Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6

Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6

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Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6 Yujia Jiang a,1, Jiasheng Lu a,1, Yang Lv a, Ruofan Wu a, Weiliang Dong a,b, Jie Zhou a,b, Min Jiang a,b,*, Fengxue Xin a,b,* a

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800, PR China b Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, 211800, PR China

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Consolidated bioprocessing (CBP) is a promising approach for hydrogen production from

Received 19 December 2018

lignocellulose owing to its lower cost and higher efficiency. In this study, the newly isolated

Received in revised form

theromphilic Thermoanaerobacterium sp. strain F6 exhibited the capability of direct utiliza-

21 January 2019

tion of various hemicellulosic and cellulosic materials for hydrogen production, including

Accepted 24 January 2019

xylan, Avicel and filter paper etc. Especially, the maximum cumulative hydrogen produc-

Available online 15 February 2019

tion reached 370.7 mmoL/L from 60 g/L of xylan. In addition, natural lignocellulosic materials, such as corn cob and sugarcane bagasse without any hydrolytic pretreatment could


also be directly utilized as the sole carbon source for hydrogen production. 1822.6 and


826.3 mL H2/L of hydrogen were produced from corn cob and sugarcane bagasse, respec-


tively. The high hydrogen production from cellulosic and hemicellulosic materials were


both benefit from its efficient secretion of hydrolytic enzymes. Thus, Thermoanaer-

Consolidated bioprocessing

obacterium sp. strain F6 is a potential candidate for effective conversion of lignocellulose to hydrogen through CBP. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Introduction Hydrogen is considered as a potential alternative energy source to fossil fuels. Hydrogen not only possesses 122 kJ/g of energy content, which is 2.75 times higher than fossil fuel, but also avoids serious negative effects on environment without

CO2 emission [1e3]. It was reported that approximately fifty million tonnes of hydrogen were traded annually worldwide, which also grows at a rate of nearly 10% annually for the time being [4]. Based on the National Hydrogen program of the United States, hydrogen will share 8e10% of total energy market by 2025 [5,6].

* Corresponding authors. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing 211800, PR China. E-mail addresses: [email protected] (M. Jiang), [email protected] (F. Xin). 1 The first two authors have contributed equally to this work. https://doi.org/10.1016/j.ijhydene.2019.01.226 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

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Biological process is regarded as an environmental friendly approach to produce hydrogen without external energy input required, which includes bio-photolysis, photo-fermentation, dark-fermentation processes and hybrid biohydrogen production by electrochemical processes [7]. Among these methods, dark fermentation is considered as the most promising one due to its higher hydrogen production rate and broader feedstock types [8,9]. As for dark fermentation process, Clostridium species are the most well studied genus for hydrogen production [10,11]. However, most Clostridium sp. could only utilize starchy based materials as the feedstocks, such as glucose etc, which will increase the cost for hydrogen production [12]. Lignocellulose is an abundant and renewable material, which is mainly composed of cellulose, hemicellulose and lignin [13]. Unfortunately, most mesophilic Clostridium sp. cannot utilize lignocellulose effectively due to their low or non expression of lignocellulose degrading enzymes [11]. Hence, pretreatment hydrolysis process should be performed through supplementation of hydrolytic enzymes or chemical method for hydrogen production when lignocellulose was used as the substrate [14]. This process will also increase the cost and hinder the commercialization of cellulosic biohydrogen production. Consolidated bioprocessing (CBP) is considered as a promising approach, which combines enzymes production, lignocellulose hydrolysis and fermentation in one step [15]. Thermophilic bacteria is considered to play an important role in CBP owing to their higher lignocellulose hydrolysis efficiency and hydrogen production rate [16,17]. Recently, thermophilic Thermoanaerobacterium genus have attracted much attention on hydrogen production through CBP, which can directly utilize hemicellulose and even few of them can degrade cellulose. For instance, hydrogen production could reach 54.0, 42.5 and 43.8 mmoL/L from 5 g/L of xylan, filter paper and cellulose, respectively by using T. thermosaccharolyticum strain M18 [18]. However, the hydrogen production from lignocellulose still kept a relatively low level. In this study, a thermophilic bacterium was first isolated and identified, which possessed the capacity of direct conversion lignocellulose into hydrogen. Furthermore, hydrogen production from various lignocellulosic substrates, including xylan, Avicel, filter paper, corn cob and sugarcane bagasse were optimized and compared, which would pave the way to produce the net energy form lignocellulosic materials through CBP.

