Bioresource Technology 102 (2011) 8432–8436

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Bioresource Technology

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Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108

Xin Zhao, Defeng Xing ⇑, Na Fu, Bingfeng Liu, Nanqi RenState Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR China

a r t i c l e i n f o

Article history:Received 27 December 2010Received in revised form 19 February 2011Accepted 21 February 2011Available online 24 February 2011

Keywords:Biohydrogen productionHydrogen-producing bacteriumClostridium beijerinckiiIsolationCulture condition

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.02.086

⇑ Corresponding author. Address: P.O. Box 2614Environmental Engineering, Harbin Institute of TecNangang District, Harbin, Heilongjiang Province 1500986282110.

E-mail addresses: dxing@hit.edu.cn, ixdf@yahoo.co

a b s t r a c t

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and iden-tified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical char-acteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 wereinvestigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highlydependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogensource for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell bio-mass yield in different culture conditions. The maximum hydrogen evolution, yield and production rateof 2209 ml H2/l medium, 1.97 mol H2/mol glucose and 104.20 ml H2/g CDW h�1 were obtained at 9 g/l ofglucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 �C, respectively. These resultsdemonstrate that C. beijerinckii can efficiently produce H2, and is another model microorganism for bio-hydrogen investigations.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction 2008), recently has been found to be capable of hydrogen produc-

Hydrogen (H2) is an environmentally friendly energy resourceand an ideal alternative to fossil fuels, and has the highest energycontent per unit weight (142 kJ/g or 61,000 Btu/lb) of all the natu-rally occurring fuels (Das, 2009; Das and Veziroglu, 2008; Renet al., 2009a). H2 can be produced by geological and biological pro-cesses in natural environments (Boyd et al., 2010; Lin et al., 2005).Biological hydrogen production using microorganisms is a promis-ing method of utilizing low-value waste as raw materials (organicwastewater and biomass) to produce an energy source whichrequires a lower energy supply and has many other benefitscompared with chemical–physical methods (Abreu et al., 2010;Chu et al., 2011; Das, 2009; Das and Veziroglu, 2008; Xing et al.,2008b).

Dark-fermentative hydrogen-producing bacteria (dHPB) such asClostridium, Ethanoligenens, Enterobacter and Bacillus have been iso-lated from bioreactors and natural environments (Das, 2009; Dasand Veziroglu, 2008; Ren et al., 2007, 2009a; Xing et al., 2006; Zhaoet al., 2010). Clostridium is one of the highly-effective hydrogen pro-ducers within the Firmicutes phylum, and many strains of whichhave been isolated and studied (Jo et al., 2010; Lin and Shei, 2008;Morimoto et al., 2005; Wang et al., 2008; Wang and Wan, 2008).Clostridium beijerinckii, which received much attention as a usefulmicroorganism for butanol production (Ezeji et al., 2003; Lee et al.,

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, School of Municipal andhnology, 73 Huanghe Road,0, PR China. Tel./fax: +86 451

m (D. Xing).

tion (Hatch and Finneran, 2008; Kim et al., 2008; Pan et al., 2008).Previous studies have shown that culture conditions or operat-

ing parameters can significantly affect cell growth and hydrogenproduction. For example, Wang and Wan (2008) studied the effectof Fe2+ on hydrogen production in a mixed culture, and found thatglucose utilization decreased with Fe2+ concentration from 0 to1500 mg/l, and a maximum hydrogen yield of 311.2 ml/g glucosewas obtained at the Fe2+ concentration of 350 mg/l. Li et al.(2008) investigated the effects of pH and glucose concentrationson hydrogen production by sludge in batch culture. Lin and Lay(2004) found that optimal concentrations of carbonate and phos-phate enhanced the hydrogen production rate by 1.9-fold. Chonget al. (2009) studied the effects of pH, glucose and iron sulfate con-centration on hydrogen yield by Clostridium butyricum EB6.Although culture conditions have been investigated widely, theoptimal culture conditions for different species or strains vary.

The aim of this study was to isolate, characterize and identify ahydrogen-producing bacterium from a continuous stirred-tankreactor (CSTR) and assess hydrogen production. To determine theoptimal conditions for maximum hydrogen production, the effectsof culture temperature, pH, substrate concentration, nitrogen sourceand inoculum volume on hydrogen production were investigated.

