- Open Access
Strain screening and optimization of biohydrogen production by Enterobacter aerogenes EB-06 from glycerol fermentation
Bioresources and Bioprocessingvolume 6, Article number: 15 (2019)
Biohydrogen technology has drawn much attention due to its many advantages. However, it is still necessary to screen much more strains with stronger hydrogen-producing capacity for future commercialization processes. In this paper, a biohydrogen-producing strain Enterobacter aerogenes EB-06 was isolated, identified, and named. It could convert glycerol to biohydrogen by microorganism fermentation. The effects of oxygen content, initial pH, initial glycerol concentration, and initial nitrogen source content on biohydrogen production process were investigated. The results have shown that biohydrogen generation was more favorable under anaerobic conditions. The optimum specific biohydrogen production rate (QH2) was obtained as 41.47 mmol H2/g DCW h at 40 g/L initial glycerol concentration. The optimum volume H2 yield (CH2) was 83.76 mmol H2/L at initial pH 7.0. It was found that nitrogen source content (0–4 g/L) could promote biohydrogen production and cell growth. The biohydrogen production of Enterobacter aerogenes EB-06 from glycerol was optimized by the orthogonal experimental design. The optimal yield coefficient of biohydrogen from glycerol fermentation (YH2/glycerol) of EB-06 was obtained as 1.07 mmol H2/ mol glycerol at 10 g/L initial glycerol concentration, initial pH 5.0, and initial C/N ratio 5/3.
Hydrogen gas is a multipurpose energy source that can change the usage of hydrocarbon-based fossil fuels since the higher energy yield (122 kJ/g per unit mass), which is 2.75-fold greater than that of fossil fuels. Only water is generated as a major by-product (Asadi and Zilouei 2017; Azman et al. 2016) after hydrogen combustion. Currently, molecular hydrogen has been mainly generated from fossil fuel-based resources. Hydrogen gas production through biological pathways from biomass is one of the rising technologies due to its eco-friendly and sustainable nature (Niu et al. 2011; Sørensen 2011; Trchounian and Trchounian 2015). Dark fermentation of biohydrogen production is a promising method because of high hydrogen-producing rate, simple fermentation equipment, and bioconversion feasibility from the recyclable resources (Dessì et al. 2018). It is essential to choose inexpensive substrates, such as glucose, xylose, glycerol, and cellulose, for the development of biohydrogen production technology (Asadi and Zilouei 2017; Pachapur et al. 2015). Recently, aviation diesel has been partly replaced by biodiesel due to the energy conservation and environmental protection (Faber and Ferreira-Leitão 2016). However, a large amount of glycerol can be produced as by-product during biodiesel production process (Sivaramakrishnan and Incharoensakdi 2018). How to use these glycerol has become an urgent problem to be solved (Jitrwung and Yargeau 2011; Pott et al. 2014). The comprehensive utilization of these glycerols has been studied extensively. A wide diversity of microorganisms from environment including archaea, cyanobacteria, and bacteria (facultative aerobic and anaerobic) have been studied. Besides, lower eukaryotes are also reported as hydrogen producers, such as Thermotoga, Rhodopseudomonas palustris, Escherichia, Enterobacter aerogenes ATCC35029, Klebsiella sp. TR17 Clostridium pasteurianum, and Chlamydomonas, which could also produce hydrogen through glycerol fermentation (Chookaew et al. 2014; Jitrwung and Yargeau 2011; Liu et al. 2015; Maru et al. 2012; Sarma et al. 2016; Zhang et al. 2015). The advantages of biohydrogen production through glycerol by microorganisms are low energy consumption, environment-friendly and high production efficiency. Ito et al. (2005) studied H2 and ethanol production through glycerol from biodiesel wastes by Enterobacter aerogenes HU-101 fermentation. It has revealed that the optimum YH2/glycerol of pure glycerol and raw glycerol are 1.05 mmol H2/ mol pure glycerol and 1.12 mmol H2/mol raw glycerol, respectively. Murarka et al. (2008) investigated the mechanism of biohydrogen production process by Escherichia coli via nuclear magnetic resonance analysis of cultures. Either 50% U-C-13-labeled or 100% unlabeled glycerol has been used. The YH2/glycerol of 0.94 mmol H2/mol glycerol was obtained. Selembo et al. (2010) obtained the YH2/glycerol of 0.28 mol H2/mol glycerol from glycerol metabolization by heat-treated mixed bacteria. (Liu and Fang 2010) has found that the YH2/glycerol of 0.53 mmol H2/mol glycerol was obtained by Klebsiella from treated biodiesel waste.