Materials and methods Microorganism and growth media Sludge sample was collected from Laoshan Nature Park, China. 1 g diluted (1:10) sludge sample was incubated at 60  C in 100-mL serum bottles with a working volume of 40 mL purged with N2 (nitrogen). The sample was cultivated in a reduced mineral salts medium, which contains 1.0 g/L NaCl, 0.5 g/L MgCl2$6H2O, 0.2 g/L KH2PO4, 0.3 g/L NH4Cl, 0.3 g/L KCl, 0.015 g/L CaCl2$2H2O. The media were also supplemented with 1 mL trace element solution, 1 mL Na2SeO3eNa2WO4 solution and 3.0 g/L yeast extract [19]. In addition, 20 mM N-


[Tris (hydroxymethyl) metyl]-2-aminopropanesulfonic acid (TES) and 10 mg resazurin were added as the pH buffer agents and oxygen indicator respectively. After six transfers in medium with cellulose as the sole carbon source, the enriched culture was diluted 105 times and screened on agar plats with Congo red staining method.

16S rRNA gene sequences The genomic DNA of the isolated strain was extracted using Gþ bacteria genomic DNA kit (ZOMANBIO, China). The extracted DNA was used as the template for PCR amplification of the 16S rDNA with a pair of universal bacterial primer 27 forward primer: 50 -AGAGTTTGATCCTGGCTCAG-30 , and 1492 reverse primer: 50 -TACGGCTACCTTGTTACGACTT-30 . The PCR process was performed using PrimeSTAR® HS DNA Polymerase (Takara, Shanghai, China), and amplification was conducted under the following conditions: 95  C for 5 min; 30 cycles of 98  C for 10 s, 55  C for 15 s, 72  C for 1 min 40 s; and finally 72  C for 10 min. The purified product of PCR was cloned into vector pMD19-T using the pMD19-T vector system I kit according to manufacturer instructions (Takara, Shanghai, China). The sequenced 16S rRNA gene was compared with the sequences in the GenBank databases by the BLASTN program. Then, Clustal X program was used to align these sequences. Finally, the phylogenetic relationship was constructed by using the neighbor-joining method using MEGA 7.0. The nucleotide sequence of strain F6 was deposited in the GenBank under an accession number of KY421592.1.

Fermentation tests of isolated strain The isolated strain F6 was cultivated anaerobically in the medium described above. The bacteria were cultivated by a single colony from an agar plate. Strain F6 should be incubated for 60 h, then added 5% v/v of inoculum into 40 mL medium in 100-mL serum bottles with pH adjusted to 6.5. The ability of isolated strain to utilize various lignocellulosic materials, including xylan, corn cob, Avicel, filter paper and sugarcane bagasse etc were investigated. Then, the effects of concentrations of these carbon sources on hydrogen production were performed. All fermentation batches were incubated at 120 rpm and 60  C. In the fermentation process, pH was adjusted to 6.5 with 3 M NaOH solution. Each experiment was carried out in triplicate. The activity of xylanase and cellulase were determined by the 3,5-dinitrosalicylic acid (DNS) method. The reaction mixture contained 1 mL of fermented supernatant and 1 mL of 1% (w/v) corresponding substrate in PBS (50 mM pH 6.5). After incubation at 60  C for 30 min, the contents of reducing sugars were calculated from the increasing absorbance at 540 nm according to standard curve. One unit (U) of enzyme activity was defined as the amount of enzyme which released 1 mmol reducing sugar per minute.

Analytical methods Metabolic products were detected by gas chromatography (GC-2010, Shimadzu Scientific Instruments, Japan) equipped with an InterCap WAX column (0.25 mm  30 m, GL Sciences


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Inc., Japan) and a flame ionization detector (FID) using the method described by Jiang et al. [19]. All samples were centrifuged at 12,000 g for 5 min at 4  C, then 50 mL HCl (2M) was added in 950 mL of samples. Isobutanol was used as internal standard. The total volume of biogas production was measured on-line through a mass flow controller, a mass flow meter (CS200-A,C,D MFC/MFM, Sevenstar, China) and a gas flow accumulator (D08-8C, Sevenstar, China). The hydrogen content was detected using a HY-OPTIMATM 700 Process Hydrogen Analyzer (H2scan, USA) [20].

