellobiose, and arabinose) and complex polysaccharides (amorphous cellulose,

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cellulose, xylan, and mixtures thereof), under the optimized conditions. The ‘Phosphoric-acid-

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swollen-cellulose’ (PASC) was used as the amorphous cellulosic substrate and was prepared by

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solubilizing cellulose in concentrated phosphoric acid and prepared as described previously [27].

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All carbon sources were added at a final loading of 5.55 g L-1 of the glucose/xylose equivalents

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unless otherwise specified. Samples were collected at the end of incubation for the determination

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of growth, final pH, substrate conversion (%) and soluble metabolic end products.

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2.10. Ethanol production from native rice straw biomass under optimum conditions

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Rice straw (Oryza sativa) was used as the lignocellulosic substrate for fermentation studies and

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was collected from the local market in Mathura district, Uttar Pradesh, India. The biomass was

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air dried at room temperature for 48 h and shredded to the particle size ∼4-5 mm by knife mill

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(Texol, Pune, India). Prior to use, rice straw was washed with water at 70 °C (the growth

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temperature of Clostridium strain DBT-IOC-DC21) for 24 h, to remove the soluble sugars that

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are likely to be present in native biomass. The collected material was dried and stored in air-

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sealed containers at 25 °C. A single lot of this substrate was used throughout this study.

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Washed native RSB was supplemented in triplicate serum bottles containing M medium and

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10% (v/v) inoculum of freshly grown culture. Bottles were incubated under the optimized

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conditions (pH 7.0, temperature 70 ºC, L-G ratio of 0.67 and 0.5- 1 % substrate loadings). Native

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RSB was added at a final loading of 5.5 g L-1 and 11 g L-1 of the xylose equivalents. Samples

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were collected after 96 h for the analysis of different parameters such as biomass solubilization

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(%), final pH, residual sugars and soluble metabolic end products. In this study, a series of

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standard protocols developed by National Renewable Energy Laboratory (NREL) were followed

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to determine compositional analysis and moisture content of the biomass samples [28, 29].

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2.11. Analytical methods

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Cell growth of Clostridium strain DBT-IOC-DC21 was determined by measuring optical

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density at 600 nm (OD600) using UV-visible spectrophotometer (UV-2450, Shimadzu,

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Japan). Initial and final pH of the culture supernatant (prepared by centrifugation at 12000

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rpm for 10 min.) was measured by pH meter (Mettler-Toledo, India). The culture

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supernatant was analyzed for residual carbohydrates (glucose, cellobiose, xylose, and

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arabinose), soluble metabolites (lactate and acetate) and inhibitors (furfural and 5-HMF)

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using High-Performance Liquid Chromatograph (HPLC) (Waters Corp. USA), following

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the same operating conditions as described previously [26].

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Ethanol was estimated using Clarus-680 Gas Chromatograph (Perkin-Elmer, USA)

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equipped with Stabilwax®-DA column (Restek), according to the operating conditions

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described previously [26]. Peak identification was performed by comparison of retention

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time with the standard area and calibration curve (the R2 value close to 0.999) was

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derived from the stock solution of each component.

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Hydrogen in the headspace of serum bottles was identified by using a Refinery Gas

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Analyser (RGA) (Agilent 6890N, USA) equipped with thermal conductivity detector

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(TCD) and a 2-meter-long molecular sieve packed column with 2 mm ID (Nucon, India).

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Nitrogen (flow rate; 40 mL min-1), was used as carrier gas. RGA was operated at 50, 100,

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and 250°C, temperatures for the oven, inlet port, and detector, respectively. The gas

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samples from serum bottles (cooled at room temperature) were collected using water

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displacement method through a sterile needle as described previously [30]. For each time

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point of analysis, separate serum bottle was sacrificed. The amount of gas (mL) produced

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was calculated from the gas composition percentage provided by the instrument’s inbuilt

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software.

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Residual biomass concentration was determined by the gravimetrical determination as

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described previously [26]. The rate of substrate conversion was calculated by estimating

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consumed sugar or sugar equivalents and expressed as the percentage (%). Soluble

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metabolite yield reported as mM was calculated using a molecular weight of 46.07 g mol-

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metabolite yield as moles of ethanol produced per mole of consumed sugar equivalents

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was calculated using the theoretical maximum equation.

