Bioethanol production potential of a novel thermophilic isolate Thermoanaerobacter sp. DBT-IOC-X2 isolated from Chumathang hot spring

Biomass and Bioenergy 116 (2018) 122–130

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Research paper

Bioethanol production potential of a novel thermophilic isolate Thermoanaerobacter sp. DBT-IOC-X2 isolated from Chumathang hot spring

T

Nisha Singha,b, Munish Purib,c,∗∗, Deepak K. Tulia, Ravi P. Guptaa, Colin J. Barrowb, Anshu S. Mathura,∗ a

DBT-IOC Centre for Advance Bioenergy Research, Research & Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad, 121007, India Centre for Chemistry and Biotechnology, Waurn Ponds, Deakin University, Victoria, 3217, Australia c Centre for Marine Bioproducts Development, College of Medicine and Public Health, Flinders University, Bedford Park 5042, Adelaide, Australia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Thermoanaerobacter Pentose-rich sugar hydrolysate Bioethanol Hot spring Rice straw

Dilute acid pretreatment of biomass generates enormous amount of hydrolysate (rich in inhibitors and pentose sugars), that remains unutilized for bioethanol production due to inadequacy of efficient C5-fermenting organisms. In this study, a predominantly pentose fermenting extremely thermophilic bacterium strain DBT-IOCX2, pertaining to the genus Thermoanaerobacter was isolated from Himalayan hot spring. Batch experiments indicated substantial inhibitor resistance (2 g dm−3 for furfural, 5-HMF, and acetic acid), substrate tolerance (∼15 g dm−3), co-sugar fermentation ability (co-production ethanol yield of 0.29 g/g), and high ethanol yield (83.57% and 91.12% of the theoretical maximum from 5 g dm−3 glucose and xylose, respectively) by the bacterium at 70 °C and pH 8.0. Here, bioethanol production process was developed using pre-treated rice straw hydrolysate (PRSH) as low-cost agro-waste and 83.47% of the total sugar conversion was obtained. This study shows that Thermoanaerobacter sp. DBT-IOC-X2 could utilize diluted PRSH efficiently to improve the overall costeffectiveness of biomass processing to bioethanol.

1. Introduction To overcome the economic and environmental issues caused by the use of petroleum-based products, development of sustainable sources of energy is necessary. Bioethanol is sought as one of the major alternatives to conventional liquid transportation fuels since it is renewable and has the potential to mitigate green house gas emissions [1]. First generation bioethanol production from sugar-rich food materials e.g. maize, sugar beet and sugarcane is not widely accepted due to its food vs. fuel conflict [2]. However, lignocellulosic biomass feedstocks such as rice straw, wheat straw, sugarcane bagasse/straw, corn stover/cob, cotton stalk etc. could serve as a promising resource, as they are generated in huge amount, doesn't compete with food security, and has worldwide acceptance [3]. Currently, enzyme-microbe based fermentation of non-food lignocellulosic biomass emerged as an attractive route and termed as second-generation bioethanol production. Second generation bioethanol production process mainly involved 4 steps: pretreatment, hydrolysis, fermentation, and distillation [4]. Biomass pretreatment is a key step essential to disintegrate complex lignocellulose matrix and release carbohydrate polymers which

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undergo hydrolysis by a mixture of cellulolytic enzymes to yield fermentable sugars. Thereafter, the released sugar is fermented to ethanol by robust fermenting microorganism having high volumetric productivity. The commercial-scale development of a cost-effective bioethanol production process faces some major bottlenecks which owes in part to biomass recalcitrance, higher cost of cellulolytic enzymes, and inhibition of both enzymes and fermenting organisms by the molecules released or produced during pretreatment [1,4]. Therefore, there is a constant search for better microorganisms having efficient cellulase production and fermentation capability. So far, filamentous fungi (for cellulases) and yeast (for C6 fermentation) have been predominantly exploited for second generation bioethanol production. Most of the current research has been focussed on; metabolic engineering of these microbes, process consolidation, and conditions optimization to achieve desired production level of bioethanol. Alternatively, thermophilic anaerobic bacteria can also be explored for bioethanol production. A microbial fermentation process employing thermophilic anaerobes is often preferred over mesophilic microorganisms due to their inherent ability to utilize both pentose and hexose sugars, natural resistance to fermentation inhibitors, and several

Corresponding author. Corresponding author. Centre for Marine Bioproducts Development, College of Medicine and Public Health, Flinders University, Bedford Park 5042, Adelaide, Australia. E-mail addresses: [email protected] (M. Puri), [email protected] (A.S. Mathur).

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https://doi.org/10.1016/j.biombioe.2018.05.009 Received 23 January 2018; Received in revised form 23 April 2018; Accepted 22 May 2018 0961-9534/ © 2018 Elsevier Ltd. All rights reserved.

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in M medium were simple sugars and complex polysaccharides, added at a concentration specified in text. Concentrated stocks of sugar solution were prepared separately and added to the autoclaved medium after filter sterilization, to avoid charring and sugar loss. Likewise, pretreated liquid hydrolysate obtained after dilute-acid pretreatment of RSB was also supplemented with M medium components (as described in section 2.4). After mixing all the solutions, desired pH of the media was adjusted just before inoculation using anaerobic stock solution of 1N HCl and 1N NaOH. Standard anaerobic culture techniques were used throughout the experimental manipulations, as previously described [14,15]. A reference culture Thermoanaerobacter ethanolicus DSM 2246 was procured from the DSMZ collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) and revived using fresh medium 640 (composition provided by DSMZ). Pure culture was stored in 30% deoxygenated glycerol at −80 °C and revived before each experiment.