Results and discussion Isolation and characterization of thermophilic cellulosic strain for hydrogen production The cellulosic cultures were enriched using cellulose as the substrate at 60  C. After six generations, diluted cultures were spread on cellulose agar plates. Congo red staining methods were used to select the efficient cellulose degradation thermophilic strains. Ten colonies with the largest clearing zones were selected for further liquid fermentation using cellulose as the substrates. Among the 10 colonies, one of them showed the best hydrogen production capacity from 10 g/L of cellulose directly at 60  C. The cumulative hydrogen production reached 1648.2 mL H2/L, accompanied with 1.17, 1.32, 0.31 and 0.12 g/L of acetate, butyrate, ethanol and butanol production. Interestingly, this thermophilic hydrogen-producing microorganism can grow on a wide variety of complex and simple carbohydrates, such as cellulose, xylan, glucose and xylose etc. Further phylogenetic analysis of the 16S rDNA (NCBI Accession Number KY421592.1) revealed that the newly isolated strain belongs to Thermoanaerobacterium genus with 98% similarity with T. thermosaccharolyticum DSM571 (formerly Clostridium thermosaccharolyticum, NCBI Accession Number CP002171.1) (Fig. 1). The phenotypical characteristics of the isolated strain were also

consistent with Thermoanaerobacterium genus, demonstrating a group of rod-shaped, motile, obligate anaerobic Gram-positive, and spore-forming bacteria. Hence, the isolated strain is named as Thermoanaerobacterium sp. strain F6.

Hydrogen production from hemicellulosic materials by Thermoanaerobacterium sp. F6 Hemicellulose generally takes account of 15e35% in lignocellulose, and xylan is the most abundant hemicelluloses [21]. Recently, relatively high yield of hydrogen from xylan has been reported. For instance, a reported strain M18, which belongs to the same genus as strain F6 could produce 54.0 mmoL/L of hydrogen from 5 g/L of xylan [18]. To explore the hydrogen production potential of strain F6, different amounts of xylan were used first. As seen in Fig. 2, strain F6 showed an effective hydrogen production from xylan, and the daily hydrogen production reached the highest at 48e96 h. The cumulative hydrogen production was enhanced obviously with the increase of the concentrations of xylan. For example, when 5 g/L of xylan was used as the substrate, hydrogen production was 18.5 mmoL/L. When xylan concentration was increases up to 60 g/L, the hydrogen production reached 370.7 mmoL/L, which is 19 times higher than that of 5 g/L xylan (Fig. 2A). Especially, the maximum daily hydrogen production reached 1326.3 mL H2/L at 96 h with the maximum hydrogen production rate of 2.02 mmoL/L/h (Fig. 2B). Finally, the cumulative hydrogen production reached the highest 370.7 mmoL/L at 288 h, which is about 3 times higher than the reported highest hydrogen production from xylan through CBP [12]. Accompanied with hydrogen production, GC analysis also showed that 1.76 g/L of ethanol, 0.66 g/L of butanol, 2.64 g/ L of acetate and 2.48 g/L of butyrate were produced by strain F6. The solvents production, especially butanol production were also higher than other reported CBP strains. Using lignocellulosic biomasses as the sole substrate for hydrogen production generally requires expensive and time-

Thermoanaerobacterium sp. strain F6 (KY421592.1) Thermoanaerobacterium aotearoense strain JW/SL-NZ613 (NR 026296.1) Thermoanaerobacterium saccharolyticum strain B6A-RI (NR 044621.1) Thermoanaerobacterium thermosulfurigenes strain 4B (NR 044622.1) Thermoanaerobacterium xylanolyticum strain LX-11 (NR 102771.1) Thermoanaerobacterium aciditolerans strain 761-119 (NR 042856.1) Thermoanaerobacterium calidifontis strain Rx1 (NR 113051.1) Thermoanaerobacterium thermosaccharolyticum strain DSM 571 (NR 074419.1) Thermohydrogenium kirishiense strain DSM 11055 (NR 117160.1) Thermotoga elfii (X80790.1) 0.050 Fig. 1 e Phylogenetic tree of Thermoanaerobacterium sp. F6 based on its 16S rDNA sequence.