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2.12. Statistical analysis

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Statistical analysis of the difference between the concentration of soluble metabolites,

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under various treatment conditions described, was performed by one-way analysis of

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variance (ANOVA) followed by Tukey’s honest significant difference (HSD) post-hoc

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tests using R-Studio®, version 1.0.136 (RStudio, Inc. Boston, MA) and differences

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considered significant at probability value less than 0.05 (p<0.05).

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for ethanol, 60.05 g mol-1 for acetate and 90.08 g mol-1 for lactate. Furthermore,

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3. Results and discussion

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3.1. Isolation and phylogenetic characterization

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The enrichment and isolation procedure yielded eight extremely thermophilic, Gram-negative,

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obligatory anaerobic bacteria for a process of direct conversion of xylan to ethanol (Table S1).

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The obtained pure colonies were round, white in colour, non-pigmented, and some of them

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formed small gas bubbles. Higher ethanol and total soluble metabolite concentrations were used

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as initial screening criteria to select the isolate best suited for hemicellulosic ethanol production.

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A comparative fermentation profiling of 8 pure cultures led to the identification of a strain,

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which we have designated as DBT-IOC-DC21, that rapidly grew on xylan and accumulated

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maximum observed ethanol (25.55 mM) and hydrogen (31.11 mmol L-1) during the fermentation

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of xylan (Table S1), thus investigated in detail in this study.

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The sequence similarity analysis of the strain DBT-IOC-DC21 with other strains available in

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GenBank database revealed its affiliation to the genus Clostridium and their evolutionary history

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is presented as a phylogenetic tree in Fig. 1. The genus Clostridium (Phylum Firmicutes)

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comprises a group of several thermophilic and solventogenic Clostridia species that are capable

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of direct fermentation of lignocellulose to fuels such as alcohol and hydrogen [31]. However,

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most solventogenic Clostridium strains still could not efficiently utilize xylan and costly

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pretreatment or enzyme treatment is necessary to produce hemicellulosic hydrolysate for

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subsequent fermentation of released sugars. Thus, the relevance of the genus Clostridium for

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direct ethanol production has not been fully explored for utilizing xylan, which is the main

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essence of this study. No enzymes were added at any stage of fermentation, emphasizing the

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effectiveness of the newly isolated strain.

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As shown in Fig. 1, strain DBT-IOC-DC21 was most closely related to Cl. stercorarium subsp.

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leptospartum strain DSM 9219T (formerly known as Thermobacteroides leptospartum) [32, 33]

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and Cl. stercorarium subsp. stercorarium strain DSM 8532T [33, 34], with 16S rRNA gene

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sequence similarities of 100% and 99%, respectively. Cl. stercorarium is a thermophilic

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anaerobe that is ubiquitously present in soil and environment where plant biomass degradation

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occurs [35]. Cl. stercorarium is also known for its range of xylanolytic enzymes produced and

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modest fermentation of cellulose compared to hemicellulose. The high genetic relatedness of the

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new isolate with different subspecies of Cl. stercorarium and cellulose utilization ability

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indicated that strain DBT-IOC-DC21 belongs to group III of Clostridia [36]. The 16S rRNA

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gene sequences of Clostridium strain DBT-IOC-D21 have been deposited in GenBank under the

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nucleotide accession number KX842077.

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3.2. Growth optimization

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The strain was unable to grow in the presence of oxygen endorsing its obligatory anaerobic

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nature. The growth temperature for strain DBT-IOC-DC21 ranged from 50-75°C, and the

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optimal temperature (Topt) for the growth was 70°C (Figure S1a) on xylose as a substrate. No

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significant growth was observed below 50°C and above 75°C defining its thermophilic nature.

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The observed growth at 70°C was slightly higher than the growth at 65°C, thus 70°C was chosen

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as optimum. This contrasted from the Topt (of 65°C) observed for its closest relative (Cl.

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stercorarium DSM 8532T) [34]. The strain had neutrophilic nature with optimal pH (pHopt) of

326

7.0, but a narrow pH range (of pH 6.0 to 8.0) with almost no growth at other pH values tested

327

(Figure S1b). As per these observations, the temperature and pH ranges observed here are in line

328

with the growth optima observed for many thermophilic Clostridium strains reported previously

329

[37, 38].

