Bio-hydrogen production from cellulose by sequential co-culture of cellulosic hydrogen bacteria of Enterococcus gallinarum G1 and Ethanoigenens harbinense B49
Microbial conversion of lignocellulose to hydrogen is a fascinating way to provide a renewable energy source. A mesophilic bacterium strain G1 that had high cellulose degradation and hydrogen production activity (2.38 mmol H2 g−1 cellulose) was isolated from rumen fluid and identified as the Enterococcus gallinarum. Hydrogen production from cellulose by using sequential co-cultures of a cellulosic-hydrolysis bacterium G1 and Ethanoigenens harbinense B49 was investigated. With an initial Avicel concentration of 5 g l−l, the sequential co-culture with G1 and strain Ethanoigenens harbinense B49 produced H2 yield approximately 2.97 mmol H2 g−1 cellulose for the co-culture system.
KeywordsHydrogen Isolation lignocellulose Sequential co-culture
Cellulosic materials mainly consist of hexose and pentose sugars with potential use for the production of fuel (Kuhad and Singh 1993; Chandel et al. 2007; Herrera, 2004). Recently, hydrogen production from cellulose has received more and more attention. In nature, cellulosic materials are degraded by mixed microbial consortia consisting of cellulose-hydrolytic, fermentative and other microorganisms depending on the substrate. For instance, rumen fluid cultures comprising cellulolytic and non-cellulolytic bacteria are ideal for cellulose degradation (Odom and Wall 1983; Lewis et al. 1988). However, most high efficient hydrogen-producing organisms are non (or low)-cellulose-degrading fermentative bacteria. A combination of high-active cellulose-hydrolyzing bacteria and hydrogen-producing bacteria could result in synergistic hydrogen production. Lay (2001) investigated the potential of producing H2 from microcrystalline cellulose under mesophilic digestion condition with heat shocked sludge as inoculum. Lo et al. (2008) determined that mixed culture of cellulosic-hydrolysis sludge and Clostridium pasteurianum produced H2 1.09 mmol H2/g cellulose from 10 g l−1 carboxymethyl cellulose.
In this study, we isolated a mesophilic bacterium from rumen fluid and identified it as Enterococcus gallinarum G1. The growth and hydrogen production from cellulose by strain G1 and its companion bacterium, a hexose-hydrogen producing Ethanoigenens harbinense B49 were investigated. We also investigated the synergistic effect of cellulose degradation and hydrogen production that occurred during sequential co-culture of these two strains.
Materials and methods
Medium and isolation of bacteria
Rumen fluid obtained from a slaughter-house in Harbin, China was used as the source for microbial isolation. The fluid was filtered through four-layers of gauze and stored in a vial purged with N2 gas prior to use. The rumen fluid (15 ml) was then inoculated into anaerobic flasks (Bellco Glass, USA) with sterilized modified 1191 Clostridium thermocellum medium (ATCC medium 1190) to enrich cellulose-degrading microorganisms under N2 atmosphere. The medium used 5 g l−1 Avicel as carbon source. The Avicel had cellulose content at 97.2% (v/v, dry basis) and a water solubility of 0.1% (w/v). The mixtures were incubated at 37°C for 100 h. Suspension samples collected from the incubated tubes were plated on agar plates with the same medium mentioned above for the isolation of cellulose-degrading and hydrogen-production bacteria.
DNA extraction and sequencing
The identification by 16S rDNA gene sequence analysis was carried out as follows. The genomic DNA was extracted from cells with the standard method (Sambrook and Russell 2001) and the 16S rDNA gene was amplified by PCR as described (Wang et al. 2008).The double-stranded PCR products were sequenced. The 16S rDNA sequence was aligned and identified best matches of the genes against existing DNA sequences in GenBank database using the BLAST program.