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Fig. 2 e Hydrogen production from hemicellulosic materials by Thermoanaerobacterium sp. F6. A. Hydrogen production from different concentrations of xylan. B. Fermentation profile for hydrogen and VFAs production from 60 g/L of xylan. C. Fermentation profile for hydrogen production from 30 g/L of corn cob.

consuming pretreatment processes to facilitate the materials more accessible to utilization. However, the pretreatment process accounted for about 20% of costs through economic analyses. Corn cob is a kind of agricultural residues rich in hemicellulose, whose contents can reach 38% [22]. Hence, hydrogen production from corn cob without any hydrolytic process was further investigated. From 30 to 60 g/L of corn cob, 41.7 and 66.7 mmoL/L of hydrogen were accumulated, respectively (Fig. 2C). The related enzyme activities were also measured aiming to analyze and confirm the hemicellulose and cellulose degradation conditions during the corn cob fermentation. When using 60 g/L of corn cob as the sole substrate, the xylanase activity increased rapidly and achieved the maximum value of 0.53 U/mL at 48 h. Whereas the cellulase activity was not obviously detected until 48 h, and the maximum level reached 0.45 U/mL at 96 h, indicating that strain F6 preferred to hemicellulose utilization. Although the final production was far less than the same concentrations of xylan, these results showed the breakthrough for hydrogen production from lignocellulosic materials without any hydrolysis pretreatment.

Hydrogen production from cellulosic materials by Thermoanaerobacterium sp. F6 Cellulose is the main component in lignocellulosic materials, which generally takes 35e50%. To further investigate the potential feedstocks for hydrogen production by using thermophlic strain F6, Avicel and filter paper were utilized as the carbon and energy sources. Compared with that using hemicellulosic materials as substrates, dark fermentation using cellulosic substrates takes a longer fermentation duration. For example, only 192 h were needed when 30 g/L of xylan was used as the substrate, however, more than 384 h were needed when using the same concentration of cellulose, resulting in much lower hydrogen production rates from Avicel (Table 1). The highest hydrogen production was obtained when 30 g/L of Avicel was used as the substrate (Fig. 3A). Especially, from 48 h to 384 h, the daily hydrogen productions were all above 10 mmoL/L, and the cumulative final hydrogen production achieved 263.7 mmoL/L, which was also 40.7% higher than using 30 g/L of xylan as the carbon source (Fig. 3B). As shown in Fig. 3B, the higher VFAs were obtained, the higher daily

Table 1 e Hydrogen production rate and hydrogen yield using different lignocellulosic feedstocks by Thermoanaerobacterium sp. F6. Subtracts Xylan Avicel Filter paper Corn cob Sugarcane

Substrate consumption (g/L) 52.63 23.20 25.57 32.33 16.67

± 0.86 ± 1.23 ± 1.22 ± 0.95 ± 0.55

H2 production (mmol/L) 370.70 ± 1.59 263.74 ± 1.73 277.82 ± 1.61 66.71 ± 1.80 30.24 ± 1.65

H2 production rate (mmol/L/h) 1.29 0.61 0.64 0.69 0.31

± 0.006 ± 0.004 ± 0.004 ± 0.019 ± 0.017

H2 yield (mmol/g substrate) 7.04 ± 0.13 11.39 ± 0.63 10.88 ± 0.53 2.07 ± 0.12 1.81 ± 0.05


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Fig. 3 e Hydrogen production from cellulosic materials by Thermoanaerobacterium sp. F6. A. Hydrogen production from different concentrations of Avicel. B. Fermentation profile for hydrogen and VFAs production from 30 g/L of Avicel. C. Hydrogen production from different concentrations of filter paper. D. Fermentation profile for hydrogen and VFAs production from 30 g/L of filter paper. E. Fermentation profile for hydrogen production from 30 g/L of sugarcane bagasse.

