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Characteristics for production of hydrogen and bioflocculant by Bacillus sp. XF-56 from marine intertidal sludge
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 0 ( 2 0 1 5 ) 1 4 1 4 e1 4 1 9
Available online at www.sciencedirect.com
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Characteristics for production of hydrogen and bioflocculant by Bacillus sp. XF-56 from marine intertidal sludge Hongyan Liu a,*, Guochao Chen a, Guangce Wang a,b a
Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science & Technology, Tianjin 300457, PR China b Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China
article info
abstract
Article history:
This study aimed to isolate the novel bacterial strains from intertidal sludge that could
Received 19 September 2014
produce hydrogen and bioflocculant simultaneously. A bacterium named as strain XF-56
Received in revised form
was isolated for high production of hydrogen and bioflocculant. The isolated strain was
15 November 2014
designated as Bacillus sp. XF-56 by 16S rRNA gene sequence analysis. Strain XF-56 could
Accepted 21 November 2014
grow using various carbon sources and glucose was found to be favorable for production of
Available online 13 December 2014
hydrogen and bioflocculant. Moreover, this strain XF-56 was able to produce hydrogen and bioflocculant at initial pH from 5.0 to 10.0, with an optimum initial pH for hydrogen pro-
Keywords:
duction at 7.0 and bioflocculant activity at 8.0 respectively. In addition, strain XF-56 showed
Intertidal sludge
the strong salt tolerance and could produce hydrogen and bioflocculant at the salt con-
Strain XF-56
centration from 0.4% to 6.0%. The hydrogen production and bioflocculant activity by strain
Hydrogen production
XF-56 were 1.47 ± 0.05 mol H2/mol glucose and 93.5% in marine culture conditions, which
Bioflocculant activity
increased to 1.79 ± 0.06 mol H2/mol glucose and 98.6% respectively in fresh ones. This result demonstrated that the hydrogen-producing bacterium XF-56 with simultaneous production of bioflocculant is beneficial for cost-effective bio-treatment process and has a wide application potential in actual fresh and marine wastewater. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Biological hydrogen production is a promising technique using organic wastewater and renewable biomass and has attracted worldwide attention [1,2]. Microorganisms utilized for biological hydrogen production were commonly divided into two main categories: photosynthetic bacteria and darkfermentative bacteria. Hydrogen production by dark-
fermentative bacteria is more widely used because it has higher hydrogen production rate with no light requirements [3]. The most dark-fermentative hydrogen-producing bacteria, such as Enterobacter, Bacillus and Clostridium species, have been reported for high hydrogen production. However, adequate hydrogen yields by these bacteria have not been achieved so far [4,5]. Regarding the fermentative hydrogen production system, finding the industry-suitable strains with high hydrogen-producing efficiency and good application potential
* Corresponding author. Tel./fax: þ86 22 60601305. E-mail address: [email protected] (H. Liu). http://dx.doi.org/10.1016/j.ijhydene.2014.11.110 0360-3199/Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
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is still of the most importance [6,7]. The hydrogen-producing bacteria that can use diverse organic wastes as feedstock are necessary for sustainable hydrogen production. Bacillus is an industrially robust organism that can grow well to high densities and compete successfully with contaminating bacteria under fermentative conditions using biowastes as feedback without sterilization [8]. Bacillus has been considered as a candidate hydrogen producer. In the study of screening efficient hydrogen-producing bacteria, we found some strains with the ability of producing hydrogen and bioflocculant simultaneously. The flocculants produced by microorganisms are used to remove suspended solids and metal ions in both aerobic and anaerobic treatment systems [9]. Bioflocculants have been attracted great research interest and widely studied in various industrial processes such as wastewater treatment, drinking water purification and other relevant industries [10,11]. Microbial-produced bioflocculants are biodegradable polymers, including proteins, polysaccharides, lipids and nucleic acid, which are not secondary pollutants and harmless [12]. However, compared with chemical flocculants, bioflocculants have not been widely used because of the high costs, which inhibit the feasibility of industrialization and practical application of bioflocculants [13]. Some efforts have been made to improve the level of microbial-produced bioflocculants, such as searching for low-cost feedstock for bioflocculant production [14]. The procedure to produce hydrogen with a byproduct of the bioflocculant helps to reduce the cost of wastewater treatment using bioflocculant. Yokoi et al. reported that a hydrogen-producing bacterium Enterobacter sp. BY-29 which can produce flocculants and hydrogen simultaneously [15]. Ren et al. isolated a hydrogen-producing bacterium Ethanoligenens harbinense with the ability of autoaggregation by producing bioflocculant [16]. Bioflocculants produced as a byproduct during the fermentative hydrogen production could be an effective procedure for realization of cost-effective practical application and has been a well developed biological treatment for the fresh wastewater [17,18]. In fact, due to the high utilization of seawater, such as mariculture, the accumulation of the high-salt organic wastewater has been becoming a prominent environmental problem in China. The techniques by hydrogen producing bacteria with bioflocculant as a byproduct could use organic contents in the wastewater to generate clean energy and also achieved the purpose of wastewater treatment and purification. However, bacterial strains which can simultaneously produce hydrogen and bioflocculant in fresh condition are not feasible to the use for biological treatment of wastewater with high salt concentration. There have been no reports for simultaneous production of hydrogen and bioflocculant by bacterial strains isolated from marine environments. Among the various marine environments, intertidal zone is the transitional position between marine and terrestrial environments, which has led to the diversity of bacterial communities. In this work, a new hydrogen producing bacterium Bacillus sp. XF-56 which can produce bioflocculant was isolated from intertidal sludge. The optimal culture conditions for production of hydrogen and bioflocculant by strain XF-56 under batch conditions were investigated, which will provide the cost-
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effective approach for application of actual high-salt organic wastewater.
Materials and methods Sludge and enrichment Sludge was collected from the intertidal zone of Bohai, in Tianjin, China (longitude of 116 and latitude of 38). The enrichment culture was set up as 10% (w/v) sludge slurry in LM-H medium. The medium (g/L) was composed of glucose 20.0, yeast extract 1.0, tryptone 4.0, beef extract 2.0, NaCl 30.0, K2HPO4 1.5, MgCl2 0.1, FeSO4,7H2O 0.1, L-cysteine 0.5, as described previously [19]. The enrichment test was performed under anaerobic condition in an incubator shaking at 120 rpm. After 3-day incubation, 1 mL culture broth was inoculated again using the same medium three times, which had led to the enrichment of the mixed microbial culture.