It has certain advantages for Enterobacter aerogenes in biohydrogen production by glycerol fermentation compared with the other bacteria. For example, a large proportion of the energy in glycerol can be converted to hydrogen energy due to a relatively high YH2/glycerol obtained by Enterobacter aerogenes. Theoretically, YH2/glycerol can reach 1.00 mol H2/mol glycerol. Genetic modification (Ito et al. 2005) and bacterial isolation (Mangayil et al. 2015, Poleto et al. 2016) were investigated to improve the biohydrogen production capacity. Therefore, bacteria with high biohydrogen production capacity still should be continuously screened and the key parameters of biohydrogen production through glycerol metabolism should also be optimized.
In this paper, several biohydrogen-producing strains could metabolize glycerol to produce hydrogen at high production efficiency were isolated by Hungate anaerobic rolling tube technology and identified by 16S rDNA gene sequence. The effects of environmental factors, including oxygen, initial glycerol concentration, initial pH, and initial nitrogen source concentration, on the biohydrogen production process from glycerol fermentation were investigated. And finally, the optimization of cultural condition was studied using orthogonal experiment. The results could provide technical support for the development of industrialization process of biohydrogen production.
Materials and methods
Strain and culture
Strain isolation and identification
The bacteria were isolated from river sludge of Shanghai Botanical Garden, Shanghai, China. Sludge samples were collected in the sterile test tubes and stored at 4 °C. The anaerobic bacteria were enriched twice in a modified Hungate technique in combination with the sera bottle technique under anaerobic conditions at 37 °C, which could inhibit the growth of aerobic bacteria. Bacterial morphology, physiology and biochemistry, molecular biology, and other characteristics were also investigated.
Preparation of culture medium
The batch culture medium (pH 6.0–6.2) was composed of glycerol 10 g/L, peptone 4 g/L, yeast extract 2 g/L, NaCl 4 g/L, beef extract 4 g/L, K2HPO4 1.5 g/L, MgCl2 0.1 g/L, FeSO4·7H2O 0.1 g/L, and trace elements 10 mL. The medium was sterilized at 121 °C for 20 min. The 50% (w/v) glycerol was sterilized at 115 °C for 30 min. The composition of trace elements included MnSO4·7H2O 0.01 g/L, ZnSO4·7H2O 0.05 g/L, H3BO3 0.01 g/L, N(CH2COOH)3 4.5 g/L, CaCl2·2H2O 0.01 g/L, Na2MnO4 0.01 g/L, CoCl2·6H2O 0.2 g/L, and KAl(SO4)2 0.01 g/L.
Biohydrogen production experiments
The isolated strain was inoculated in the sterile medium and cultured in a 250 mL sealed flask with working volume of 100 mL. Batch fermentations were carried out in one 500 mL anaerobic sera bottle with 50 mL of working volume by shaking at 180 rpm. The sera bottle was sealed by a rubber stopper after inoculation and flushed with N2 for 30 min. The biogas was collected by a syringe and measured by gas chromatography immediately. The controlled pH was measured by a pH meter (FE20, Mettler-Toledo, Shanghai, PR China) at every sampling interval. All experiments were carried out in triplicate.
The bacteria concentration was evaluated by the turbidimetric method. The optical density (OD) of cell growth was measured by a spectrophotometer (752 N, Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, PR China) at 660 nm. The DCW was calculated through the correlation between OD600 and DCW. One OD660 unit was estimated as 0.41 g/L dry cell weight, as shown in the following equation:
Gas-phase products (mainly H2 and CO2) were analyzed by gas chromatography (GC, 900C, Shanghai Sky Spectrum Analysis Instrument Co., Ltd., Shanghai, China). High purity nitrogen (99.999%) was used as carrier gas. The column model was TDX-01 and the detector was TCD thermal conductivity detector. The temperatures of the chromatographic column, sampler, and detector were 80 °C, 100 °C, and 100 °C, respectively. During the test, the headspace gas of the culture bottle was sampled to analyze the composition and content. The amount of hydrogen accumulated was calculated by the percentage content of hydrogen and volume of headspace gas.
High-performance liquid chromatography (SHIMADZU 10A, Shimadzu International Treading Co. Ltd., Japan) was used for the analysis of composition of biohydrogen fermentation broth. The model of liquid chromatographic column was Aminex HPX-87 h. The temperature of chromatographic column was 65 °C. The flow phase was 5 M dilute sulfuric acid with the flow rate of 0.6 mol/min. The detector model was RID-10A, 8.6 Pa. The amount of sampling was 20 μL. The UV spectrophotometric was used to determine the glycerol concentration.