associated process benefits offered by high temperature conditions (> 70 °C) [5]. Previously, high ethanol yield on both simple sugars and complex biomass have been reported by several thermophilic anaerobic bacteria belonging to the genus Clostridium, Thermoanaerobacter, and Thermoanaerobacterium [6]. In addition to above mentioned sources, waste effluents derived from various pretreatment processes could be a source for bioethanol production, where waste valorization to fuel would be an advantage [7–9]. The slurry obtained after dilute-acid pretreatment of biomass contains a cellulose rich solid fraction and liquid hydrolysate rich in C5 sugars and inhibitors. This liquid hydrolysate is usually separated or detoxified to prevent the inhibition of enzymes and fermenting organisms in subsequent steps, which leads to sugar loss and cost escalation. Thus, the effective utilization of this sugar-rich stream by efficient thermophilic anaerobic bacteria can improve the economic value of the whole process. Anderson et al., recently reviewed and reported most efficient ethanolic fermentation (> 90% of the theoretical maximum) of such C5-rich liquid fraction (comprising glucose, xylose, arabinose, and furfural) derived from dilute acid pre-treatment of wheat straw using genetically engineered Thermoanaerobcater italicus Pentocrobe 411X [7]. Bioethanol production from C5-rich liquid hydrolysate derived from pre-treated biomass has not been fully explored. In present study, a potent bacterial isolate which can produce bioethanol directly from pretreated lignocellulosic hydrolysate was isolated and its biochemical characteristics were explored. An undisturbed thermal hot spring site situated at utmost location was chosen for isolating such bacteria. To the best of our knowledge, this is the first study presenting isolation and characterization of a predominantly pentose fermenting thermoanaerobe from Chumathang hot spring site. Rice straw biomass (RSB) was chosen for this study as one of the most abundant feedstock in the world. Around 650–975 million tons of rice straw is produced annually worldwide, which could generate around 250 billion litres of bioethanol based on its high holocellulose (32–47% cellulose and 19–27% hemicellulose) content [10]. However, the structure of RSB is severely complex due to high degree of cellulose polymerization and high silica content that slow down its biodegradation by hydrolytic organisms and enzymes [11]. Therefore, pretreatment of RSB is essential to breakdown its recalcitrant structure. Here, pre-treated rice straw hydrolysate (PRSH) was used in batch mode fermentation to evaluate the applicability of the isolate for bioethanol production from real substrates. In addition to this, fermentation performances on simple sugars and sugar mix (C6 and C5) were also explored. We have chosen to focus on the simultaneous co-sugar fermentation and inhibitor tolerance of the newly isolated strain, in order to acquire a basic knowledge which could be applicable for the fermentation of mixed sugars derived from real substrates. Process parameters such as pH, temperature, and initial concentration of substrates were optimized to achieve maximum production of bioethanol. Here, a comparison has been made between the fermentation performance of newly isolated hot spring strain with already established and closely related Thermoanaerobacter ethanolicus DSM 2246 [12].

2.2. Isolation and identification of C5-fermenting thermophilic anaerobic bacteria Bacterial strain DBT-IOC-X2 used in this study was isolated from hot water and bacterial mat samples collected from Chumathang hot spring site (N33°21′37.11”; E78°19′24.50″, altitude 3950 m), located 150 km southeast of Leh district, North West Himalayan, India. The enrichment and isolation using hot spring samples was performed under strict anaerobic conditions, according to the procedure published earlier [14]. M medium (50 cm3, pH 7.0) with 10 g dm−3 xylose as a sole carbon source was inoculated anaerobically with hot spring samples to selectively enrich and purify C5-fermenting thermophilic anaerobic bacteria. Enrichment cultures were incubated at 70 °C, as the suggestive temperature optima for most sugar-fermenting thermoanaerobes, without shaking till positive growth was evidenced in the form of turbidity, pH drop, and gas production. The stable enrichment culture responsible for maximum ethanol production was selected. Individual colonies were purified from this enrichment culture using Hungate roll tube technique of culture purification, as described previously [14]. The best C5-fermenting isolate used in this study was selected on the basis of maximum ethanol production and designated as strain DBTIOC-X2. Total genomic DNA of the purified bacterial isolate DBT-IOC-X2 was extracted using DNeasy blood and tissue kit (Qiagen India Pvt. Ltd) following the manufacturer's instruction. The 16S rRNA was amplified by PCR using bacterial domain-specific universal set of primers: 27F ( 5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTT ACGACTT-3′) in accordance to the conditions described previously [14]. The sequencing was carried out by Institute of Microbial Technology, Chandigarh, India. Gene sequences obtained were then analyzed for evolutionary history as described previously [14] and references therein. 2.3. Strain characterization

2. Materials and methods The effect of temperature, initial pH, and varying concentration of substrates and inhibitors on the growth of strain DBT-IOC-X2 was studied in triplicate in serum bottles containing 50 cm3 M medium and 10 g dm−3 xylose as the sole carbon source. Each experiment set was inoculated with 5% (v/v) inoculum from a freshly grown culture (OD600∼0.8–1.0) prepared by passaging thrice on M medium (pH 7.0) containing 10 g dm−3 xylose. Influence of initial pH of the medium on the growth and maximum ethanol production by strain DBT-IOC-X2 was studied with the pH range of 3.0–10.0, at 70 °C for 48 h. The initial pH of the medium was adjusted with 1N HCl and 1N NaOH solutions prepared anaerobically. To investigate the effect of temperature, inoculated bottles were incubated at ranges of temperature from 45 °C to 85 °C with 5 °C intervals,

2.1. Growth medium and reference strain A chemically defined minimal medium (M), used for all the enrichment, isolation and fermentation studies was in accordance to Sizova et al., [13]. M medium was prepared by boiling under a constant flow of nitrogen gas to remove dissolved oxygen. Medium was cooled and dispensed into serum bottles (125 cm3, Wheaton) inside a Coy anaerobic chamber (USA) having a headspace of N2:CO2:H2 (90:5:5). The bottles were sealed using butyl rubber stopper (Bellco, USA) and aluminium crimp to ensure anaerobic conditions. Sealed bottles were autoclaved for 15 min at 121 °C and reduced using a concentrated stock solution of L-cysteine HCl. Moreover, the carbon sources supplemented 123