Isolation and identification The enrichment culture was diluted and spread onto agar plates containing the following sterilized medium (g/L): glucose 10.0, yeast extract 0.5, NaCl 30.0, KH2PO4 2.0, K2HPO4 5.0, MgSO4,7H2O 0.2, urea 0.5, agar 20.0. The pH of the medium was adjusted initially to 7.2 ± 0.2. After cultivation 2 days at 35 C, single colonies with morphology of mucoid and ropy were picked and inoculated to liquid media for 24 h at 35 C. The hydrogen production and bioflocculant activity by isolated strains were determined respectively. The strains having the ability to produce hydrogen and bioflocculant simultaneously were selected and spread onto agar plates for 48 h at 35 C. Five cycles of replating onto the agar plates were conducted to ensure the purity of the strain with the highest production of hydrogen and bioflocculant. The morphological characteristic of the strain was observed with a light microscope (Olympus, Japan). The 16S rRNA of the strain was determined by PCR amplification and sequenced directly by a cycle sequencing system (Sangon, China). The PCR conditions were 30 cycles of 30 s at 94 C, 30 s at 58 C, 60 s at 72 C and then final extension for 10 min at 72 C. The sequences of the closely related strains were retrieved from GenBank database using BLAST search.
Culture condition The strain with the highest production of hydrogen and bioflocculant was grown in 250 mL serum bottles (working volume 100 mL) under anaerobic conditions at 35 C with agitation speed of 120 rpm. In batch tests, the operations were conducted at different initial pH (5.0e10.0) and NaCl concentration (0.4e6.0%) using various carbon sources such as starch, sucrose, lactose, glucose, xylose and fructose fixed at 10 g/L. The biomass accumulation was determined by measuring the optical absorbance at 600 nm using a spectrophotometer (UV757CRT, China). All tests were performed in triplicate to assure data reproducibility.
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Measurement of hydrogen production Biogas production was collected using the drainage method. The hydrogen composition in the gaseous product was determined by a gas chromatograph (Model 6820 Agilent, China) according to the same method as described previously [19]. In brief, the chromatograph was equipped with a thermal conductivity detector. Nitrogen was used as the carrier gas at a flow rate of 30 mL/min. The oven, injector and detector were kept at 40, 200 and 200 C, respectively.
Measurement of bioflocculant activity The bioflocculant activity was measured by kaolin suspension using a previous method [20]. 0.5 mL of fermentation culture liquor and 0.15 mL of 10% CaCl2 solution were added to 100 mL kaolin suspension which was prepared by adding 5 g kaolin clay in 1000 mL deionized water. The mixed solution was stirred at 120 rpm for 30 s, and held still for 10 min. The optical density of the upper solution and the blank control without bioflocculant was measured at 550 nm. The bioflocculant activity was calculated as follows: bioflocculant activity¼ (ODAODB)/ODA 100, where ODA and ODB represents the absorbance of the control and the sample, respectively.
environment, which can simultaneously flocculant as a byproduct to our knowledge.
produce
bio-
Production of hydrogen and bioflocculant by strain XF-56 The growth curve of strain XF-56 with the simultaneous production of hydrogen and bioflocculant is shown in Fig. 1. Strain XF-56 cells were in logarithm growth after 16 h and arrived at the stationary phase at 32 h. Most of hydrogen was produced between 40 and 48 h. The process of hydrogen production and cell growth was not synchronous, which indicated hydrogen generation was not a preferable to the biomass accumulation. Strain XF-56 cells produced bioflocculant along with their growth (Fig. 1). The bioflocculant activity by strain XF-56 was increased rapidly with cultivation time. Since 48 h, the absorbance values at 600 nm was slightly reduced, cells were in death phase. However, the bioflocculant activity was still increased in the death phase, which indicated that the bioflocculant was the release of intracellular substances and was not used as a carbon source by strain XF-56. The phenomenon that increased bioflocculant activity during the cultivation was consistent with the report of Agrobacterium sp. M503 [22].
Production of hydrogen and bioflocculant from various carbon sources
Results and discussion Isolation and identification of strain XF-56 A strain with high production of hydrogen and bioflocculant was isolated from intertidal sludge and named as XF-56. Strain XF-56 was a facultative aerobe. Microscopic examination showed that it was a Gram-positive bacterium in shortrod shape. PCR was amplified using 16S rRNA universal primers 27f/1541r and 1464 bp sequence was determined. According to the similarity of the 16S rRNA gene, the strain was found to be analogous to Bacillus subtills (99%). The 16S rRNA sequence of strain XF-56 was deposited in the GenBank database with the accession number of KM555037. Based on its morphological and 16S rRNA BLAST result, the strain was belonging to Bacillus sp. and designated as Bacillus sp. XF-56. There was no difference in the morphology by light microscope observation between strain XF-56 and the same fresh strains. Based on the 16S rDNA sequence analysis, the sequence similarity between strain XF-56 and the same fresh strains was 99%. Thus, strain XF-56 may be originated from terrestrial environment and has been adapted to the marine environment. During the process of evolution, salt tolerance mechanism will be activated, which can grow and breed in marine environment [21]. Bacillus, belong to facultative anaerobes without the demand for strictly anaerobic conditions, has an advantage over the obligate ones on the cultivation. So many Bacillus sp. strains have been examined for their potential in hydrogen production. The reported hydrogen yields by Bacillus sp. under dark fermentative conditions vary in the range of 0.54e1.96 mol/mol hexose [8]. The Bacillus sp. XF-56 is the first hydrogen-producing strain isolated from marine
The effects of carbon sources on production of hydrogen and bioflocculant by strain XF-56 were investigated. The medium contained starch, sucrose, lactose, glucose, xylose and fructose as the sole carbon source fixed at 10 g/L. As shown in Table 1, strain XF-56 could utilize various carbon sources and had the highest hydrogen production from glucose with 1.47 ± 0.05 mol H2/mol glucose in marine condition. Strain XF-56 could grow and produce bioflocculant with the different bioflocculant activities, when glucose, sucrose, maltose and lactose were used as carbon source respectively. The result was consisted with the studies reported that carbon sources have an important impact on the production of bioflocculant [23]. The optimum carbon source for bioflocculant
Fig. 1 e Variation of OD600, hydrogen production and bioflocculant activity by strain XF-56 during the fermentation (n ¼ 3, Error bars ¼ s.e.m.).