2. Methods

2.1. Isolation of hydrogen-producing bacterium

Anaerobic sludge was collected from a continuous stirred-tankreactor (CSTR) used for biohydrogen production. A bacterial

Fig. 1. Phylogenetic tree showing the relationship between strain RZF-1108 and related species based on 16S rRNA gene sequences.

X. Zhao et al. / Bioresource Technology 102 (2011) 8432–8436 8433

suspension was produced by vortexing the sludge in an anaerobictube containing 10 ml of sterile anaerobic 1% NaCl and glass beads(2 mm diameter). After serial dilution of the bacterial suspension,isolation was performed by the Hungate roll-tube technique(Hungate, 1969) with PYG medium (1 l) containing 10 g glucose,3 g peptone, 1 g yeast extract, 21.5 g Na2HPO4�12H2O, 8.1 gKH2PO4�2H2O, 0.1 g MgCl2�6H2O, 0.2 g FeSO4�7H2O, 0.1 g L-cysteine,10 ml mineral salt solution (containing 0.01 g/l MnSO4�7H2O,0.05 g/l ZnSO4�7H2O, 0.01 g/l H3BO3, 0.01 g/l CaCl2�2H2O, 0.01 g/l Na2MoO4, 0.2 g/l CoCl2�6H2O, 0.01 g/l AlK(SO4)2�12H2O, 0.001 g/lNiCl�6H2O, 2.0 g/l N[(CH2)COO]3) and 10 ml vitamin solution (con-taining 0.01 g/l cobalamin, 0.025 g/l vitamin C, 0.025 g/l riboflavin,0.02 g/l citric acid, 0.05 g/l pyridoxal, 0.01 g/l folic acid, 0.025 g/lcreatine and 0.01 g/l P-aminobenzoic acid).

Single colonies were transferred to the same broth and incu-bated at 35 �C for 2 days. The roll-tube procedure was repeatedseveral times until a pure culture was obtained. Routine cultivationwas in anaerobic tubes (18 � 150 mm) sealed with butyl rubberstoppers under a gaseous atmosphere of 99.999% N2 (200 kPa) at35 �C.

2.2. Morphological test

Gram staining was performed as previously described(Beveridge, 2001). Morphological examinations were performedwith a light microscope BX51 (Olympus, Japan), an atomic forcemicroscope (AFM) (DI Bioscope, Veeco, USA) and a transmissionelectron microscope (TEM) (TECNAIG2, Philips). A small drop of10 times diluted RZF-1108 suspension was placed and thensmeared on the ethanol and HCl pretreated glass slide using aninoculating loop, and examined using AFM after air drying. A 5 ll

cell suspension of RZF-1108 was placed on a 200 mesh formvarcarbon-coated copper grid, and wicked off after 3 min. The samplewas soaked in 5 ll of uranyl acetate (2%) for 30 s, then drained, air-dried and examined using TEM.

2.3. Identification and phylogenetic analysis

Total genomic DNA was extracted from strain RZF-1108 usingthe Bacterial DNA Mini Kit (Watson Biotechnologies Inc., China).The 16S rRNA gene was amplified as previously described (Xinget al., 2006). The PCR product was purified using the Gel ExtractionMini Kit (Watson Biotechnologies Inc, China), and cloned with thepMD19-T plasmid vector system (TaKaRa, Dalian, China). The 16SrRNA gene sequence was aligned and identified against existing se-quences in the GenBank database using the BLAST program.

2.4. Hydrogen production in batch culture

The PYG medium, as described above, was dispensed anaerobi-cally into 100 ml anaerobic culture bottles. Prior to testing, thebottles containing 50 ml liquid medium were flushed with ultrahigh-pure nitrogen gas (99.999%) for 5 min, sealed with rubberplugs, and autoclaved at 121 �C for 15 min. The bottles were sha-ken in an air bath at 140 rpm. The gas was collected by the drainingmethod after culturing for 20 h. To determine the effects of cultureconditions on hydrogen production, temperature, pH value, sub-strate concentration, nitrogen source and inoculum volume werealternately varied. The culture temperature of 25–40 �C and initialpH of 4–10 (adjusted by 1 mol/l HCl or 1 mol/l NaOH solution)were set. The glucose concentration of 5–15 g/l was fixed in theexperiment in order to study the effect of substrate concentration.

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Fig. 2. The effect of culture temperature on hydrogen production by C. beijerinckiiRZF-1108.