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(glucose, cellobiose, xylose, and arabinose) and inhibitors (furfural and 5-HMF) in the culture supernatant was analyzed by HPLC using the same operating conditions as previously described [14]. Identification of peaks was performed by comparison of retention times with standards area. Calibration curves were derived from stock solutions of each component and standard showed R2 value close to 0.999 was accepted. Ethanol was estimated using Clarus-680 Gas Chromatograph (PerkinElmer, USA) equipped with a flame ionization detector (Perkin-Elmer, USA) and a capillary column (Stabilwax®-DA 30 m × 0.25 mm inner diameter, Restek), using the same operating conditions as described previously [14]. All samples were appropriately diluted and filtered through a 0.2 μm filter before chromatographic analysis. The rate of substrate conversion was calculated by estimating consumed sugar and expressed in percentage (%). Metabolite yield reported as g dm−3 converted to moles using a molecular weight of 46.07 g mol−1 for ethanol, 60.05 g mol−1 for acetate and 90.08 g mol−1 for lactate. Further, metabolite yield as mol of ethanol produced per mol of consumed sugar was calculated using the theoretical maximum equation. In case of sugar mix and PRSH, the ethanol yield was calculated by diving gram of ethanol produced divided by gram of total sugar consumed.

at selected pH value for 48 h. The optimized pH and temperature condition determined was thereafter used in all experiments performed. Substrate utilization spectrum was tested using different carbon sources (cellobiose, glucose, xylose, arabinose, mannose, galactose, fructose, maltose, lactose, carboxy methyl cellulose sodium salt, sucrose, starch, xylan, and microcrystalline cellulose) at 5 g dm−3 concentration in M medium (pH 8.0), at 70 °C for 24–96 h. For each substrate triplicate reactions and control without inoculation were included. Influence of various concentration of sugars (5–30 g dm−3) and inhibitors [furfural (0–0.8%), 5-hydroxyl methyl furfural (0–0.8%), and acetic acid (0–3%)] on ethanol production was also investigated in batch experiment as described above. 2.4. Dilute-acid pretreatment of rice straw biomass and preparation of PRSH Rice straw was pre-treated by dilute sulfuric acid pretreatment in a 250 kg biomass per day capacity continuous pilot scale plant, in accordance to the procedure described previously [14]. After pretreatment the biomass slurry was collected in the slurry tank, cooled and transferred through a peristaltic pump to a high speed centrifuge for separating residual solids (cellulose) and C5-rich liquid fractions. The collected liquid part was stored at 4 °C throughout the study. The inhibitors and carbohydrate composition of the hydrolysate was determined using high performance liquid chromatography (HPLC) as described in the analytical method section. The PRSH was then supplemented with same concentration of remaining M medium components [13], except any carbon source. After purging with nitrogen gas for an hour, prepared stream-waste medium was added to each bottle as a filter sterilized solution, under strict anaerobic conditions. The medium was adjusted to pH 8.0 and appropriately diluted to optimal sugar concentration before inoculation. All the bottles were incubated in dark without shaking for 24–48 h and analyzed for fermentation end products. Duplicate control without inoculation was included as negative control.

2.8. Statistical analysis The statistical significance of the difference between the concentration of soluble metabolite produced in various treatment conditions was determined by using the one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference (HSD) post hoc tests. Statistical analyses were performed using R-Studio®, version 1.0.136 (RStudio, Inc. Boston, MA). The concentration of all soluble metabolites were expressed as average with error bars ( ± ) showing standard deviation and differences considered significant at probability value less than 0.05 (p < 0.05). 3. Results and discussion

2.5. Ethanol production from sugar mix under optimum conditions

3.1. Characteristics of strain DBT-IOC-X2

Kinetics of co-sugar fermentation from a mixture of glucose and xylose at two different concentrations (i.e. 10 g dm−3 and 20 g dm−3) was studied for strain DBT-IOC-X2, under the optimized conditions (pH 8.0 and temperature 70 °C in 50 cm3 M medium). Samples were collected at different time intervals for a period of 48 h. At each time interval three bottles along with control were sacrificed for the analysis of different parameters such as changes in pH, growth, soluble metabolites production, and sugar conversion.

A novel C5-fermenting strain Thermoanaerobacter sp. DBT-IOC-X2 was isolated from an alkaline thermal hot spring (∼80 °C, pH 8) located at the Himalayan region of India. This strain grew rapidly on xylose and presented maximum ethanol yield, thus investigated in detail for further characterization and fermentation properties (Supplementary Table S1). 3.1.1. Phylogenetic characterization Phylogenetic and sequence similarity analysis of the strain DBTIOC-X2 and other strains available in GenBank database revealed its affiliation to the genus Thermoanaerobacter and their evolutionary relationship is presented as a phylogenetic tree (Supplementary Fig. S1). The genus Thermoanaerobacter contains several species of strictly anaerobic, spore forming and carbohydrate fermenting thermophilic anaerobic bacteria that produces ethanol along with lactate, acetate, carbon dioxide, and hydrogen by mixed acid fermentation pathway [16]. The 16S rRNA gene sequences of strain DBT-IOC-X2 revealed its 99% identity to the 16S rRNA gene of Thermoanaerobacter pseudethanolicus strain 39E (formerly known as Clostridium thermohydrosulfuricum and Thermoanaerobacter ethanolicus) [17,18] and Thermoanaerobacter thermohydrosulfuricus strain E100-69 (formerly known as Clostridium thermohydrosulfuricum) [17,19]. Other species of the genus Thermoanaerobacter had above 90% sequence identity with strain DBT-IOC-X2. Nucleotide sequence of Thermoanaerobacter sp. DBT-IOC-X2 are submitted to the NCBI GenBank under the accession number; KY056821. The pertinence of genus Thermoanaerobacter for bioethanol production has not been fully explored for utilizing C5 and inhibitor rich stream derived from dilute-acid pre-treated RSB, which is the main