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Table 1 e Hydrogen production and bioflocculant activity by strain XF-56 from various carbon sources in the initial pH of 7.0 Carbon sources Glucose Fructose Xylose Sucrose Lactose Starch a
Hydrogen production (mol H2/mol glucose) 1.47 1.29 0.29 1.14
± 0.05a ± 0.04 ± 0.02 ± 0.03 e e
Bioflocculant activity (%) 93.5 ± 6.78 82.3 ± 5.12 64.5 ± 4.34 89.6 ± 5.67 e e
Mean ± S.E., n ¼ 3.
production by strain XF-56 was glucose with the highest bioflocculant activity of 93.5%. Strain XF-56 hardly produced bioflocculant using starch as a carbon source. Whereas, for Bacillus sp. DP-152, starch was good carbon source for the production of bioflocculant [24]. There is still no consensus on the optimal carbon source for bioflocculant production used by different bioflocculant producing bacteria [25].
Production of hydrogen and bioflocculant under different initial pH Initial pH is an important factor that influences fermentative hydrogen production. Accumulated hydrogen production of strain XF-56 at different pHs in the media was determined as shown in Fig. 2. Strain XF-56 could produce hydrogen from glucose at pH 5.0e10.0. The optimum initial pH value for hydrogen production was 7.0 with the maximum hydrogen production yield of 1.47 ± 0.05 mol H2/mol glucose. There is existed the disagreement on optimum pH for hydrogen production by different bacteria. The reported optimum pH for hydrogen production was ranged at 6.0e8.0 [26]. Strain XF-56 could grow and produce bioflocculant under wide ranges of pH (5.0e10.0). The optimum initial pH for biomass accumulation and bioflocculant production by strain XF-56 was at 8.0, which was consistent with the pH of seawater. Several Bacillus strains and their bioflocculant had been studied previously in different fields. Bacillus agaradhaerens C9 was isolated from alkaline lake and had highest bioflocculant rate of 90.87% at pH 10.2 [27]. Whereas for Bacillus subtilis MSBN17 isolated from the southeast coast of India, the highest bioflocculant activity of 90.02% was observed at pH 7.0 [28]. Strain XF-56 was isolated from intertidal sludge and was adapted to marine environment. Therefore, strain XF-56 can be considered to be an engineering strain for bioflocculant production in treatment of marine wastewater. In addition to the initial pH in the medium, the pH of kaolin suspension is also known to be a key factor influencing bioflocculant activity [29]. The kaolin suspension was set at different pH values (4.0e10.0). The bioflocculant activity increased almost linearly in an acidic pH range and with the maximum bioflocculant activity (99.5%) at pH 6.0. The bioflocculant activity by strain XF-56 was sensitive to the increase of pH in kaolin suspension and decreased significantly in alkaline conditions with 36.8% at pH 10.0. This may be due to fact that flocculation easily lead to inter-particle bridging between kaolin particles in an acidic pH range and has
Fig. 2 e Effect of initial pH in the medium on OD600, hydrogen production and bioflocculant activity by strain XF-56 (n ¼ 3, Error bars ¼ s.e.m.). the poor stability in alkaline environment without the settlement of the suspended particles in alkaline environment [30].
Production of hydrogen and bioflocculant under different salt concentration Strain XF-56 was isolated from intertidal sludge. The properties of producing-hydrogen and bioflocculant by strain XF-56 under different salt concentrations were investigated (Fig. 3). Strain XF-56 had the highest hydrogen production with 1.79 ± 0.06 mol H2/mol glucose at the NaCl concentration 0.4%. In marine condition (at the NaCl concentration 3.0e6.0%), strain XF-56 could grow and produce hydrogen with the maximum hydrogen production of 1.47 ± 0.05 mol H2/mol glucose at the NaCl concentration 3.0%. The results indicated the salt-tolerant strain XF-56 was able to produce hydrogen effectively under either fresh or marine condition. Bacillus strains can withstand adverse environmental conditions through sporulation under dark-fermentation and have been widely studied for hydrogen production [31]. Strain XF-56 had
Fig. 3 e Effect of salt concentration on OD600, hydrogen production and bioflocculant activity by strain XF-56 (n ¼ 3, Error bars ¼ s.e.m.).
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a similar hydrogen production as that of other bacteria in fresh condition, such as Bacillus thuringiensis EGU45, Bacillus sp. FS2011, Bacillus coagulans IIT-BTS1 [31e33]. Strain XF-56 was able to produce bioflocculant at all salt concentration ranges tested. The bioflocculant activity by strain XF-56 was different under marine and fresh conditions. With a rise of NaCl concentration, bioflocculant activity by strain XF-56 decreased and was obviously weak when NaCl concentration was 6%. The strain XF-56 showed the property of adaptation to salinity variation. The highest bioflocculant activity by strain XF-56 in marine condition was 93.5%, whereas it increased to 98.6% in fresh condition. Although strain XF-56 isolated from marine environment, the strain was able to produce hydrogen and bioflocculant at all salt concentration ranges tested. The production of hydrogen and bioflocculant was higher under fresh than marine condition. Bacillus is a well-known robust organism and has been exploited the novel biotechnological applications such as the production of biofuels, biopolymers, and antimicrobial agents [34,35]. Bacillus has been recognized for its biotechnological applications on an industrial scale [8]. Pure strains of Bacillus can compete successfully with contaminating bacteria and ensure the sustainability of the bioprocess with the biowastes. Therefore, Bacillus sp. XF-56 can be considered to be an engineering strain for application of the procedure to produce hydrogen and bioflocculant simultaneously, which is useful for realization of the cost-effective biotreatment process of actual fresh or marine wastewater.
Conclusions A hydrogen-producing bacterium which could produce bioflocculant simultaneously was isolated and identified as Bacillus sp. XF-56 (Gene Bank accession no. KM555037). This study examined the ability of hydrogen and bioflocculant produced by strain XF-56. Optimum conditions for production of hydrogen and bioflocculant was at 35 C in the medium containing glucose as a carbon source and salt concentration of 0.4% under the initial pH of 7.0(8.0) in batch test. The highest production of hydrogen and bioflocculant by Bacillus sp. XF-56 was 1.79 ± 0.06 mol H2/mol glucose and 98.6% respectively. This study revealed that the hydrogen-producing strain XF-56 could simultaneously produce bioflocculant as a byproduct, which will largely reduce productive costs of hydrogen and bioflocculants and increase the application in actual fresh and marine wastewater treatments.