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8434 X. Zhao et al. / Bioresource Technology 102 (2011) 8432–8436

The nitrogen sources at a concentration of 4 g/l, included peptone,yeast extract, beef extract or urea which was supplemented inthe medium. The inoculum volume varied in the range of 2–15%(v/v).

2.5. Analytical methods

Glucose was measured by a glucose assay kit (RongshengBiotech, Shanghai, China) using the peroxidase method. Biomasswas measured as cell dry weight (CDW) according to a previouslydescribed method (Ren et al., 2009b). The hydrogen content, vola-tile fatty acids and alcohol were analyzed by gas chromatography(GC, Agilent 4890) as previously described (Xing et al., 2008a).

3. Results and discussion

3.1. Identification of a hydrogen-producing isolate

A strain designated as RZF-1108 was isolated from the anaero-bic sludge in the CSTR. RZF-1108 is a gram-positive motile rod witha terminal flagellum. The bacterial cells occurred singly or in pairsand had a diameter of 0.7–1.0 lm and a length of 3.0–5.0 lm (seesupplemental material Fig. S1). Spores were observed when theconditions were unfavorable for RZF-1108. A colony of strainRZF-1108 was milk-white in color, circular, opaque and slightlyconcave with a ring margin, and had a diameter of about 2.0 mmafter cultivation on agar medium at 35 �C for 24 h. Its main byprod-ucts from glucose fermentation were acetic acid, butyric acid, H2,CO2, and lower concentrations of ethanol and butanol.

The 16S rRNA gene sequence of 1504 bp (GenBank accessionnumber GQ375085) was amplified from the genomic DNA of strainRZF-1108, and showed 99% identity with C. beijerinckii NCIMB8052(Fig. 1). The physiological and morphological characters were alsoconsistent with C. beijerinckii. Thus, RZF-1108 was identified as anew strain within the species C. beijerinckii RZF-1108 on thebasis of the 16S rRNA gene analysis and physiobiochemicalcharacteristics.

3.2. Effect of culture temperature on hydrogen production

Growth and hydrogen production of strain RZF-1108 were ob-served at temperatures ranging from 25 �C to 40 �C with an opti-mal temperature of 35 �C. The hydrogen production and yield,and cell dry weight (CDW) increased with increasing temperature(from 25 �C to 35 �C) and decreased at temperatures >35 �C. Themaximum hydrogen yield and CDW were 1.86 mol H2/mol glucoseand 1.07 g/l at 35 �C, respectively (Fig. 2). Volatile fatty acids(VFAs) showed the same trend as cell biomass and hydrogen pro-duction. Final pH corresponded negatively with VFAs production.The hydrogen production rate was low at 25 �C, similar to hydro-gen evolution, yield, CDW and VFAs, then increased and changedslightly when the temperature was between 30 and 38 �C, then de-creased. Lower or higher temperatures inhibited the growth andhydrogen production of strain RZF-1108.

3.3. Effect of initial pH on hydrogen production

Hydrogen production and bacterial growth were not observedat the initial pH of 4.0. When the initial pH increased from 5.0 to7.0, the hydrogen yield increased from 0.53 mol to 1.75 mol H2/

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X. Zhao et al. / Bioresource Technology 102 (2011) 8432–8436 8435

mol glucose, and then decreased gradually when pH was increasedfurther (Fig. 3). The CDW, accumulative hydrogen production andVFAs had a similar trend to that of hydrogen yield. The hydrogenproduction rate was increased with increasing initial pH, with amaximum hydrogen production rate of 187.72 ml H2/g CDW�h�1

at pH 10.0. The final pH values in different initial pH were almostin an oblique line as hydrogen production rate, with the exceptionof the initial pH of 7.0. The maximum hydrogen yield of 1.75 molH2/mol glucose and maximum CDW of 0.95 g/l were obtainedwhen the initial pH was 7.0 and 6.5, respectively. These resultswere consistent with previous studies in which hydrogen produc-tion and cell growth were dependent on initial pH (Li et al., 2008;Li and Fang, 2007; Pan et al., 2008). Low pH usually results in alower level of ATP in the cell and inhibition of bacterial growthand activity (Bowles and Ellefson, 1985; Ferchichi et al., 2005).