330

3.3. Substrate utilization

331

For an ideal bioethanol production process, a strain with an ability to utilize a broad range of

332

carbohydrates related to lignocellulosic biomass is essential [1, 39]. Strain DBT-IOC-DC21

333

presented positive growth on various substrates including glucose, xylose, cellobiose, arabinose,

334

maltose, mannose, carboxymethyl cellulose, lactose and polysaccharide such as microcrystalline

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cellulose and xylan (summarized in Table 1). However, strain did not grow on fructose, sucrose,

336

and pectin even after prolonged incubation for 7 days. With an ability to grow on both hexose

337

and pentose sugars along with complex polysaccharides, strain DBT-IOC-DC21 presented a

338

sharp contrast in its substrate spectrum compared to the model CBP candidate Cl. thermocellum

339

which was unable to grow on xylose. Here, the broad substrate spectrum endorses the suitability

340

of strain DBT-IOC-DC21 for direct biomass fermentation to bioethanol, termed as consolidated

341

bioprocessing (CBP). CBP is so far suggested as the most cost-effective means of biomass

342

processing [39]. In the later part of this study, the potential of this strain to produce bioethanol

343

from untreated rice straw biomass was invested.

344

Despite the very high genetic relatedness, when the new isolate compared for different

345

characteristics with the type strain of Cl. stercorarium, some marked differences in their

346

substrate spectrum were observed (Table 1). Arabinose fermentation differentiated strain DBT-

347

IOC-DC21 from two of its closest relatives, Cl. stercorarium subsp. leptospartum and Cl.

348

stercorarium subsp. thermolacticum. In addition, strain DBT-IOC-DC21 also differed in fructose

349

and sucrose utilization ability compared to Cl. stercorarium subsp. thermolacticum (Table 1).

350

Based on the results presented above, it can be concluded that strain DBT-IOC-DC21 is the

351

subspecies of the Cl. stercorarium similar to other reported strains.

352

3.4. Ethanol tolerance

353

Despite the high cellulolytic/hemicellulolytic potential, lower ethanol tolerance is one of the

354

prime challenges limiting the industrial application of thermophilic Clostridia for bioethanol

355

production [40]. The increased membrane fluidity due to changes in fatty acid composition at

356

high temperature is the prime reasons for decreased cell growth thus lower ethanol tolerance by

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high-temperature anaerobes [41]. In order to evaluate ethanol tolerance level of strain DBT-IOC-

358

DC21, the influence of varying concentration of exogenously added ethanol on cell growth was

359

tested during batch fermentation in the medium containing xylose and xylan (Table 2).

360

During growth on xylose-containing medium, strain tolerated up to 3.0 % (v/v) added ethanol.

361

The observed growth (OD600 reached = 0.80 ± 0.01) at this concentration was comparative to

362

what observed in medium without ethanol (OD600 reached = 1.33 ± 0.05). However, drastic

363

growth inhibition was observed when medium contained 3.5% (v/v) or higher ethanol level,

364

suggesting that 3% (v/v) was the threshold level of tolerance by the strain. The ethanol tolerance

365

of strain DBT-IOC-DC21 was found to be comparable to most wild-type Clostridia. However,

366

strain improvement efforts are necessary to endorse suitability of this strain for industrial

367

application. In industrial practice, ethanol concentrations could potentially be kept low due to in

368

situ distillation as a way of side stepping the lower ethanol tolerance. Interestingly, during xylan

369

fermentation, the growth of this strain stopped when ethanol concentration was higher than 2%

370

(v/v). The reason for substrate selective tolerance to ethanol by strain DBT-IOC-DC21 is not

371

clear. However, it can be related to pH decline and stressful conditions encountered during

372

closed batch fermentation, preventing further substrate utilization, thus no growth.

373 374 375

3.5. Effect of liquid to headspace ratio on ethanol production Thermophilic Clostridia can consume different carbon sources under obligatory anaerobic

376

conditions and produces hydrogen in its gaseous phase as a by-product of mixed acid

377

fermentation pathway [31]. The inhibitory effect of hydrogen accumulation leads to a high

378

partial pressure of hydrogen (PH2) had been demonstrated for several hydrogen-producing

379

thermophilic anaerobic bacteria [42-44]. It is now well-known fact that, culture conditions such

380

as liquid-gas (headspace) [L-G] volume ratio affects this partial pressure of hydrogen (PH2),

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which plays a predominant role in determining metabolic flux distribution during dark

382

fermentation by ethanol and hydrogen-producing thermoanaerobes [44]. Thus, a proper medium

383

volume, particularly during closed batch fermentation, is an important factor to be considered

384

while studying ethanolic fermentation by gas-producing bacteria. In this study, the influence of

385

varying medium volumes (10, 30, 50, 70, and 90 mL) on ethanol and hydrogen production was

386

investigated at lower xylan concentration of 5 g L-1 using Clostridium strain DBT-IOC-DC21

387

(Fig. 2). A significant difference in the concentration of soluble metabolic end products and

388

hydrogen accumulation was observed at varying L-G ratio, as shown by ANOVA (Fig. 2).