Acknowledgments The authors acknowledge the financial support by the Natural Science Foundation of Tianjin, China (12JCQNJC04200) and the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry (No. 201302).
references
[1] Oh SE, Ginkel SV, Logan BE. The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production. Environ Sci Technol 2003;37:5186e90. [2] Xing DF, Ren NQ, Rittmann BE. Genetic diversity of hydrogen-producing bacteria in an acidophilic ethanol-H2coproducing system, analyzed using the [Fe]-hydrogenase gene. Appl Environ Microbiol 2008;74:1232e9. [3] Hallenbech PC, Benemann JR. Biological hydrogen production: fundamentals and limiting process. Int J Hydrogen Energy 2000;27:1185e93. [4] Zhao L, Cao GL, Wang AJ, Guo WQ, Liu BF, Ren HY, et al. Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier. Int J Hydrogen Energy 2012;37:162e6. [5] Nath K, Das D. Improvement of fermentative hydrogen production: various approaches. Appl Microbiol Biotechnol 2004;65(5):520e9. [6] Kotay SM, Das D. Biohydrogen as a renewable energy resourceeprospects and potentials. Int J Hydrogen Energy 2008;33:258e63. [7] Liu HY, Wang GC. Characteristics of hydrogen production by an aciduric transposon-mutagenized strain of Pantoea agglomerans BH18. Int J Hydrogen Energy 2013;38:13192e7. [8] Kumar P, Patel SKS, Lee JK, Kalia VC. Extending the limits of Bacillus for novel biotechnological applications. Biotechnol Adv 2013;31(8):1543e61. [9] Houghton JI, Quarmby J. Biopolymers in wastewater treatment. Curr Opin Biotechnol 1999;10:259e62. [10] Yim JH, Kim JS, Anh SH, Lee HK. Characterization of a novel bioflocculant, p-KG03, from a marine dinoflagellate, Gyrodinium impudicum KG03. Bioresour Technol 2007;98:361e7. [11] You Y, Ren NQ, Wang AJ, Ma F, Gao L, Peng YZ, et al. Use of waste fermenting liquor to produce bioflocculants with isolated strains. Int J Hydrogen Energy 2008;33:3295e301. [12] Labille J, Thomas F, Milas M, Vanhaverbeke C. Flocculation of colloidal clay by bacterial polysaccharides: effect of macromolecule charge and structure. J Colloid Interface Sci 2005;1:149e56. [13] He J, Zhen QW, Qiu N, Liu ZD, Wang BJ, Shao ZZ, et al. Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a0 using response surface methodology. Bioresour Technol 2009;100:5922e7. [14] Li Y, He N, Guan H, Du G, Chen J. A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation. Appl Microbiol Biotechnol 2003;63:200e6. [15] Yokoi H, Aratake T, Hirose J, Hayashi S, Takasaki Y. Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29. World J Microb Biotechnol 2001;17:609e13. [16] Ren NQ, Xie TH, Xing DF. Composition of extracellular polymeric substances influences the autoaggregation capability of hydrogen-producing bacterium Ethanoligenens harbinense. Bioresour Technol 2009;100:5109e13. [17] Xing DF, Ren NQ, Wang AJ, Li QB, Feng YJ, Ma F. Continuous hydrogen production of auto-aggregative Ethanoligenens harbinense YUAN-3 under non-sterile condition. Int J Hydrogen Energy 2008;33:1489e95. [18] Jia BJ, Yu JM. The research status and development trend of microbial flocculant. Phys Procedia 2012;24:425e8. [19] Liu HY, Wang GC, Zhu DL, Pan GH. Improvement of hydrogen production by transposon-mutagenized strain of Pantoea agglomerans BH18. Int J Hydrogen Energy 2012;37:8282e7.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 0 ( 2 0 1 5 ) 1 4 1 4 e1 4 1 9
[20] Liu ZY, Hu ZQ, Wang T, Chen YY, Zhang JB, Yu JR, et al. Production of novel microbial flocculants by Klebsiella sp. TG1 using waste residue from the food industry and its use in defecating the trona suspension. Bioresour Technol 2013;139:265e71. [21] Liu HY, Wang GC. Hydrogen production of a salt tolerant strain Bacillus sp.B2 from marine intertidal sludge. World J Microb Biot 2012;28(1):31e7. [22] Li Q, Liu HL, Qi QS, Wang FS, Zhang YZ. Isolation and characterization of temperature and alkaline stable bioflocculant from Agrobacterium sp. M-503. New Biotechnol 2010;27(6):789e94. [23] Salehizadeh H, Shojaosadati SA. Extracellular biopolymeric flocculants recent trends and biotechnological importance. Biotechnol Adv 2001;19:371e85. [24] Suh HH, Kwon GS, Lee CH, Kim HS, Oh HM, Yoon BD. Characterization of bioflocculant produced by Bacillus sp. DP152. J Ferment Bioeng 1997;84(2):108e12. [25] Wang L, Ma F, Lee DJ, Wang AJ, Ren NQ. Bioflocculants from hydrolysates of corn stover using isolated strain Ochrobactium ciceri W2. Bioresour Technol 2013;145:259e63. [26] Merugu R, Girisham S, Reddy SM. Bioproduction of hydrogen by Rhodobacter capsulatus KU002 isolated from leather industry effluents. Int J Hydrogen Energy 2010;35:9591e7. [27] Liu C, Wang K, Jiang JH, Liu WJ, Wang JY. A novel bioflocculant produced by a salt-tolerant, alkaliphilic and biofilm-forming strain Bacillus agaradhaerens C9 and its application in harvesting Chlorella minutissima UTEX2341. Biochem Eng J 2015;93:166e72.