3.4. Effect of glucose concentration on hydrogen production

The initial glucose concentrations affected hydrogen productionand growth of strain RZF-1108 (Fig. 4). The hydrogen yield and pro-duction rate increased with increasing glucose supplementationfrom 5 g/l to 9 g/l, reached a maximum of 1.80 mol H2/mol glucoseand 117.30 ml H2/g CDW�h�1 at 9 g/l, and then decreased. Thehydrogen evolution and CDW showed the same trend as that ofhydrogen yield and production rate, however, the maximum of1891 ml H2/l medium and 0.99 g/l were obtained when the glucoseconcentration was 10 g/l. Substrate utilization and final pH de-creased with increasing glucose concentration, and some substrateremained when the glucose concentration was over 9 g/l. Enhance-ment of cell growth and VFAs slowed when the glucose concentra-tion was higher than 10 g/l, implying that pH decreased due to the

accumulation of VFAs, and inhibited hydrogen production whenthe concentration was over a certain threshold.

3.5. Effect of nitrogen source on hydrogen production

C. beijerinckii can utilize various organic and inorganic nitrogensources such as beef extract, peptone, yeast extract, urea, NH4NO3

and NaNO3 (Pan et al., 2008). The medium supplemented withyeast extract showed the highest hydrogen yield of 1.26 mol H2/mol glucose, hydrogen evolution of 1417 ml H2/l medium, CDWof 1.14 g/l, VFAs, and lowest final pH of 4.81 of the four nitrogensources tested (Fig. 5). The hydrogen yield and hydrogen evolutionwhen cultured with beef extract were better than those obtained inpeptone and urea. Our experimental results indicated that a suit-able nitrogen source could facilitate cell growth and hydrogen evo-lution, and yeast extract was found to be a favorable organicnitrogen source for cell growth and hydrogen production in strainRZF-1108. The lower hydrogen yield in the presence of single nitro-gen source indicated that combined nitrogen sources may be ben-efit for enhancing cell growth and hydrogen production. The effectsof different nitrogen sources combination need to be further inves-tigated in the future.

3.6. Effect of inoculum volume on hydrogen production

The inoculum volume in the range of 2–15% (v/v) influenced thehydrogen yield, hydrogen evolution and CDW. Although the VFAs,final pH and CDW changed little with different inoculum volumes,glucose utilization increased about 10% with the inoculum volumeincreased from 2% to 15% (see supplemental material Fig. S2). This

8436 X. Zhao et al. / Bioresource Technology 102 (2011) 8432–8436

implied that excessive inoculum volume led to substrate over-consumption and cell overgrowth in a short time, which resultedin a rapid decrease in pH and hydrogen production. The hydrogenyield and evolution were a little higher when the inoculum volumeranged from 6% to 10%, and the highest hydrogen yield of 1.91 molH2/mol glucose occurred with an inoculum volume of 8% (v/v).

3.7. Hydrogen production under optimal conditions

A hydrogen evolution of 2209 ml H2/l medium, a yield of1.97 mol H2/mol glucose and a production rate of 104.20 ml H2/gCDW�h�1 were obtained under the following optimal conditions:9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and35 �C. The CDW and final pH were 1.06 g/l and 4.68, respectively.The hydrogen yield of C. beijerinckii RZF-1108 was comparable withthat obtained by other clostridia (Chen et al., 2001; Heyndrickxet al., 1986; Pan et al., 2008; Zhang et al., 2006).

4. Conclusion

A hydrogen-producing strain was isolated from anaerobicsludge in a continuous stirred-tank reactor (CSTR), and was identi-fied as C. beijerinckii RZF-1108. Yeast extract was the most favor-able nitrogen source for cell growth and hydrogen production instrain RZF-1108. The maximum hydrogen yield of 1.97 mol H2/mol glucose and production rate of 104.20 ml H2/g CDW�h�1 wereobtained under optimal condition of 9 g/l of glucose, 35 �C, initialpH of 7.0 and an inoculum volume of 8% (v/v).

Acknowledgements

This research was supported by the Program for New CenturyExcellent Talents in University (No. NCET-10-0066), NationalNatural Science Foundation of China (Nos. 30870037, 30900046),State Key Laboratory of Urban Water Resource and Environment(Harbin Institute of Technology) (No. 2010TS09), and theFoundation for Innovative Research Groups of the National NaturalScience Foundation of China (No. 50821002).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2011.02.086.

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