2.6. Ethanol production with various carbon sources The ability of Thermoanaerobacter sp. DBT-IOC-X2 for ethanol production from different carbon sources was investigated under the optimized conditions (pH 8.0 and temperature 70 °C). The carbon sources; glucose, xylose, and PRSH (composed of mainly xylose) were used in the study at a final concentration of 10 g dm−3 unless otherwise specified. Each experiment set was performed in triplicate, inoculated with 5% (v/v) inoculum and incubated in dark without shaking. Samples were collected after 48 h for the determination of growth, final pH, sugar conversion, and fermentation products. 2.7. Analytical methods Growth of Thermoanaerobacter sp. DBT-IOC-X2 was monitored by determination of the optical density at 600 nm (OD600) using UV visible spectrophotometer (UV-2450, Shimadzu, Japan). Initial and final pH was measured by pH meter (Mettler-Toledo, India). The concentration of soluble metabolites (lactate and acetate), residual carbohydrates 124

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bottom line of this study. One of the potential sugar fermenting member of this genera; Thermoanaerobacter ethanolicus DSM 2246, was procured from DSMZ for comparison studies and cultivated in the same medium as used for strain DBT-IOC-X2. 3.1.2. Physiological characterization The strain DBT-IOC-X2 isolated during this study was gram negative, rod shape, obligatory anaerobic and extremely thermophilic anaerobic bacteria and showed optimal growth at temperature 70 °C. No significant growth was observed below 55 °C defining its thermophilic nature (Supplementary Fig. S2A). This isolate was able to grow on a wide range of pH from 5.0 to 9.0, however, maximum growth was observed at pH 7.0 and 8.0 (Supplementary Fig. S2B). The ability of strain to grow in a broad pH range is highly desirable characteristics that enable its application for fermentation of industrial waste effluents that are acidic or alkaline [20]. When 5 g dm−3 glucose and xylose were used as substrate for strain DBT-IOC-X2, an ethanol yield of 83.57% and 91.12% of the theoretical maximum respectively, was achieved (shown in Supplementary Table S2). The following stoichiometric equation of soluble metabolites was observed: 1.0 Glucose 1.0 Xylose

Fig. 1. Fermentation profile of Thermoanaerobacter sp. DBT-IOC-X2 at different initial pH. Each data value represents average with error bars ( ± ) showing standard deviation calculated from triplicate fermentations. Lowercase letters represents the level of significance [ANOVA Tukey's test; ***p < 0.001, * = 0.05 > p > 0.01, and ** = 0.01 > p > 0.001] in the treatment conditions described.

1.67 Ethanol +0.12 Lactate +0.30 Acetate

effect of culture pH on metabolic flux redistribution. It was found that during xylose fermentation, both sugar conversion (%) and ethanol production are primarily dependent on the initial pH of the medium. An increase in the pH from 4.0 to 6.0 resulted in increase in xylose conversion from 17.75% to 48.56%. Further increase in the pH to 7.0 and 8.0 resulted in an impressive fermentation performance and more than 90% of xylose conversion (Fig. 1). However, when cultured with an initial pH above 8.0, both growth and xylose conversion was decreased by more than 70%. The strain exhibited maximum ethanol yield of 1.25 mol-ethanol/mol-xylose consumed (75% of theoretical maximum) at pH 8.0 (Supplementary Table S4). During closed batch conditions, initiation of fermentation at higher pH values provided longer time to reach the final pH (below 5.5), which resulted in more cell growth thus higher ethanol production. Similar to present study, more favoured ethanol production at neutral to alkaline pH values was observed for Thermoanaerobacter ethanolicus [12] and Thermoanaerobacter strain AK68 [22]. Other soluble metabolites produced at various pH were mainly acetic acid and lactic acid (Fig. 1). The influence of initial pH on acetic acid and lactic acid production was different. The production of acetic acid was more favoured when initial culture pH was neutral to alkaline, compared to other pH values tested. Maximum acetic acid (0.47 molacetate/mol-xylose consumed) was produced at initial pH 9.0. In contrast, no significant difference in lactate yield (0.05–0.07 mol-lactate/ mol-xylose consumed) was observed at an initial pH range of 6.0–9.0, suggesting lactic acid production was minimally affected at various pH values. Since highest ethanol yield was observed at pH 8.0, the same pH was used for all the subsequent experiments.

1.52 Ethanol +0.04 Lactate +0.26 Acetate

Clearly the strain is a potential ethanol producer with the highest ethanol yield so far from xylose among the known wild-type thermophilic anaerobic bacteria belonging to the genera Thermoanaerobacter, presented in Supplementary Table S3. A near stoichiometric ethanol yield of 1.65 mol ethanol/mol of xylose (98.8% of the theoretical maximum) was reported from a xylanolytic Thermoanaerobacterium strain NTUO1 at equivalent substrate concentration [21], suggesting the highest yield report on xylose by Thermoanaerobacter sp. DBT-IOCX2, from this particular genus. 3.1.3. Biochemical characteristics Ethanol producing bacteria growing at high temperature (> 60 °C), are naturally capable of fermenting a wide range of carbohydrates and their polymers [5]. Strain DBT-IOC-X2 was also investigated for this key characteristic under optimized growth conditions and positive growth was considered by measuring OD600, pH decrease and gas production on these substrates. Among all the tested substrates, strain DBT-IOC-X2 utilized only restricted range of sugars and did not grow on any polysaccharide including cellulose, pectin and xylan. Despite the high genetic similarities of the isolate with Thermoanaerobacter thermohydrosulfuricus strain E100-69, a few differences remain in their substrate spectrums (Supplementary Table S1). Xylan and pectin utilization is a characteristic property for strain E100-69 [17], while strain DBT-IOC-X2 could ferment only simple sugars, suggesting the pure sugar fermenting nature of the isolate. In addition to this, strain DBTIOC-X2 was isolated from natural thermophilic environment i.e. hot spring, while strain E100-69 was isolated from a sugar factory. Nevertheless, strain DBT-IOC-X2 differs from Thermoanaerobacter pseudethanolicus strain 39E with respect to lactose and maltose utilization.