Physiological and biochemical characteristics of isolates
The bacterial physiological and biochemical characteristics were analyzed to identify the isolated strain. The extraction of bacterial genomic DNA, electrophoretic detection, PCR amplification, and the analysis of 16S rDNA gene sequence were performed by Shanghai Major biological Medicine Technology Co. Ltd. The sequence was compared with the NCBI database. Sequence alignment was performed with phylogenetic analysis which was accomplished using the MEGA 6.0 software (Zhang and Sun 2008).
Effect of environmental factors on biohydrogen production
The effects of the key environmental factors, including oxygen, initial glycerol concentration, initial pH, and initial nitrogen source content, on the biohydrogen production process through glycerol fermentation were investigated.
Orthogonal experimental design was used to examine the interaction among factors. The orthogonal experiments of three factors and three levels were conducted to investigate the effects of the interaction of factors on the bacterial growth and the biohydrogen production under the anaerobic condition. The experimental error of all data in this paper was no more than 5%.
Results and discussion
Results of strains screening
For the primary isolation, 108 strains were isolated and numbered through initial enrichment, anaerobic plate culture, and anaerobic fermentation of glucose (to expand the substrate utilization range of hydrogen-producing microorganisms). It was observed that 98 strains could produce gas through the anaerobic fermentation of glucose.
For the secondary isolation, 16 strains with high biohydrogen production capacity were screened through the results of glucose fermentation from the 98 strains. The amounts of biohydrogen production of No. 6, No. 63, and No. 104 were higher than those of others. Therefore, they were selected for further investigation and named as EB-06, EB-63, and EB-104. The results of biohydrogen production of them are shown in Table 1.
It is demonstrated that the volume of biohydrogen production per milliliter of fermentation broth (CH2) from EB-06 was the largest in the three experimental groups. Therefore, EB-06 strain would be studied in detail as the model strain for biohydrogen production through glycerol fermentation in this paper.
Identification of strain
The bacterial morphological characteristics, physiology and biochemistry, were analyzed in this paper. It was observed that EB-06 was a facultative anaerobe, Gram-negative, rod-shaped, moving, no spore, single or paired, as shown in Fig. 1. According to the results of Petri dish experiment, it was shown that the colony was milky white, neat, round, smooth, and opaque, as shown in Fig. 1a. It was pink or mauve colony on the EMB identification medium, as shown in Fig. 1b.
The results of the physiological and biochemical characteristics of EB-06, EB-104, and EB-63 are shown in Table 2. It was demonstrated that the physiological and biochemical characteristics of EB-06 were consistent with that of Enterobacter aerogenes.
The results of sequence of 16S rDNA of EB-06 indicated that there were 99% homology similarity between the 16S rDNA sequences of Enterobacter aerogenes LN623608,1. The phylogenetic tree of EB-06 based on 16S rDNA sequences was shown in Fig. 2. According to the physiological and biochemical characteristics, and the phylogenetic tree of EB-06, the target strain EB-06 was identified as Enterobacter aerogenes and named as Enterobacter aerogenes EB-06.
Characteristics of biohydrogen production by Enterobacter aerogenes EB-06 through glycerol fermentation
The Enterobacter aerogenes EB-06 strain could produce biohydrogen through glycerol fermentation. The experiment was conducted in a 500 mL sera bottle under anaerobic conditions at 37 °C, 180 rpm. The initial glycerol concentration was 10 g/L. The initial pH was 6.0.
The change of amount of biohydrogen per liter of broth (CH2), pH, amount of biomass per liter of broth (Cx), and the residual glycerol concentration (Cglycerol) during the biohydrogen production process by EB-06 through glycerol fermentation were shown in Fig. 3. It was shown that CH2 and Cx in the process increased with time, while Cglycerol decreased and pH maintained constantly, which means that the EB-06 strain could utilize glycerol under anaerobic conditions, accompanied by the generation of biohydrogen and acidic metabolites in the fermentation broth. In addition, the properties of biohydrogen production through glucose anaerobic fermentation by EB-06 in batch culture were also shown in Fig. 4.
It was demonstrated that EB-06 was able to utilize glycerol to produce more hydrogen and biomass than glucose from Figs. 3 and 4. Therefore, the characteristics of biohydrogen production of EB-06 by glycerol fermentation were worthy of further study. As shown in Fig. 3, it was demonstrated that CH2, pH, Cx, and Cglycerol changed apparently from the 4th to the 8th cultural hour. After the 8th hour, the Cx, pH, and Cglycerol tended to be invariant. However, CH2 continued to increase, which means that biohydrogen production was lagging behind the growth of EB-06 and glycerol consumption.