Co-culture tests were performed using the same medium as that in the cultivation tests. Briefly, medium (150 ml) was mixed with 15 ml inoculum (0.016 g dry cells) and was kept at 37°C for 100 h. The isolated strain that had the highest hydrogen production potential among those isolates from Avicel suspensions was tested further. Strain Ethanoigenens harbinense B49 (AF481148 in NCBI), a H2-producing, fermentative bacteria isolated (Ren et al.2007) was utilized as a partner for co-culture with the isolated strain G1 in fermentation tests. Strain B49 does not hydrolyze cellulose, but produced hydrogen from glucose with a maximum hydrogen production rate of 25 mmol H2 h−1 g−1 dry cells and a hydrogen yield of 1,810 ml l−1 medium (Wang et al. 2008).
In the co-culture tests, G1 was cultured individually as either control or sequential co-culture with B49 at the same culture volumes at a total biomass quantity of 0.0184 g dry cells, namely B49 was inoculated into the same medium after 20 h that of G1 at 37°C, considering nearly 20 h gap of doubling time between G1 and B49.
Three replicates of culture tubes were used at each experimental sampling point. The time zero samples were collected immediately after inoculation and used as controls.
The cell dry weight was measured based on centrifuging the filtered culture broths without cellulose (3,000 g, 10 min), washing twice with distilled water and drying at 105°C until constant. The gas composition was measured as described by Wang et al. (2008). The Avicel concentration in the medium was determined by phenol–H2SO4 method after removal of cell mass as described by Minato et al. (1962).
Results and discussions
Isolation and characterization of the cellulosic bacterial strains
Hydrogen yields, cellulose degradation ratio and metabolites from Avicel with different strains
Substrate 5 g l−1
YH2 (ml L−1)
Metabolites (mg l−1)
Cellulose degradation and hydrogen production of G1 started after the 5 h lag phase. The entire fermentation process was completed in 55–60 h. The corresponding hydrogen yield, maximum hydrogen production rate and cellulose hydrolysis ratio reached 107.5 ml l−1 medium, 1.16 mmol H2 h−1 g−1 dry cell, and 42.6% (v/v), respectively. Hence, strain G1 has a good capability to hydrolyze Avicel and then convert it into hydrogen.
Growth and corresponding changes in pH during sequential co-culture
H2 production in sequential co-culture
From the above results, it was clear that G1 and B49 formed stable synergistic co-cultures system. When G1 was co-cultured with B49, B49 utilized partial reducing sugars as a carbon source to produce H2, organic acids and ethanol. The cellulases produced by the strain G1 continued to decompose cellulose as B49 removed the reduced saccharides quickly.
Conclusions and discussion
In this research, a mesophilic anaerobic bacterium named as Enterococcus gallinarum G1 was isolated to effectively decompose cellulose and produce hydrogen, At 37°C and pH 6.5, the mono-culture tests with G1 revealed that cellulose hydrolysis and hydrogen production occurred with a 5 h time lag. The cellulose was then promptly hydrolyzed and hydrogen was produced in the following 60 h. The corresponding hydrogen yield, maximum hydrogen production rate, and cellulose hydrolysis ratio reached 107.5 ml l−1 medium, 1.16 mmol H2 h−1 g−1 dry cell, and 42.6% (v/v), respectively. Equivalently, the hydrogen yield was 2.38 mmol H2 g−1 cellulose. The end liquid products were primarily acetate, propionate and butyrate.
Comparison of H2 production performance using cellulosic material as substrate under mesphilic condition
H2 yield (mmol H2 g−1 substrate)
X9 + B49
MC (10 g l−1)
Wang et al. (2007)
Stream-exploded corn stover (15 g l−1)
Wang et al. (2008)
Anaerobic digested sludge
MC (12.5 g l−1)
Sludge + Clostridium pasteurianum
CMC (10 g l−1)
Lo et al. (2008)
G1 + B49
Avicel (5 g l−1)
Rumen microbes and its co-culture companion for cellulose degradation and hydrogen production activity have been demonstrated in this study. However, further study on the optimization of the co-culture system is required, such as the different cellulase systems in rumen microorganism. Understanding optimization parameters and cellulase systems can potentially lead to significant enhancement of hydrogen production from cellulosic biomass.
This project was supported by Natural Science Foundation of China (NSFC 50678049, 50878062, and by Program for New Century Excellent Talents in University (NECT-2005)).
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