3.3. Effect of initial substrate loadings Most wild-type thermophilic ethanologens have limited tolerance towards increasing concentration of sugars [8,9,22–25], which is one of the prime hurdles preventing their commercial application. In this study, effect of various concentration of glucose and xylose (ranging from 5 to 30 g dm−3) on cell growth and ethanol production by Thermoanaerobacter sp. DBT-IOC-X2 was studied at initial pH 8.0 in batch fermentation for 48 h. It was observed that both sugar conversion and soluble metabolite production was significantly affected when strain DBT-IOC-X2 was subjected to increasing concentration of glucose and xylose, as shown by ANOVA (Fig. 2A and B). From this study, it was found that decreased substrate conversion

3.2. Effects of initial pH on fermentation performance Initial culture pH plays an important role in determining optimal cell growth and ethanol production by thermophilic anaerobic bacteria [6]. The influence of initial pH (pH 4.0 to 10.0) on ethanol production by Thermoanaerobacter sp. DBT-IOC-X2 was examined at 70 °C for 48 h using xylose (10 g dm−3) as the sole carbon source (Fig. 1). For all the initial pH values tested, a significant difference in the ratio of final end products was observed as shown by ANOVA (Fig. 1), suggesting an 125

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3.4. Effect of inhibitors on ethanol production 3.4.1. Impact of acetate Acetate is one of the major end products observed during the mixed acid fermentation of sugars by Thermoanaerobacter species. It is also a common inhibitor generated as a result of hemicellulose (acetylxylan) hydrolysis during the dilute acid pretreatment of lignocellulosic biomass [27]. Thus, it was important to study the impact of acetate on cell growth and ethanol production by the strain DBT-IOC-X2. To characterize the influence of acetate on the fermentation performance of strain DBT-IOC-X2, various concentration (0–0.3%) of sodium acetate was added to the medium containing xylose (10 g dm−3) as the sole carbon source (Table 1). It was observed that an increase in the acetate concentration beyond 0.25% resulted in complete growth inhibition. As the concentration of acetate increased, ethanol production decreased except at the lowest concentration of acetate where an improved ethanol yield of 3.54 g dm−3 (1.18 mol-ethanol/mol-xylose) was observed. This suggested a stimulatory effect of acetate addition on ethanol production. Similar effect was observed during xylose fermentation by Thermoanaerobacterium thermosaccharolyticum strain W16, where cell growth and ethanol production was stimulated in the presence of sodium acetate concentration of 6 g dm−3 [28]. He et al., also reported that acetate had a consistent stimulatory effect on ethanol production up to higher acetate concentrations of 150 mM (on xylose) and 75 mM (on glucose) [29]. In the presence of acetic acid, cells are forced to generate more ATP to maintain intracellular pH and produced ethanol at the cost of cell growth and thus production increased [30]. However, this increase was possible only at lower concentration of acetate up to 0.05%, in case of strain DBT-IOC-X2. Addition of acetate also resulted in changes in end product distribution even at the lowest concentration (0.05%) tested. The production of lactic acid was quenched by > 50% by the addition of acetate and almost no lactic acid produced when higher concentration of acetate was added in the medium (Table 1). The reason for similar xylose conversion but lower ethanol production at 0.1, 0.15 and 0.2% (w/v) concentration of acetate can be explained by the possible redirection of metabolic flux towards the production of gaseous products such as hydrogen or carbon dioxide.

Fig. 2. Effect of initial concentration of glucose (A) and xylose (B) on fermentation performance of Thermoanaerobacter sp. DBT-IOC-X2. Lowercase letters represents the level of significance [ANOVA Tukey's test; ***p < 0.001, * = 0.05 > p > 0.01, and ** = 0.01 > p > 0.001] in the treatment conditions described.

3.4.2. Impact of 2-furfural and 5-HMF The primary breakdown products of hemicellulose are pentoses and their further degradation releases furfural, while 5-HMF is a degradation product of hexoses and generated from cellulose [27]. Therefore, these products are essentially present in the slurry of dilute-acid pretreated biomass. These compounds act as strong inhibitor of fermentation by many microorganisms including yeast and bacteria [28]. Moreover, it can be seen that only few studies have focussed on the influence of 2-furfural and 5-HMF on ethanol fermentation by high temperature anaerobes. In this study, effect of varying concentration of 2-furfural (0–0.8%) and 5-HMF (0–0.8%) on cell growth and ethanol production by strain DBT-IOC-X2 was studied using xylose (10 g dm−3) as the carbon source at initial pH 8.0 for 48 h. As shown in Table 1, strain DBT-IOC-X2 was highly sensitive to increasing 2-furfural and 5-HMF concentrations. Both xylose conversion and ethanol concentrations were significantly affected by increased inhibitor concentrations, as shown by ANOVA (Table 1). An addition of 0.2% or higher concentration of 2-furfural and 5-HMF, caused more than 85% and 64% decrease in xylose conversion respectively, which is accompanied by less growth (OD600). Palmquist et al., suggested a similarity in the inhibitory mechanism of 5-HMF and 2-furfural, which is consistent with what we observed with strain DBT-IOC-X2 [27]. These observation suggested that strain DBT-IOC-X2 possess the inherent ability to tolerate particular concentration of these inhibitors likely to be present in the liquid part of pre-treated biomass.