Figure 5 shows the trend of μ (the specific growth rate, h−1), Qglycerol (the specific rate of glycerol consumption, g/g DCW h), and QH2 (the specific rate of hydrogen production, mmol/g DCW h) with cultural time. It was observed that the trend of Qglycerol, μ, and QH2 was similar during whole biohydrogen production process. All of them increased first and then decreased. The corresponding time of the maximum value of them was different.
The corresponding time of the maximum of QH2 significantly lagged behind those of the maximum of Qglycerol and μ. It was indicated that the biohydrogen production was lagging behind the bacteria growth process. The corresponding time of the maximum Qglycerol appeared earlier than those of growth and biohydrogen generation. It was indicated that a large amount of glycerol was consumed in advance for the preparation of bacterial growth and biohydrogen production during the 0–2nd hours of cultural time.
Effect of oxygen on biohydrogen production by EB-06 through glycerol fermentation
To study biohydrogen production characteristics of Enterobacter aerogenes under anaerobic and aerobic conditions, the effect of oxygen on the biohydrogen production by EB-06 through glycerol fermentation was explored. Figure 6 shows the change of Cx and CH2 with time by EB-06 through glycerol fermentation under anaerobic and aerobic conditions, respectively. It was observed that Cx and CH2 increased with time under both the anaerobic and aerobic conditions. The YH2/glycerol under anaerobic conditions was calculated as 0.81 mol H2/mol glycerol, which was 4.5 times higher than that under aerobic conditions.
It seems that CH2 under anaerobic conditions was much smaller than that under aerobic conditions shown in Fig. 6. It is well known that there were two biohydrogen-producing pathways in the genus of Enterobacter, i.e., formic acid hydrogen production pathway and NADH hydrogen production pathway (Kurokawa and Tanisho 2005; Leonhartsberger et al. 2002). For the former, the activity of the formate dehydrogenase could be inhibited by oxygen (Vidal-Limón et al. 2017). For the latter, NADH was converted to hydrogen through hydrogenase. And the oxygen was used as the electron acceptor, which could efficiently provide more NADH reduction capacity and further produce more biohydrogen. Thus, the presence of oxygen will have the opposite effect on these two biohydrogen production pathways (Liu and Fang 2010). Moreover, it could also be deduced that the amount of biohydrogen produced by the formic acid pathway was different from NADH pathway. The formic acid biohydrogen production pathways might play a major role under anaerobic conditions.
On the other hand, the formate dehydrogenase activity was inhibited by O2, so that the hydrogen-producing pathway of formic acid was heavily blocked under aerobic condition (Selembo et al. 2010). Similarly, the hydrogenase could also be inhibited partly by O2. Meanwhile, NADH hydrogen-producing pathway was also affected. The electrons from NADH pathway could not flow to hydrogen production pathway, but to biomass (Chu et al. 2013). However, a small amount of biohydrogen was still produced under aerobic conditions, as shown in Fig. 6. This phenomenon might due to the fact that the activities of the above two enzymes were severely inhibited in the presence of oxygen rather than being completely inactivated.
According to the results shown in Fig. 6, Cx was 0.33 g/L under anaerobic conditions, while it was 0.60 g/L under aerobic conditions. It means that EB-06 could grow better under aerobic condition than that of anaerobic condition just because more NADH and ATP might be obtained for the biosynthetic pathway of EB-06 when O2 was the electron acceptor.
The trends of μ and QH2 during the biohydrogen production by EB-06 glycerol fermentation under aerobic and anaerobic conditions were shown in Fig. 7. It was observed that the trends of them with time were similar, whether it is under the condition of aerobic or anaerobic. All of them increased first and then decreased. It was also shown that the presence of oxygen was beneficial to the growth of EB-06, while was harmful to the biohydrogen production capacity of EB-06.
The μ and Qglycerol were shown in Fig. 8 during the biohydrogen production by EB-06 glycerol fermentation under aerobic and anaerobic conditions. The presence of oxygen was harmful to the consumption of glycerol by EB-06 due to the Pasteur effect.
Effects of initial pH on biohydrogen production by EB-06 through glycerol fermentation
The fermentative biohydrogen production could be effected by different initial pH (Nikhil et al. 2014). To investigate the effects of pH on biohydrogen production process by EB-06 through glycerol fermentation, CH2, Cx, pH, and Cglycerol under different initial pH were studied with results shown in Fig. 9.