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[28] Sathiyanarayanan G, Kiran GS, Selvin J. Synthesis of silver nanoparticles by polysaccharide bioflocculant produced from marine Bacillus subtilis MSBN17. Colloids Surf B Biointerfaces 2013;102:13e20. [29] Liu LF, Cheng W. Characteristics and culture conditions of a bioflocculant produced by Penicillium sp. Biomed Environ Sci 2010;23:213e8. [30] Bartley ML, Boeing WJ, Corcoran AA, Holguin FO, Schaub T. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and invading organisms. Biomass Bioenerg 2013;54:83e8. [31] Patel SKS, Singh M, Kalia VC. Hydrogen and polyhydroxybutyrate producing abilities of Bacillus spp. from glucose in two stage system. Indian J Microbiol 2011;51:418e23. [32] Song ZX, Li WW, Li XH, Dai Y, Peng XX, Fan YT, et al. Isolation and characterization of a new hydrogen-producing strain Bacillus sp. FS2011. Int J Hydrogen Energy 2013;38:3206e12. [33] Kotay SM, Das D. Novel dark fermentation involving bioaugmentation with constructed bacterial consortium for enhanced biohydrogen production from pretreated sewage sludge. Int J Hydrogen Energy 2009;34:7489e96. [34] Schallmey M, Singh A, Ward OP. Developments in the use of Bacillus species for industrial production. Can J Microbiol 2004;50:1e17. [35] Patel SKS, Kumar P, Kalia VC. Enhancing biological hydrogen production through complementary microbial metabolisms. Int J Hydrogen Energy 2012;37:10590e603.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/he
Characteristics for production of hydrogen and bioflocculant by Bacillus sp. XF-56 from marine intertidal sludge Hongyan Liu a,*, Guochao Chen a, Guangce Wang a,b a
Tianjin Key Laboratory of Marine Resources and Chemistry, College of Marine Science and Engineering, Tianjin University of Science & Technology, Tianjin 300457, PR China b Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China
article info
abstract
Article history:
This study aimed to isolate the novel bacterial strains from intertidal sludge that could
Received 19 September 2014
produce hydrogen and bioflocculant simultaneously. A bacterium named as strain XF-56
Received in revised form
was isolated for high production of hydrogen and bioflocculant. The isolated strain was
15 November 2014
designated as Bacillus sp. XF-56 by 16S rRNA gene sequence analysis. Strain XF-56 could
Accepted 21 November 2014
grow using various carbon sources and glucose was found to be favorable for production of
Available online 13 December 2014
hydrogen and bioflocculant. Moreover, this strain XF-56 was able to produce hydrogen and bioflocculant at initial pH from 5.0 to 10.0, with an optimum initial pH for hydrogen pro-
Keywords:
duction at 7.0 and bioflocculant activity at 8.0 respectively. In addition, strain XF-56 showed
Intertidal sludge
the strong salt tolerance and could produce hydrogen and bioflocculant at the salt con-
Strain XF-56
centration from 0.4% to 6.0%. The hydrogen production and bioflocculant activity by strain
Hydrogen production
XF-56 were 1.47 ± 0.05 mol H2/mol glucose and 93.5% in marine culture conditions, which
Bioflocculant activity
increased to 1.79 ± 0.06 mol H2/mol glucose and 98.6% respectively in fresh ones. This result demonstrated that the hydrogen-producing bacterium XF-56 with simultaneous production of bioflocculant is beneficial for cost-effective bio-treatment process and has a wide application potential in actual fresh and marine wastewater. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Biological hydrogen production is a promising technique using organic wastewater and renewable biomass and has attracted worldwide attention [1,2]. Microorganisms utilized for biological hydrogen production were commonly divided into two main categories: photosynthetic bacteria and darkfermentative bacteria. Hydrogen production by dark-
fermentative bacteria is more widely used because it has higher hydrogen production rate with no light requirements [3]. The most dark-fermentative hydrogen-producing bacteria, such as Enterobacter, Bacillus and Clostridium species, have been reported for high hydrogen production. However, adequate hydrogen yields by these bacteria have not been achieved so far [4,5]. Regarding the fermentative hydrogen production system, finding the industry-suitable strains with high hydrogen-producing efficiency and good application potential
* Corresponding author. Tel./fax: þ86 22 60601305. E-mail address: [email protected] (H. Liu). http://dx.doi.org/10.1016/j.ijhydene.2014.11.110 0360-3199/Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
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is still of the most importance [6,7]. The hydrogen-producing bacteria that can use diverse organic wastes as feedstock are necessary for sustainable hydrogen production. Bacillus is an industrially robust organism that can grow well to high densities and compete successfully with contaminating bacteria under fermentative conditions using biowastes as feedback without sterilization [8]. Bacillus has been considered as a candidate hydrogen producer. In the study of screening efficient hydrogen-producing bacteria, we found some strains with the ability of producing hydrogen and bioflocculant simultaneously. The flocculants produced by microorganisms are used to remove suspended solids and metal ions in both aerobic and anaerobic treatment systems [9]. Bioflocculants have been attracted great research interest and widely studied in various industrial processes such as wastewater treatment, drinking water purification and other relevant industries [10,11]. Microbial-produced bioflocculants are biodegradable polymers, including proteins, polysaccharides, lipids and nucleic acid, which are not secondary pollutants and harmless [12]. However, compared with chemical flocculants, bioflocculants have not been widely used because of the high costs, which inhibit the feasibility of industrialization and practical application of bioflocculants [13]. Some efforts have been made to improve the level of microbial-produced bioflocculants, such as searching for low-cost feedstock for bioflocculant production [14]. The procedure to produce hydrogen with a byproduct of the bioflocculant helps to reduce the cost of wastewater treatment using bioflocculant. Yokoi et al. reported that a hydrogen-producing bacterium Enterobacter sp. BY-29 which can produce flocculants and hydrogen simultaneously [15]. Ren et al. isolated a hydrogen-producing bacterium Ethanoligenens harbinense with the ability of autoaggregation by producing bioflocculant [16]. Bioflocculants produced as a byproduct during the fermentative hydrogen production could be an effective procedure for realization of cost-effective practical application and has been a well developed biological treatment for the fresh wastewater [17,18]. In fact, due to the high utilization of seawater, such as mariculture, the accumulation of the high-salt organic wastewater has been becoming a prominent environmental problem in China. The techniques by hydrogen producing bacteria with bioflocculant as a byproduct could use organic contents in the wastewater to generate clean energy and also achieved the purpose of wastewater treatment and purification. However, bacterial strains which can simultaneously produce hydrogen and bioflocculant in fresh condition are not feasible to the use for biological treatment of wastewater with high salt concentration. There have been no reports for simultaneous production of hydrogen and bioflocculant by bacterial strains isolated from marine environments. Among the various marine environments, intertidal zone is the transitional position between marine and terrestrial environments, which has led to the diversity of bacterial communities. In this work, a new hydrogen producing bacterium Bacillus sp. XF-56 which can produce bioflocculant was isolated from intertidal sludge. The optimal culture conditions for production of hydrogen and bioflocculant by strain XF-56 under batch conditions were investigated, which will provide the cost-
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effective approach for application of actual high-salt organic wastewater.
Materials and methods Sludge and enrichment Sludge was collected from the intertidal zone of Bohai, in Tianjin, China (longitude of 116 and latitude of 38). The enrichment culture was set up as 10% (w/v) sludge slurry in LM-H medium. The medium (g/L) was composed of glucose 20.0, yeast extract 1.0, tryptone 4.0, beef extract 2.0, NaCl 30.0, K2HPO4 1.5, MgCl2 0.1, FeSO4,7H2O 0.1, L-cysteine 0.5, as described previously [19]. The enrichment test was performed under anaerobic condition in an incubator shaking at 120 rpm. After 3-day incubation, 1 mL culture broth was inoculated again using the same medium three times, which had led to the enrichment of the mixed microbial culture.