was correlated with increasing substrate concentration, thus lower ethanol yield. As the concentration of glucose increased from 5 to 30 g dm−3, a decrease in ethanol yield from 1.67 mol-ethanol/molglucose to 0.38 mol-ethanol/mol-glucose was accompanied by more glucose accumulation. Here, the observed inhibitory effect perhaps due to limited buffer capacity of the medium [8,23], accumulation of hydrogen [22,26] or disturbances in the medium osmolarity [22]. The maximum ethanol production during glucose (2.77 g dm−3) and xylose (3.38 g dm−3) fermentation was observed at 15 g dm−3 initial substrate concentration (Supplementary Table S2). These results suggested that the wild-type Thermoanaerobacter sp. DBT-IOC-X2 possess substantial tolerance to higher concentration of glucose and xylose during batch culture, compared to other Thermoanaerobacter strains reported. Many sugar fermenting ethanologens showing near stoichiometric yield got inhibited by as low as 5 g dm−3 concentration, while severe inhibition was observed at higher sugar concentration [25]. Nevertheless, strain DBT-IOC-X2 also presented improved utilization of xylose than glucose at higher concentration of sugars as well, which positions this isolate as a ‘potential C5-fermenting strain’.

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Table 1 Effect of furfural, 5-HMF, and acetic acid on fermentation profile and xylose conversion by Thermoanaerobacter sp. DBT-IOC-X2. Inhibitors

Fermentation products (g dm−3) Lactate

5-HMF 0% 5-HMF 0.2% 5-HMF 0.4% 5-HMF 0.6% 5-HMF 0.8% Furfural 0% Furfural 0.2% Furfural 0.4% Furfural 0.6% Furfural 0.8% Acetic acid 0% Acetic acid 0.05% Acetic acid 0.1% Acetic acid 0.15% Acetic acid 0.2% Acetic acid 0.25% Acetic acid 0.3%

∗∗∗

0.29 ± 0.05 0.2 ± 0.03 N N N 0.28 ± 0.03 N N N N 0.29 ± 0.03 0.07 ± 0.01abc 0.09 ± 0.03ade 0.12 ± 0.03bdf 0.10 ± 0.03cef N N

Xylose conversion (%) Acetate 0.87 N N N N 0.92 0.22 N N N 0.92 1.08 1.28 2.47 2.76 3.07 3.18

∗∗∗

Ethanol

± 0.10

± 0.04 ± 0.02

± ± ± ± ± ± ±

0.12gh* 0.14gi 0.09h* 0.07ij 0.15jkl* 0.11km 0.14l*m

3.26 0.17 N N N 3.31 0.03 N N N 3.36 3.54 2.69 2.34 1.31 0.28 0.11

∗∗∗

± 0.13 ± 0.02

± 0.13 ± 0.02

± ± ± ± ± ± ±

0.08n 0.14n 0.16° 0.22° 0.02 0.03p 0.00p

100 ± 0.00 36.77 ± 2.82 6.32 ± 1.04 2.17 ± 1.64 1.60 ± 0.27 100 ± 0.00 15.11 ± 2.84 1.67 ± 0.28 1.20 ± 0.35 1.31 ± 0.11 99.60 ± 0.45 96.37 ± 0.86 99.55 ± 0.45 99.56 ± 0.38 98.90 ± 0.63 63.42 ± 0.97 48.14 ± 2.46

a)

Values are presented as average with error bars ( ± ) showing standard deviation of the samples prepared in triplicate. Fermentation of xylose by uninoculated controls was less than 10%. Lowercase letters represents the level of significance [ANOVA Tukey's test; ***p < 0.001, ** = 0.01 > p > 0.001, and * = 0.05 > p > 0.01] in the treatment conditions described. c) Xylose conversion (%) is based on the relative initial sugar concentration values. b)

concentration of xylose was higher than glucose [7]. The simultaneous co-utilization of hexose and pentose sugar represents the absence of carbon catabolite repression [35] in Thermoanaerobacter sp. DBT-IOCX2 and also demonstrates its potential suitability for fermentation of mixed sugars derived from lignocellulosic biomass.

3.5. Co-sugar fermentation by strain DBT-IOC-X2 under the optimum conditions For an ideal second generation bioethanol production process, simultaneous uptake and fermentation of both C6 and C5 sugars is desired [31]. Traditional fermenting organisms for example, Saccharomyces cerevisiae and Zymomonas mobilis cannot achieve this co-fermentation naturally and often engineered to perform this task [32,33]. Thus, achieving the efficient fermentation of complex sugar mixtures resulting from the hydrolysis of lignocellulosic biomass is still challenging. Based on the results of optimization experiments, ethanol production under optimum conditions (pH 8.0 and temperature 70 °C) was studied by Thermoanaerobacter sp. DBT-IOC-X2 for 48 h using (i) mixture of 5 g dm−3 glucose and 5 g dm−3 xylose (low concentration) and (ii) mixture of 10 g dm−3 glucose and 10 g dm−3 xylose (high concentration), as the carbon source. Fig. 3A and B depicted growth and sugar utilization by the strain DBT-IOC-X2 at different time intervals. The concentration of soluble metabolites was found to be significantly affected when strain was subjected to increasing concentration of sugar mixtures, as shown by ANOVA (Fig. 3A and B). When low concentration of sugar mixture was used as substrate for the strain DBT-IOC-X2, nearly complete sugar consumption (> 90%) was achieved and ethanol (2.94 g dm−3), acetate (0.54 g dm−3), and lactate (0.39 g dm−3) were the major soluble metabolites produced (Fig. 3A). At higher concentration of mixed sugar, ethanol (2.87 g dm−3), acetate (0.75 g dm−3), and lactate (0.34 g dm−3) were the major soluble metabolites produced and 62.45% of maximum sugar conversion achieved (Fig. 3B). Of total sugar consumed; 80% was for xylose and 49.2% was for glucose, indicating that most of the metabolite yield was achieved through xylose utilization. Only few previous studies explained the effect of mixed sugars on the redistribution of metabolic flux and selective carbohydrate preferences by thermophilic ethanologenic bacteria in batch and continuous culture [21,28,31,34]. Here, co-utilization of both sugars was reflected by the simultaneous lowering of available glucose and xylose at different time intervals. However, xylose was metabolized at a faster rate than glucose and this effect was more prominent at higher sugar levels. A similar preference of xylose over glucose was demonstrated recently for a genetically engineered strain of Thermoanaerobacter Pentocrobe 411X, when the