hydrogen production was achieved, indicating the relevance between VFAs and hydrogen production (Fig. 3B), and the similar results have also referred in the previous reports [23]. Finally, 1.76 g/L of acetate, 1.98 g/L of butyrate, 1.06 g/L of ethanol and 0.21 g/L of butanol were produced from 30 g/L of Avicel. It should also be noticed that substrate types would affect hydrogen ratio. For example, the hydrogen to carbon dioxide was 1.2:1 when using Avicel as the substrate; whereas higher hydrogen to carbon dioxide ratio of 1.4:1 was found when xylan was used as the substrate. To elucidate the biodegradation characteristics of strain F6 on cellulosic feedstocks, filter paper was also utilized as the

sole carbon source. Rapid filter paper degradation was observed, as 277.8 mmoL/L of hydrogen production with 1.81 g/L of acetate, 2.28 g/L of butyrate, 1.03 g/L of ethanol and 0.28 g/L of butanol were found from 30 g/L of filter paper after 432 h (Fig. 3D). The hydrogen and metabolic products production were similar to those using the same concentration of Avicel. The hydrogen production rate from filter paper was also similar to that of the Avicel. However, it is interesting that when the concentrations of cellulosic materials were above 30 g/L, the hydrogen production was not enhanced any more (Fig. 3C). The hydrogen production from Avicel and filter paper were higher than reported studies. For example, 31.9 mmoL/L

Table 2 e Biofuels and chemicals production by microbial consortia. Strains T. thermosaccharolyticum KKU19 T. thermosaccharolyticum KKU19 T. thermosaccharolyticum KKU19 T. thermosaccharolyticum M18 T. thermosaccharolyticum M18 T. thermosaccharolyticum M18 T. thermosaccharolyticum M18 Thermoanaerobacterium sp. F6 Thermoanaerobacterium sp. F6 Thermoanaerobacterium sp. F6 Thermoanaerobacterium sp. F6 Thermoanaerobacterium sp. F6


H2 production (mmol/L)

Xylan Cellulose powder a-Cellulose Xylan Filter paper Cellulose Corn cob Xylan Avicel Filter paper Corn cob Sugarcane

92.5 31.9 28.9 54.0 42.5 43.8 9.7 370.7 263.7 277.8 66.7 30.2

References [12] [12] [12] [18] [18] [18] [18] This This This This This

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of hydrogen was produced from 10 g/L of cellulose powder by using T. thermosaccharolyticum KKU19 [12] (Table 2). To further investigate the hydrogen production from real lignocellulosic feedstock, sugarcane bagasse was utilized as the sole carbon source for hydrogen production without any hydrolysis pretreatment. In this study, sugarcane bagasse was mainly composed of 44% of cellulose and 34% of hemicellulose. Strain F6 could grow well in the medium containing sugarcane bagasse, indicating it possess effective hemicellulose and cellulose degrading enzyme systems. However, much lower hydrogen production was obtained, as only 30.2 mmoL/ L of hydrogen was obtained from 30 g/L of sugarcane bagasse after 96 h (Fig. 3E). In addition, VFAs (acetate and butyrate) and biofuel (ethanol and butanol) were all lower than those of pure cellulose and hemicellulose. Future pretreatments are still needed to remove lignin, which will facilitate cellulose and hemicellulose degradation and improve the final hydrogen production.

Conclusions A newly isolated CBP thermophilic hydrogen-producing bacterium, Thermoanaerobacterium sp. strain F6 was isolated and identified, which has the capacity of producing hydrogen directly from various pure hemicellulosic and cellulosic materials, such as xylan, Avicel and filter papers. In addition, strain F6 could also grow well in natural lignocellulosic feedstocks, such as corn cob and sugarcane bagasse, but a lower hydrogen production was achieved than that of pure substrates. Taken together, strain F6 was more preference to hemicellulosic feedstocks, and higher maximum hydrogen production was obtained from hemicellulosic materials than that of cellulosic ones. The results of this study offer a promising candidate strain with the relatively higher hydrogen production from lignocellulosic substrates directly, but the improvement of hydrogen yield should also be further studied.

Acknowledgements This work was supported by National Natural Science Foundation of China (No. 21706125, No. 21727818, No. 21706124, No. 31700092), the Jiangsu Province Natural Science Foundation for Youths (BK20170993, BK20170997, BK20180712), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1109), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture of China.


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