Isolation and identification The enrichment culture was diluted and spread onto agar plates containing the following sterilized medium (g/L): glucose 10.0, yeast extract 0.5, NaCl 30.0, KH2PO4 2.0, K2HPO4 5.0, MgSO4,7H2O 0.2, urea 0.5, agar 20.0. The pH of the medium was adjusted initially to 7.2 ± 0.2. After cultivation 2 days at 35 C, single colonies with morphology of mucoid and ropy were picked and inoculated to liquid media for 24 h at 35 C. The hydrogen production and bioflocculant activity by isolated strains were determined respectively. The strains having the ability to produce hydrogen and bioflocculant simultaneously were selected and spread onto agar plates for 48 h at 35 C. Five cycles of replating onto the agar plates were conducted to ensure the purity of the strain with the highest production of hydrogen and bioflocculant. The morphological characteristic of the strain was observed with a light microscope (Olympus, Japan). The 16S rRNA of the strain was determined by PCR amplification and sequenced directly by a cycle sequencing system (Sangon, China). The PCR conditions were 30 cycles of 30 s at 94 C, 30 s at 58 C, 60 s at 72 C and then final extension for 10 min at 72 C. The sequences of the closely related strains were retrieved from GenBank database using BLAST search.
Culture condition The strain with the highest production of hydrogen and bioflocculant was grown in 250 mL serum bottles (working volume 100 mL) under anaerobic conditions at 35 C with agitation speed of 120 rpm. In batch tests, the operations were conducted at different initial pH (5.0e10.0) and NaCl concentration (0.4e6.0%) using various carbon sources such as starch, sucrose, lactose, glucose, xylose and fructose fixed at 10 g/L. The biomass accumulation was determined by measuring the optical absorbance at 600 nm using a spectrophotometer (UV757CRT, China). All tests were performed in triplicate to assure data reproducibility.
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Measurement of hydrogen production Biogas production was collected using the drainage method. The hydrogen composition in the gaseous product was determined by a gas chromatograph (Model 6820 Agilent, China) according to the same method as described previously [19]. In brief, the chromatograph was equipped with a thermal conductivity detector. Nitrogen was used as the carrier gas at a flow rate of 30 mL/min. The oven, injector and detector were kept at 40, 200 and 200 C, respectively.
Measurement of bioflocculant activity The bioflocculant activity was measured by kaolin suspension using a previous method [20]. 0.5 mL of fermentation culture liquor and 0.15 mL of 10% CaCl2 solution were added to 100 mL kaolin suspension which was prepared by adding 5 g kaolin clay in 1000 mL deionized water. The mixed solution was stirred at 120 rpm for 30 s, and held still for 10 min. The optical density of the upper solution and the blank control without bioflocculant was measured at 550 nm. The bioflocculant activity was calculated as follows: bioflocculant activity¼ (ODAODB)/ODA 100, where ODA and ODB represents the absorbance of the control and the sample, respectively.
environment, which can simultaneously flocculant as a byproduct to our knowledge.
produce
bio-
Production of hydrogen and bioflocculant by strain XF-56 The growth curve of strain XF-56 with the simultaneous production of hydrogen and bioflocculant is shown in Fig. 1. Strain XF-56 cells were in logarithm growth after 16 h and arrived at the stationary phase at 32 h. Most of hydrogen was produced between 40 and 48 h. The process of hydrogen production and cell growth was not synchronous, which indicated hydrogen generation was not a preferable to the biomass accumulation. Strain XF-56 cells produced bioflocculant along with their growth (Fig. 1). The bioflocculant activity by strain XF-56 was increased rapidly with cultivation time. Since 48 h, the absorbance values at 600 nm was slightly reduced, cells were in death phase. However, the bioflocculant activity was still increased in the death phase, which indicated that the bioflocculant was the release of intracellular substances and was not used as a carbon source by strain XF-56. The phenomenon that increased bioflocculant activity during the cultivation was consistent with the report of Agrobacterium sp. M503 [22].
Production of hydrogen and bioflocculant from various carbon sources
Results and discussion Isolation and identification of strain XF-56 A strain with high production of hydrogen and bioflocculant was isolated from intertidal sludge and named as XF-56. Strain XF-56 was a facultative aerobe. Microscopic examination showed that it was a Gram-positive bacterium in shortrod shape. PCR was amplified using 16S rRNA universal primers 27f/1541r and 1464 bp sequence was determined. According to the similarity of the 16S rRNA gene, the strain was found to be analogous to Bacillus subtills (99%). The 16S rRNA sequence of strain XF-56 was deposited in the GenBank database with the accession number of KM555037. Based on its morphological and 16S rRNA BLAST result, the strain was belonging to Bacillus sp. and designated as Bacillus sp. XF-56. There was no difference in the morphology by light microscope observation between strain XF-56 and the same fresh strains. Based on the 16S rDNA sequence analysis, the sequence similarity between strain XF-56 and the same fresh strains was 99%. Thus, strain XF-56 may be originated from terrestrial environment and has been adapted to the marine environment. During the process of evolution, salt tolerance mechanism will be activated, which can grow and breed in marine environment [21]. Bacillus, belong to facultative anaerobes without the demand for strictly anaerobic conditions, has an advantage over the obligate ones on the cultivation. So many Bacillus sp. strains have been examined for their potential in hydrogen production. The reported hydrogen yields by Bacillus sp. under dark fermentative conditions vary in the range of 0.54e1.96 mol/mol hexose [8]. The Bacillus sp. XF-56 is the first hydrogen-producing strain isolated from marine
The effects of carbon sources on production of hydrogen and bioflocculant by strain XF-56 were investigated. The medium contained starch, sucrose, lactose, glucose, xylose and fructose as the sole carbon source fixed at 10 g/L. As shown in Table 1, strain XF-56 could utilize various carbon sources and had the highest hydrogen production from glucose with 1.47 ± 0.05 mol H2/mol glucose in marine condition. Strain XF-56 could grow and produce bioflocculant with the different bioflocculant activities, when glucose, sucrose, maltose and lactose were used as carbon source respectively. The result was consisted with the studies reported that carbon sources have an important impact on the production of bioflocculant [23]. The optimum carbon source for bioflocculant
Fig. 1 e Variation of OD600, hydrogen production and bioflocculant activity by strain XF-56 during the fermentation (n ¼ 3, Error bars ¼ s.e.m.).