389

As shown in Fig. 2, ethanol production increased with increasing medium volume from 10 to 90

390

mL and maximum ethanol accumulation of 28.54 mM and total soluble metabolite production of

391

49.25 mM, was observed at the L-G ratio of 0.67. By lowering the L-G ratio from 2.57 to 0.09,

392

the ethanol production decreased while hydrogen accumulation increased except at the lowest L-

393

G ratio. Maximum hydrogen accumulation of 41.16 mmol L-1 was observed at a lower L-G ratio

394

of 0.32 (Table S2). This is suggesting that L-G ratio of 0.67 was the optimum liquid-to-

395

headspace ratio for ethanol production by Clostridium strain DBT-IOC-DC21, based on the

396

maximum ethanol concentration obtained. The production of acetate increased from 9.08 mM to

397

15.54 mM at L-G ratio of 0.09 and 0.32 respectively, which eventually remained constant to 12-

398

13 mM at other L-G ratios tested (Fig. 2). Thus, acetate production was not much affected by

399

varying L-G ratios. In contrast, the most predominant effect of increasing L-G ratio on xylan

400

fermentation by strain DBT-IOC-DC21 was observed as redirection of metabolic flux away from

401

lactate production. From these results, we concluded that the initial pH, temperature, and L-G

402

ratio of 7.0, 70°C, and 0.67, respectively, was the optimum conditions for ethanol production

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from xylan by Clostridium strain DBT-IOC-DC21 and the same was followed in this study for

404

subsequent batch fermentation studies.

405

3.6. Effect of xylan concentration on ethanol production

406

It is evident from previous studies that initial substrate concentration is an important factor that

407

affects fermentation performance of thermophilic anaerobic bacteria [13, 45]. The effect of xylan

408

concentration (5, 10, 15, 20, 25, and 30 g L-1) on ethanol production was performed at the

409

optimum initial pH of 7.0, the optimum temperature of 70°C and the L-G ratio of 0.67. A change

410

in initial xylan concentration significantly affected the concentration of metabolic end products,

411

as shown by ANOVA (Fig. 3). Analysis of the end products revealed that ethanol was the

412

primary soluble metabolite followed by acetate and lactate with other end products including

413

hydrogen (Table S3).

414

As shown in Fig. 3, the concentration of soluble metabolites increased with an increase in xylan

415

concentration and reached a maximum concentration (105.11 mM) at 25 g L-1 xylan. The

416

concentration of ethanol increased from 25.27 mM to 67.29 mM with an increase in xylan

417

concentration up to 25 g L-1. However, the concentration of ethanol decreased by 33.64 % when

418

xylan concentration increased from 25 g L-1 to 30 g L-1 (Table S3). Maximum ethanol

419

accumulation of 67.29 mM was observed at a xylan concentration of 25 g L-1. In this regard,

420

strain DBT-IOC-DC21 seemed to have the ability to grow at higher substrate concentration

421

compared to other similar thermophilic Clostridia where often a lower substrate concentration of

422

2 to 5 g L-1 is inhibitory [1, 26, 46]. Similar to ethanol an increase in acetate production from 11

423

mM to 32 mM was observed at higher xylan concentration of 20-25 g L-1. In contrast, lactate

424

production was variable and ranged from 3.02 mM to 8.10 mM at various xylan concentrations

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tested. Maximum hydrogen production of 53.91 mmol L-1 was observed at the highest xylan

426

concentration tested (Table S3). These results suggested that a change in the initial xylan

427

concentration remarkably affected the ethanol production.