3.6. Effect of different carbon sources on ethanol production While the higher ethanol production was evident by glucose and xylose fermentation using strain DBT-IOC-X2, the significance of this yield needs to be compared with other thermophilic ethanol producing bacteria. Therefore, fermentation studies were carried out using a potential sugar fermenting and ethanologenic bacteria, Thermoanaerobacter ethanolicus DSM 2246, under the experimental conditions described in this study. Strain DSM 2246 was selected as one of the phylogenetically closest relative (> 99% identity) to the strain DBT-IOC-X2 and a most thoroughly described thermophilic ethanologen in literature. Different types of carbon sources have been reported as feedstock for bioethanol production. Therefore, various carbon sources were examined for strain DBT-IOC-X2 and DSM 2246 including simple sugars (glucose, xylose) and sugar mixture obtained from pretreated biomass material (rice straw). In all the cases, a significant difference in sugar consumption and end product profile between strain DBT-IOC-X2 and DSM 2246 was observed, as shown by ANOVA (Table 2). 3.6.1. Profile of end products in glucose fermentation During the fermentation of 10 g dm−3 glucose, an ethanol yield of 1.23 mol-ethanol/mol glucose (61.91% of the theoretical maximum) and 89.84% glucose conversion was observed for strain DBT-IOC-X2 (Table 2). Although lesser glucose conversion (62.44%) and slightly higher ethanol yield of 1.48 mol-ethanol/mol-glucose (74.65% of theoretical maximum) was observed for strain DSM 2246. The ethanol yield obtained in this study for the strain DSM 2246 was lower than the enhanced yield of 1.9 mol-ethanol/mol-glucose reported in previous study, however, at glucose concentration below 10 g dm−3 [12]. At 10 g dm−3 substrate concentration strain DSM 2246 was reported to produce an ethanol yield of 0.75 mol-ethanol/mol-glucose with only 37% glucose conversion [23]. Thus, it can be anticipated that the cultivation conditions described in this paper are more favourable 127

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for strain DSM 2246 to achieve a higher glucose consumption and ethanol yield at 10 g dm−3 substrate concentration. Notably, strain DBT-IOC-X2 and DSM 2246 exhibited distinct profiles from glucose fermentation, under similar growth conditions. Strain DBT-IOC-X2 is accompanied by the formation of ethanol and acetate as major products followed by lactate in lesser quantity while DSM 2246 produced higher amounts of both acetate and lactate (Table 2). This is contrary to some previous reports where lower lactate formation during growth on glucose by DSM 2246 was observed [23,36]. In this study, an increased ethanol yield by the ethanologenic strain DBT-IOC-X2, illustrates the potential importance of this strain for ethanolic fermentation of glucose as well. 3.6.2. Profile of end products in xylose fermentation In a defined medium with 10 g dm−3 xylose, strain DBT-IOC-X2 exhibited enhanced growth and ethanol production compared to glucose, suggesting that strain DBT-IOC-X2 is a predominantly C5-fermenting ethanologen. Ethanol was the main fermentation product yielding 1.12 mol-ethanol/mol-xylose corresponding to 67.56% of the theoretical maximum (Table 2) with nearly complete xylose conversion (97.96%). However, xylose utilization was incomplete by DSM 2246 and a lower ethanol yield of 0.84 mol-ethanol/mol-xylose (50.63% of the theoretical maximum) was observed. A comparison of the soluble metabolites produced and ethanol yield on glucose and xylose showed that glucose was the more preferred substrate for DSM 2246. This is in agreement with the previous reports suggesting more glucose utilization by DSM 2246. Although by mutation and genetic modification some derived strains of DSM 2246 could consume higher xylose concentration with increased amount of ethanol production [36]. 3.6.3. Fermentation of PRSH by Thermoanaerobacter sp. DBT-IOC-X2 Batch fermentation was carried out to test the comparative performance of strain DBT-IOC-X2 and DSM 2246 on PRSH at 70 °C for 48 h. The composition of the PRSH was (in g dm−3): xylose (38.7), glucose (8.2), cellobiose (2.5), arabinose (8.6), 5-HMF (1.23) and 2-furfural (1.11) (Supplementary Table S5). Considering the substrate tolerance level of strain DBT-IOC-X2, the obtained liquid hydrolysate was diluted appropriately to attain the final concentration equivalent to 15–20 g dm−3 of total sugars, and used as a carbon source in the formulation of bioethanol production medium (pH 8.0). End product profile revealed that both strains were able to utilize various sugars present in PRSH. However, maximum ethanol production (ethanol yield of 0.19 g ethanol/g sugar consumed) and total sugar conversion (83.47%) was observed for strain DBT-IOC-X2 compared to strain DSM 2246 (Table 2). Data analysis showed that the concentration of soluble metabolites [ethanol (2.83 g dm−3), acetate (0.67 g dm−3),

Fig. 3. Time course of sugar uptake and product formation as a function of two different concentration of sugar mixtures: A; (5 g dm−3 glucose plus 5 g dm−3 xylose) and B; (10 g dm−3 glucose plus 10 g dm−3 xylose). Lowercase letters represents the level of significance [ANOVA Tukey's test; ***p < 0.001, * = 0.05 > p > 0.01, and ** = 0.01 > p > 0.001] in the treatment conditions described.