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Table 1 e Hydrogen production and bioflocculant activity by strain XF-56 from various carbon sources in the initial pH of 7.0 Carbon sources Glucose Fructose Xylose Sucrose Lactose Starch a
Hydrogen production (mol H2/mol glucose) 1.47 1.29 0.29 1.14
± 0.05a ± 0.04 ± 0.02 ± 0.03 e e
Bioflocculant activity (%) 93.5 ± 6.78 82.3 ± 5.12 64.5 ± 4.34 89.6 ± 5.67 e e
Mean ± S.E., n ¼ 3.
production by strain XF-56 was glucose with the highest bioflocculant activity of 93.5%. Strain XF-56 hardly produced bioflocculant using starch as a carbon source. Whereas, for Bacillus sp. DP-152, starch was good carbon source for the production of bioflocculant [24]. There is still no consensus on the optimal carbon source for bioflocculant production used by different bioflocculant producing bacteria [25].
Production of hydrogen and bioflocculant under different initial pH Initial pH is an important factor that influences fermentative hydrogen production. Accumulated hydrogen production of strain XF-56 at different pHs in the media was determined as shown in Fig. 2. Strain XF-56 could produce hydrogen from glucose at pH 5.0e10.0. The optimum initial pH value for hydrogen production was 7.0 with the maximum hydrogen production yield of 1.47 ± 0.05 mol H2/mol glucose. There is existed the disagreement on optimum pH for hydrogen production by different bacteria. The reported optimum pH for hydrogen production was ranged at 6.0e8.0 [26]. Strain XF-56 could grow and produce bioflocculant under wide ranges of pH (5.0e10.0). The optimum initial pH for biomass accumulation and bioflocculant production by strain XF-56 was at 8.0, which was consistent with the pH of seawater. Several Bacillus strains and their bioflocculant had been studied previously in different fields. Bacillus agaradhaerens C9 was isolated from alkaline lake and had highest bioflocculant rate of 90.87% at pH 10.2 [27]. Whereas for Bacillus subtilis MSBN17 isolated from the southeast coast of India, the highest bioflocculant activity of 90.02% was observed at pH 7.0 [28]. Strain XF-56 was isolated from intertidal sludge and was adapted to marine environment. Therefore, strain XF-56 can be considered to be an engineering strain for bioflocculant production in treatment of marine wastewater. In addition to the initial pH in the medium, the pH of kaolin suspension is also known to be a key factor influencing bioflocculant activity [29]. The kaolin suspension was set at different pH values (4.0e10.0). The bioflocculant activity increased almost linearly in an acidic pH range and with the maximum bioflocculant activity (99.5%) at pH 6.0. The bioflocculant activity by strain XF-56 was sensitive to the increase of pH in kaolin suspension and decreased significantly in alkaline conditions with 36.8% at pH 10.0. This may be due to fact that flocculation easily lead to inter-particle bridging between kaolin particles in an acidic pH range and has
Fig. 2 e Effect of initial pH in the medium on OD600, hydrogen production and bioflocculant activity by strain XF-56 (n ¼ 3, Error bars ¼ s.e.m.). the poor stability in alkaline environment without the settlement of the suspended particles in alkaline environment [30].
Production of hydrogen and bioflocculant under different salt concentration Strain XF-56 was isolated from intertidal sludge. The properties of producing-hydrogen and bioflocculant by strain XF-56 under different salt concentrations were investigated (Fig. 3). Strain XF-56 had the highest hydrogen production with 1.79 ± 0.06 mol H2/mol glucose at the NaCl concentration 0.4%. In marine condition (at the NaCl concentration 3.0e6.0%), strain XF-56 could grow and produce hydrogen with the maximum hydrogen production of 1.47 ± 0.05 mol H2/mol glucose at the NaCl concentration 3.0%. The results indicated the salt-tolerant strain XF-56 was able to produce hydrogen effectively under either fresh or marine condition. Bacillus strains can withstand adverse environmental conditions through sporulation under dark-fermentation and have been widely studied for hydrogen production [31]. Strain XF-56 had
Fig. 3 e Effect of salt concentration on OD600, hydrogen production and bioflocculant activity by strain XF-56 (n ¼ 3, Error bars ¼ s.e.m.).
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a similar hydrogen production as that of other bacteria in fresh condition, such as Bacillus thuringiensis EGU45, Bacillus sp. FS2011, Bacillus coagulans IIT-BTS1 [31e33]. Strain XF-56 was able to produce bioflocculant at all salt concentration ranges tested. The bioflocculant activity by strain XF-56 was different under marine and fresh conditions. With a rise of NaCl concentration, bioflocculant activity by strain XF-56 decreased and was obviously weak when NaCl concentration was 6%. The strain XF-56 showed the property of adaptation to salinity variation. The highest bioflocculant activity by strain XF-56 in marine condition was 93.5%, whereas it increased to 98.6% in fresh condition. Although strain XF-56 isolated from marine environment, the strain was able to produce hydrogen and bioflocculant at all salt concentration ranges tested. The production of hydrogen and bioflocculant was higher under fresh than marine condition. Bacillus is a well-known robust organism and has been exploited the novel biotechnological applications such as the production of biofuels, biopolymers, and antimicrobial agents [34,35]. Bacillus has been recognized for its biotechnological applications on an industrial scale [8]. Pure strains of Bacillus can compete successfully with contaminating bacteria and ensure the sustainability of the bioprocess with the biowastes. Therefore, Bacillus sp. XF-56 can be considered to be an engineering strain for application of the procedure to produce hydrogen and bioflocculant simultaneously, which is useful for realization of the cost-effective biotreatment process of actual fresh or marine wastewater.
Conclusions A hydrogen-producing bacterium which could produce bioflocculant simultaneously was isolated and identified as Bacillus sp. XF-56 (Gene Bank accession no. KM555037). This study examined the ability of hydrogen and bioflocculant produced by strain XF-56. Optimum conditions for production of hydrogen and bioflocculant was at 35 C in the medium containing glucose as a carbon source and salt concentration of 0.4% under the initial pH of 7.0(8.0) in batch test. The highest production of hydrogen and bioflocculant by Bacillus sp. XF-56 was 1.79 ± 0.06 mol H2/mol glucose and 98.6% respectively. This study revealed that the hydrogen-producing strain XF-56 could simultaneously produce bioflocculant as a byproduct, which will largely reduce productive costs of hydrogen and bioflocculants and increase the application in actual fresh and marine wastewater treatments.