428

3.7. Comparative fermentation performance on various substrates

429

The xylan fermentation potential and ability to grow on various substrates related to

430

lignocelluloses was evident by the xylan tolerance and wide substrate spectrum of strain DBT-

431

IOC-DC21. However, to investigate its potential for consolidated bioprocessing, comparative

432

fermentation performance was investigated during batch fermentation on various substrates

433

including ; predominant sugars present in lignocellulosic materials (i.e. glucose, xylose,

434

cellobiose and arabinose), amorphous cellulose, complex polysaccharides (i.e. cellulose and

435

xylan) and their mixture and untreated RSB, under optimized conditions for 48-96 h. All

436

substrates were loaded at equivalent substrate loadings of 5.55 g L-1, expressed as glucose/xylose

437

equivalents unless otherwise specified.

438

Fermentation of sugars

439

As shown in Figure 4A and Table S4, strain DBT-IOC-DC21 presented significant variation in

440

sugar conversion (%), soluble metabolite profile and hydrogen production during the

441

fermentation of various sugars, as shown by ANOVA. The major soluble metabolic end products

442

were ethanol and acetate while lactate was the minor end product obtained irrespective of the

443

type of sugar tested. During the fermentation of glucose and cellobiose, strain presented efficient

444

conversion of both the sugars (>85%). However, the amount of ethanol produced during

445

cellobiose fermentation (35.42 mM) was higher than the concentration achieved during glucose

446

fermentation (27.39 mM). This observation suggested that cello and oligoglucoses are

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preferentially consumed by the strain DBT-IOC-DC21 responsible for more cellobiose

448

fermentation compared to glucose [47]. Here, glucose fermentation resulted in an ethanol yield

449

of 0.91 mol ethanol/mol of glucose consumed (45.51% of the theoretical maximum yield).

450

Although, slightly higher ethanol yield of 2.59 mol ethanol/mol of cellobiose consumed (64.83%

451

of theoretical maximum yield) was observed during cellobiose fermentation. In contrast to

452

ethanol, production of acetate and lactate was not much affected and only slight increment was

453

observed during growth on cellobiose compared to glucose (Table S4). Similar and contrasting

454

end product profile on glucose and cellobiose fermentation was suggested in previous studies

455

involving Cl. stercorarium strains [25, 32, 34, 48].

456

During xylose fermentation, the strain DBT-IOC-DC21 demonstrated 100 % conversion of

457

xylose to end products with a maximum ethanol concentration of 43.63 mM (Table S4). Here, an

458

ethanol yield of 1.18 mol ethanol/mol xylose consumed (70.83% of the theoretical maximum)

459

was observed. In contrast to xylose, 43.88% of arabinose remained unutilized, suggesting that

460

arabinose is not the preferred substrate for the growth and fermentation of strain DBT-IOC-

461

DC21. However, even at a lower extent, arabinose fermentation is an important characteristic

462

that distinguishes our new isolate from the type strain of Cl. stercorarium that cannot degrade

463

arabinose (Table 1). The higher ethanol yield and maximum hydrogen accumulation (30.72

464

mmol L-1) during xylose fermentation compared to other sugars defines that strain DBT-IOC-

465

DC21 is a potent pentose-fermenting ethanologen. This agrees with the previous study reports

466

suggesting more xylose utilization by Cl. stercorarium strains [7, 33].

467

Fermentation of complex polysaccharides and their mixture

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Batch fermentation of two complex polysaccharides of plant cell wall (cellulose and xylan) and

469

amorphous cellulose was performed at a final loading of 5.55 g L-1 of the glucose/xylose

470

equivalents, under the optimized conditions described above. As shown in Figure 4B and Table

471

S4, strain DBT-IOC-DC21 grew better in the presence of xylan as the carbon source compared to

472

cellulose and amorphous cellulose with a final pH drop to 5.74. The concentration of soluble

473

metabolic end products produced and hydrogen accumulation by the strain on these two

474

substrates varied significantly, as shown by ANOVA (Figure 4B).

475

The soluble metabolic end products generated by avicel fermentation using strain DBT-IOC-

476

DC21 were 2.22 mM lactate, 4.54 mM acetate, and 8.97 mM ethanol, whereas the end products

477

generated by amorphous cellulose fermentation were 2.41 mM lactate, 7.12 mM acetate, and

478

10.53 mM ethanol, suggesting that cellulosic fermentation performance was improved slightly

479

when less crystalline cellulose was used. In contrast to both the cellulosic substrates, during

480

fermentation of xylan 5.22 mM lactate, 9.72 mM acetate, and 26.61 mM ethanol were produced

481

(Figure 4B). The modest cellulose fermentation ability of the new isolate is in line with previous

482

reports where the type strain and different subspecies of Cl. stercorarium also presented