Table 2 Comparative fermentation performance of strain DBT-IOC-X2 and DSM 2246 on various substrates at 70 °C. Strain

Substrate

Fermentation products (g dm-3)*** Ethanol

Acetate

Lactate

Final pH

Growth (OD600)

Ethanol yield (g/g)#

Substrate conversion (%)##

DBT-IOC-X2

Glucose Xylose Diluted PRSH∗

2.82 ± 0.09j**l 3.30 ± 0.17 2.83 ± 0.17k**l

0.89 ± 0.04h 0.86 ± 0.04h 0.67 ± 0.02f

0.26 ± 0.01a 0.33 ± 0.02b*c 0.37 ± 0.01c

5.54 ± 0.06 5.52 ± 0.06 5.23 ± 0.08

2.05 ± 0.00 2.03 ± 0.03 ND

0.32 ± 0.01 0.34 ± 0.01 0.19 ± 0.01

89.84 ± 0.21 97.96 ± 0.18 83.47 ± 0.13

DSM 2246

Glucose Xylose Diluted PRSH∗

2.44 ± 0.05ij**k** 1.79 ± 0.04 2.37 ± 0.09i

0.56 ± 0.01def 0.49 ± 0.05eg 0.48 ± 0.03dg

0.45 ± 0.01 0.14 ± 0.02 0.28 ± 0.01ab*

5.67 ± 0.04 6.01 ± 0.10 6.3 ± 0.09

1.75 ± 0.03 1.41 ± 0.04 ND

0.38 ± 0.03 0.26 ± 0.00 0.21 ± 0.01

62.44 ± 1.11 69.91 ± 1.65 63.89 ± 0.82

a)

Results are presented as average with error bars ( ± ) showing standard deviation of the samples prepared in triplicate. Fermentation of various substrates by uninoculated controls was less than 10%. Lowercase letters represents the level of significance [ANOVA Tukey's test; ***p < 0.001, ** = 0.01 > p > 0.001, and * = 0.05 > p > 0.01] in the treatment conditions described. c)# Yields were calculated as gram of ethanol produced per gram of total sugars consumed. d)## Substrate conversion (%) is based on relative initial sugar concentration values. [Abbreviations: PRSH, Pre-treated rice straw hydrolysate; OD600, Optical density at 600 nm; ND, Not determined]. b)

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and lactate (0.37 g dm−3)] achieved by strain DBT-IOC-X2 was somewhat higher compared to strain DSM 2246 [ethanol (2.37 g dm−3), acetate (0.48 g dm−3), and lactate (0.28 g dm−3)]. Apart from this, both the strains presented different pattern of sugar (glucose, xylose, cellobiose and arabinose) utilization during the fermentation of PRSH. Higher xylose conversion (92.4%) was achieved by the strain DBT-IOCX2 compared to only 59.16% conversion achieved by the strain DSM 2246. However, arabinose (48%) and cellobiose (63.65%) conversion was higher by strain DSM 2246 compared to only 37.43% and 50.5% conversions respectively, achieved by strain DBT-IOC-X2. With very low initial concentrations of glucose both strains presented 100% conversion. On the basis of these results the carbohydrate utilization preference was Glucose > Xylose > Cellobiose > Arabinose for strain DBT-IOC-X2 and Glucose > Cellobiose > Xylose > Arabinose for strain DSM 2246. Due to limited application of un-detoxified C5 and inhibitor rich waste stream for bioethanol production, we focussed on its effective utilization by our natural isolate. The ethanol yield obtained from batch experiments can be enhanced further by using fed-batch or continuous operation providing better pH control and improved utilization of sugars for ethanol generation. Most studies involving wild-type thermophilic ethanologen, investigated the fermentation of pentose and hexose sugar rich-hydrolysates obtained after pretreatment and enzymatic hydrolysis of different lignocellulosic biomass such as; hemp [8], barley straw [8,9], wheat straw [24,37,38], and rice straw [21]. However, there are limited reports available on ethanol production by thermophilic ethanologens utilizing pre-treated liquid hydrolysate as such. There are also no reports so far on the utilization of rice straw hemicellulosic liquid pre-hydrolysate for bioethanol production by Thermoanaerobacter species and the current study is first such report.

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4. Concluding remarks A novel C5-fermenting bacteria Thermoanaerobacter sp. DBT-IOC-X2, was isolated from thermal hot spring and exhibited highest ethanol yield from xylose among the known member of the genus Thermoanaerobacter. This isolate has the potential to achieve simultaneous uptake and fermentation of both C5 and C6 sugars to produce ethanol. In this study due to the broad substrate spectrum and inhibitor tolerance, strain DBT-IOC-X2 degraded all types of C5 and C6 sugars present in the waste stream directly, emphasizing the likely cost-effectiveness of the process. However, further optimization work is needed before proposing its application for industrial ethanol production. The lower substrate tolerance of the strain revealed the need to further develop the fermentation process, involving use of controlled bioreactor conditions, cultivation of strain in fed-batch or continuous fermentation mode, and genetic engineering of the strain to redirect the metabolic flux to the desired end product i.e. bioethanol. Acknowledgement The authors thank Deakin University and DBT-IOC Centre for Advance Bioenergy Research, Indian Oil R & D centre, India for supporting collaborative research. One of the authors NS thanks the DIRI program of Deakin University for providing a scholarship to pursue this research work. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.biombioe.2018.05.009. Conflicts of interest The authors declare no financial or commercial conflict of interest. 129

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