Acknowledgments The authors acknowledge the financial support by the Natural Science Foundation of Tianjin, China (12JCQNJC04200) and the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry (No. 201302).
references
[1] Oh SE, Ginkel SV, Logan BE. The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production. Environ Sci Technol 2003;37:5186e90. [2] Xing DF, Ren NQ, Rittmann BE. Genetic diversity of hydrogen-producing bacteria in an acidophilic ethanol-H2coproducing system, analyzed using the [Fe]-hydrogenase gene. Appl Environ Microbiol 2008;74:1232e9. [3] Hallenbech PC, Benemann JR. Biological hydrogen production: fundamentals and limiting process. Int J Hydrogen Energy 2000;27:1185e93. [4] Zhao L, Cao GL, Wang AJ, Guo WQ, Liu BF, Ren HY, et al. Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier. Int J Hydrogen Energy 2012;37:162e6. [5] Nath K, Das D. Improvement of fermentative hydrogen production: various approaches. Appl Microbiol Biotechnol 2004;65(5):520e9. [6] Kotay SM, Das D. Biohydrogen as a renewable energy resourceeprospects and potentials. Int J Hydrogen Energy 2008;33:258e63. [7] Liu HY, Wang GC. Characteristics of hydrogen production by an aciduric transposon-mutagenized strain of Pantoea agglomerans BH18. Int J Hydrogen Energy 2013;38:13192e7. [8] Kumar P, Patel SKS, Lee JK, Kalia VC. Extending the limits of Bacillus for novel biotechnological applications. Biotechnol Adv 2013;31(8):1543e61. [9] Houghton JI, Quarmby J. Biopolymers in wastewater treatment. Curr Opin Biotechnol 1999;10:259e62. [10] Yim JH, Kim JS, Anh SH, Lee HK. Characterization of a novel bioflocculant, p-KG03, from a marine dinoflagellate, Gyrodinium impudicum KG03. Bioresour Technol 2007;98:361e7. [11] You Y, Ren NQ, Wang AJ, Ma F, Gao L, Peng YZ, et al. Use of waste fermenting liquor to produce bioflocculants with isolated strains. Int J Hydrogen Energy 2008;33:3295e301. [12] Labille J, Thomas F, Milas M, Vanhaverbeke C. Flocculation of colloidal clay by bacterial polysaccharides: effect of macromolecule charge and structure. J Colloid Interface Sci 2005;1:149e56. [13] He J, Zhen QW, Qiu N, Liu ZD, Wang BJ, Shao ZZ, et al. Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a0 using response surface methodology. Bioresour Technol 2009;100:5922e7. [14] Li Y, He N, Guan H, Du G, Chen J. A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation. Appl Microbiol Biotechnol 2003;63:200e6. [15] Yokoi H, Aratake T, Hirose J, Hayashi S, Takasaki Y. Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29. World J Microb Biotechnol 2001;17:609e13. [16] Ren NQ, Xie TH, Xing DF. Composition of extracellular polymeric substances influences the autoaggregation capability of hydrogen-producing bacterium Ethanoligenens harbinense. Bioresour Technol 2009;100:5109e13. [17] Xing DF, Ren NQ, Wang AJ, Li QB, Feng YJ, Ma F. Continuous hydrogen production of auto-aggregative Ethanoligenens harbinense YUAN-3 under non-sterile condition. Int J Hydrogen Energy 2008;33:1489e95. [18] Jia BJ, Yu JM. The research status and development trend of microbial flocculant. Phys Procedia 2012;24:425e8. [19] Liu HY, Wang GC, Zhu DL, Pan GH. Improvement of hydrogen production by transposon-mutagenized strain of Pantoea agglomerans BH18. Int J Hydrogen Energy 2012;37:8282e7.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 0 ( 2 0 1 5 ) 1 4 1 4 e1 4 1 9
[20] Liu ZY, Hu ZQ, Wang T, Chen YY, Zhang JB, Yu JR, et al. Production of novel microbial flocculants by Klebsiella sp. TG1 using waste residue from the food industry and its use in defecating the trona suspension. Bioresour Technol 2013;139:265e71. [21] Liu HY, Wang GC. Hydrogen production of a salt tolerant strain Bacillus sp.B2 from marine intertidal sludge. World J Microb Biot 2012;28(1):31e7. [22] Li Q, Liu HL, Qi QS, Wang FS, Zhang YZ. Isolation and characterization of temperature and alkaline stable bioflocculant from Agrobacterium sp. M-503. New Biotechnol 2010;27(6):789e94. [23] Salehizadeh H, Shojaosadati SA. Extracellular biopolymeric flocculants recent trends and biotechnological importance. Biotechnol Adv 2001;19:371e85. [24] Suh HH, Kwon GS, Lee CH, Kim HS, Oh HM, Yoon BD. Characterization of bioflocculant produced by Bacillus sp. DP152. J Ferment Bioeng 1997;84(2):108e12. [25] Wang L, Ma F, Lee DJ, Wang AJ, Ren NQ. Bioflocculants from hydrolysates of corn stover using isolated strain Ochrobactium ciceri W2. Bioresour Technol 2013;145:259e63. [26] Merugu R, Girisham S, Reddy SM. Bioproduction of hydrogen by Rhodobacter capsulatus KU002 isolated from leather industry effluents. Int J Hydrogen Energy 2010;35:9591e7. [27] Liu C, Wang K, Jiang JH, Liu WJ, Wang JY. A novel bioflocculant produced by a salt-tolerant, alkaliphilic and biofilm-forming strain Bacillus agaradhaerens C9 and its application in harvesting Chlorella minutissima UTEX2341. Biochem Eng J 2015;93:166e72.
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[28] Sathiyanarayanan G, Kiran GS, Selvin J. Synthesis of silver nanoparticles by polysaccharide bioflocculant produced from marine Bacillus subtilis MSBN17. Colloids Surf B Biointerfaces 2013;102:13e20. [29] Liu LF, Cheng W. Characteristics and culture conditions of a bioflocculant produced by Penicillium sp. Biomed Environ Sci 2010;23:213e8. [30] Bartley ML, Boeing WJ, Corcoran AA, Holguin FO, Schaub T. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and invading organisms. Biomass Bioenerg 2013;54:83e8. [31] Patel SKS, Singh M, Kalia VC. Hydrogen and polyhydroxybutyrate producing abilities of Bacillus spp. from glucose in two stage system. Indian J Microbiol 2011;51:418e23. [32] Song ZX, Li WW, Li XH, Dai Y, Peng XX, Fan YT, et al. Isolation and characterization of a new hydrogen-producing strain Bacillus sp. FS2011. Int J Hydrogen Energy 2013;38:3206e12. [33] Kotay SM, Das D. Novel dark fermentation involving bioaugmentation with constructed bacterial consortium for enhanced biohydrogen production from pretreated sewage sludge. Int J Hydrogen Energy 2009;34:7489e96. [34] Schallmey M, Singh A, Ward OP. Developments in the use of Bacillus species for industrial production. Can J Microbiol 2004;50:1e17. [35] Patel SKS, Kumar P, Kalia VC. Enhancing biological hydrogen production through complementary microbial metabolisms. Int J Hydrogen Energy 2012;37:10590e603.