483

inefficient fermentation of crystalline cellulose [7, 34, 35], emphasizing their high genetic

484

relatedness. A recent genome sequence and central metabolic pathway analysis of the type strain

485

of Cl. stercorarium DSM 8532T revealed that it is comprised of a simple two-component

486

cellulose hydrolyzing enzyme system while a suite of free hemicellulose hydrolyzing enzymes

487

[7, 35]. Thus, it can be anticipated that strain DBT-IOC-DC21 may comprise a similar or more

488

efficient free hemicellulase enzyme system due to which a “preferential hemicellulose

489

hydrolysis” observed by our new isolate, was obvious. The amount of ethanol produced during

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xylan fermentation by the strain DBT-IOC-DC21 was comparative to the ethanol concentration

491

reported for other xylan fermenting thermophilic anaerobic bacteria [8, 10, 11].

492

In this study, a mixture of complex polysaccharides (to mimic real lignocellulosic biomass) was

493

also used as a substrate to assess the fermentation performance of strain DBT-IOC-DC21, before

494

testing its potential for native RSB fermentation. In the simultaneously performed fermentation

495

on a mixture of avicel and xylan, increased in ethanol production was observed. Here, the

496

mixture of avicel and xylan was loaded at a final concentration of 5 g L-1 (low) and 10 g L-1

497

(high). At 5 g L-1 loading of the polysaccharide mixture, the pattern of soluble metabolite profile

498

was more or less similar to that observed during the fermentation of xylan alone and maximum

499

24.40 mM ethanol was produced (Table S4). However, production of acetate and lactate was

500

decreased. The higher loading of polysaccharide mixture resulted in an increase ethanol

501

production of 40.23 mM (Table S4). The accumulation of hydrogen was also increased to 30.28

502

mmol L-1 at higher concentration of polysaccharide mixture. However, production of acetate

503

(9.62 mM) and lactate (3.35 mM) was not much affected. Here, no significant enhancement in

504

the fermentation performance of strain was observed in the presence of complex polysaccharide

505

mixture. This could be attributed to decrease in the initial pH of the medium at the end of

506

fermentation. From these results, it is clear that strain DBT-IOC-DC21 is specialized in the

507

utilization of hemicellulosic part of lignocellulosic biomass (i.e. xylan, xylose, and arabinose).

508

Fermentation of natural lignocellulosic biomass

509

Native RSB was used as the substrate for bioethanol production by the strain DBT-IOC-DC21

510

under the optimized conditions, described in section 3.2 and 3.5. RSB (on a dry weight basis)

511

was found to contained 36.9% glucan, 20% xylan, 3.5% arabinan, 13.4% lignin, 7.3% ash, 1.1%

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acetic acid and 17.8% extractives, as determined by composition analysis. Native RSB was used

513

at 28 g L-1 (equivalent to 5.7 g L-1 of the xylose equivalents) and 56.4 g L-1 (equivalent to 11.4 g

514

L-1 of the xylose equivalents) concentration, calculated after moisture correction. Here, loading

515

was performed essentially on the basis of xylan content only (expressed as g L-1 of the xylose

516

equivalents), considering the high hemicellulose fermentation potential of the strain DBT-IOC-

517

DC21 compared to crystalline cellulose.

518

As presented in Figure 4C, strain DBT-IOC-DC21 grew and fermented native RSB directly and

519

produced ethanol and acetate as the main soluble metabolic end products while lactate was

520

produced as the minor end product. Metabolites production by strain DBT-IOC-DC21 was found

521

to be affected when subjected to different concentrations of RSB, but statistically significant as

522

shown by ANOVA (Figure 4C). The concentration of ethanol produced were 19.48 mM and

523

11.26 mM at lower and higher concentration of RSB, respectively. The accumulation of

524

hydrogen at lower RSB concentration was 8.43 mmol L-1, similar to what observed during the

525

fermentation of complex polysaccharide mixture when loaded at 5 g L-1 concentration. A slight

526

increase in hydrogen production (12.81 mmol L-1) was observed at the higher concentration of

527

RSB (Table S4). The reduced performance of strain on this substrate compared to pure xylan can

528

be accounted to the high crystallinity of raw biomass and unavailability of the exposed

529

hemicellulosic portion. Despite the fact, the ethanol production at a lower concentration of RSB

530

was comparable to the ethanol produced during fermentation of xylan. Nevertheless, the ability

531

of this strain to grow directly on untreated biomass and subsequently to convert resulting sugars

532

into ethanol indicates the presence of active hemicellulolytic enzymes. Only a few previous

533

studies demonstrated the direct microbial fermentation of untreated lignocellulosic feedstocks

534

such as by high-temperature anaerobes [22-24]. The untreated biomass offered no inhibition at a

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lower loading of biomass. No enzymes were added, emphasizing the significance of the strain

536

DBT-IOC-DC21 as a CBP candidate for the production of bioethanol from hemicellulose rich

537

substrates.

538

4. Conclusion

539

Compost is a good target for isolating hemicelluloses-fermenting thermophilic anaerobic

540

bacteria. In this study, a strictly anaerobic thermophilic bacteria Clostridium strain DBT-IOC-

541

DC21, isolated from a composting facility was capable of utilizing xylan as the sole carbon

542

source with ethanol as the dominant end product. Clostridium strain DBT-IOC-DC21 is a

543

potential CBP candidate due to its; (i) broad substrate spectrum including the major component

544

of lignocellulosic biomass (glucose, mannose, galactose, xylose, arabinose, cellobiose, cellulose,

545

and xylan); (ii) preferential utilization of the hemicellulose fermentation; and (iii) single step

546

bioconversion of untreated lignocellulosic biomass to ethanol. This preliminary study shows the

547

potential of new isolate for bioconversion of hemicellulosic biomass to ethanol. Further work is

548

already underway to test the performance of this strain on other untreated biomass and

549

optimization of process parameters for enhanced performance.

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Acknowledgement

552

The authors thank Deakin University and DBT-IOC Centre for Advance Bioenergy Research,

553

Indian Oil R & D centre, India for supporting collaborative research. One of the authors NS

554

thanks the DIRI program of Deakin University for providing a scholarship to pursue this research

555

work.

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Declarations

558

Ethical Approval and Consent to participate: Not applicable

559

Consent for publication: All authors approved the manuscript.

560

Availability of supporting data: Not applicable

561

Competing interests: The authors declare that they have no competing interests.

562

Funding: Not applicable.

563

Authors’ contributions: NS carried out the research work and drafted the manuscript. MP, CJB,

564

RPG, and DKT provided suggestions to strengthen the manuscript. ASM conceived the study

565

and participated in its design and coordination. ASM helped to draft the manuscript. All authors

566

read and approved the final manuscript.

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[33] M.L. Fardeau, B. Ollivier, J.L. Garcia, B.K. Patel, Transfer of Thermobacteroides leptospartum and Clostridium thermolacticum as Clostridium stercorarium subsp. leptospartum subsp. thermolacticum subsp. nov., comb. nov. and C. stercorarium subsp. thermolacticum subsp. nov., comb. nov., Int. J. Syst. Evol. Microbiol. 51 (2001) 1127-31.

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[34] R.H. Madden, Isolation and characterization of Clostridium stercorarium sp. nov., cellulolytic thermophile, Int. J. Syst. Evol. Microbiol. 33 (1983) 837-40.

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[38] W. Lv, Z. Yu, Isolation and characterization of two thermophilic cellulolytic strains of Clostridium thermocellum from a compost sample, J. Appl. Microbiol. 114 (2013) 1001-7.

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[46] L.D. Ellis, E.K. Holwerda, D. Hogsett, S. Rogers, X. Shao, T. Tschaplinski, et al., Closing the carbon balance for fermentation by Clostridium thermocellum ATCC 27405, Bioresour.Technol. 103 (2012) 293-9.

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[47] T. Rydzak, D.B. Levin, N. Cicek, R. Sparling, End product induced metabolic shifts in Clostridium thermocellum ATCC 27405, Appl. Microbiol. Biotechnol. 92 (2011) 199-209.

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[48] P. Le Ruyet, H.C. Dubourguier, G. Albagnac, Thermophilic fermentation of cellulose and xylan by methanogenic enrichment cultures: preliminary characterization of main species, Syst. Appl. Microbiol. 5 (1984) 247-53.

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Figure captions

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Figure 1. Phylogeny of isolated hemicellulose-fermenting thermophilic anaerobic bacteria based

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on 16S rRNA gene sequence analysis.

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Footnote: Bootstrap values are based on 1,000 replicates. GenBank accession numbers are given

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in parentheses.

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Figure 2 Fermentation profile of Clostridium strain DBT-IOC-DC21 at varying liquid to

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headspace ratio.

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Footnote: Each data value represents average with error bars (±) showing standard deviation

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calculated from triplicate fermentations. Lowercase letters represents the level of significance

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[ANOVA Tukey’s test; *=0.05>p>0.01, **= 0.01>p >0.001 and ***p<0.001] in the treatment

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conditions described. Fermentation by uninoculated duplicate controls was less than 10%.

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Figure 3 Effect of xylan concentrations on fermentation performance of Clostridium strain DBT-

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IOC-DC21.

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Footnote: Each data value represents average with error bars (±) showing standard deviation

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calculated from triplicate fermentations. Lowercase letters represents the level of significance

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[ANOVA Tukey’s test; *=0.05>p >0.01, **= 0.01>p >0.001 and ***p<0.001] in the treatment

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conditions described. Fermentation of various xylan concentrations by uninoculated duplicate

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controls was less than 10%.

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Figure 4 Comparative fermentation profile of Clostridium strain DBT-IOC-DC21 on various

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Footnote: Each data value represents average with error bars (±) showing standard deviation

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calculated from triplicate fermentations. Lowercase letters represents the level of significance

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[ANOVA Tukey’s test; *=0.05>p>0.01, **= 0.01>p>0.001 and ***p<0.001] in the treatment

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conditions described. Fermentation of various substrates by uninoculated duplicate controls was

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less than 10%.

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Table 1 Salient features of Clostridium strain DBT-IOC-DC21 and phylogenetically related species

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Clostridium strain DBTIOC-DC21 -

ATCC 35414, DSM 8532T Compost Gram-negative Rods NA/65/NA NA/7.3/NA

DSM 2910T Compost Gram variable Rods 50/60-65/70 6.4/7/7.8

Not deposited Compost Gram-negative Rods 45/70/75 4/7/8

+ + + + + + + NA + NA + G: E, L, A

+ + + + + + + + + + NA + G: E, L, A, CO2, H2

+ + + NA + + + + + + + + + G, x: E, L, A, CO2, H2

+ + + + + + + + + + + G, x, X: E, L, A, CO2, H2

[33, 34]

[33]

This study

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Clostridium stercorarium subsp. thermolacticum Clostridium thermolacticum

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Accession number Habitat Gram reaction Morphology Tmin/Topt/Tmax (°C) pHmin/pHopt/ pHmax Substrate spectrum Glucose Xylose Mannose Galactose Arabinose Fructose Maltose Sucrose Lactose Cellobiose Starch Cellulose Pectin Xylan End Products of fermentation References

Clostridium stercorarium subsp. stercorarium Clostridium stercorarium

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Former name

Clostridium stercorarium subsp. leptospartum Thermobacteroides leptospartum DSM 9219T Cattle manure Gram variable Rods 45/60/71 6.7/7.5/8.9

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Footnote: Positive; indicates growth, Negative; indicates no growth, NA; Not available, G; Glucose, x; Xylose, X; Xylan, E; Ethanol, A; Acetate, L; Lactate, H2; Hydrogen, CO2; Carbon dioxide. 33

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Table 2 Ethanol tolerance of Clostridium strain DBT-IOC-DC21 Xylose (OD600) 1.33 ± 0.05 1.19 ± 0.06 1.06 ± 0.04 0.91 ± 0.01 0.81 ± 0.01 0.98 ± 0.02 0.80 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

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Footnote: Growth was determined based on the optical density of sample minus the optical density of uninoculated control samples, measured at 600 nm.

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Xylan (OD600) 2.00 ± 0.16 1.65 ± 0.03 0.86 ± 0.05 0.83 ± 0.05 0.43 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

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Ethanol % (v/v) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

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Figure 2

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Lactate Acetate Ethanol Hydrogen

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Lactate Acetate

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Ethanol Hydrogen

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Hemicellulose-fermenting thermophilic anaerobic bacteria isolate, Clostridium strain DBT-IOC-X2, from compost

•

Broad substrate spectrum [Glucose, xylose, cellulose and xylan fermentation]

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Maximum ethanol yield of 45.51%, 64.83%, 70.82%, and 56.11 % of the theoretical maximum from glucose, cellobiose, xylose, and arabinose, respectively

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Maximum 67.29 mM ethanol produced from high concentration (25 g L-1) of xylan

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Direct microbial conversion of untreated rice straw biomass

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