The CH2 under different initial pH are shown in Fig. 9a. It was found that CH2 increased with time under different pH. The CH2 increased first and then decreased with pH. They were significantly higher at pH 6 and 7 than that of others. It is indicated that amount of biohydrogen generated by EB-06 would be heavily inhibited at too lower or too higher pH. The optimum CH2 was 83.76 mmol H2/L, and the YH2/glycerol was 0.78 mol H2/mol glycerol under initial pH 7.0.
The effects of initial pH values on biomass of EB-06 were shown in Fig. 9b. It was found that Cx in all the experimental groups increased with time, and reached to a stable stage after the 10th cultural hour. However, Cx were first increased and then decreased with the increasing of initial pH values. The optimum Cx was obtained as 1.34 g/L at pH 6.0.
The influence of initial pH on the pH of the fermentation broth could be seen in Fig. 9c. It was shown that the pH of broth had gradually decreased over time. In addition, the pH in the broth increased gradually with the increase of the initial pH. It was indicated that acidic metabolites gradually produced with the glycerol/glucose fermentation progresses.
The major liquid metabolites and concentrations in biohydrogen production process from EB-06 fermentation of glucose and glycerol are shown in Table 3. It could be seen that the major liquid metabolites were lactic acid and acetic acid for glucose fermentation, while ethanol for glycerol fermentation. Therefore, the pH of the fermentation broth during the biohydrogen production through glycerol fermentation was more stable than those of glucose fermentation.
It was found that Cx increased without any biohydrogen produced from 0 to 2nd cultural hour at pH 6.0 and 7.0 from Fig. 9. The Cglycerol was decreased and approached zero from 8th to 12th cultural hour. However, CH2 continued to increase even at the 12th cultural hour. It was reported that formic acid might be an important intermediate metabolite (Ghimire et al. 2015). Thus, to a certain extent, the amount of intracellular formic acid was likely to accumulate before hydrogen could be produced.
Another possible explanation was that there existed biohydrogen-producing step and the biohydrogen-releasing step for biohydrogen production process. The hydrogen would be released to outside of the cell when the intracellular hydrogen accumulated to a certain level (Ghimire et al. 2015).
The curves of QH2 with time under different initial pH are shown in Fig. 10. All of these QH2 increased first and then decreased with time. However, the maximum QH2 and the corresponding time under different initial pH were different. The optimum of QH2 was obtained as 22.11 mmol H2/g DCW h at initial pH 6.
In regards to the biohydrogen production of EB-06 through glucose fermentation. The optimum Cx, CH2 and YH2/glycerol was obtained as 1.42 g/L, 46.13 mmol H2/L and 0.71 mmol/mol glycerol at initial pH 8.0, respectively.
It could be found that the effects of the optimum initial pH on biohydrogen production process were different when glycerol or glucose was used as fermentation substrate. Glucose fermentation tends to occur under alkaline conditions, while glycerol fermentation tends to occur under acidic or neutral conditions.
Effect of initial glycerol concentration on biohydrogen production by EB-06 through glycerol fermentation
The effects of different initial glycerol concentrations on biohydrogen production by EB-06 were studied by batch culture at initial pH 6.0. The effects of different initial glycerol concentration on CH2, Cx, pH, and Cglycerol are shown in Fig. 11.
As shown in Fig. 11a, CH2 increased gradually and eventually became invariant with time during the whole fermentation process. However, CH2 increased first and then decreased with initial glycerol concentration. The optimum CH2 was obtained as 87.24 mmol H2/L at the initial glycerol concentration of 10 g/L. It was also reported that CH2 would be increased with substrate concentration at the low concentration range of substrate. However, it was inhibited at high concentrations of substrate.
The cell growth curve is also shown in Fig. 11b. It was shown that the Cx was gradually increased during whole fermentation process. However, the Cx increased first and then decreased with the initial glycerol concentration. The growth of EB-06 was inhibited by the high initial glycerol concentrations. The optimum biomass concentration was about 1.10 g/L at the initial glycerol concentration of 10 g/L. It was found that the Han–Levenspiel model could well describe the effect of substrate concentration on biohydrogen production.
The pH in the fermentation broth decreased with the fermentation time, as shown in Fig. 11c. Among them, the pH in the fermentation broth was decreased when the initial glycerol concentration was 10 g/L, which indicated that the amount of acidic metabolites produced were relatively large.
The Cglycerol would gradually decrease due to substrate consumption, as shown in Fig. 11d. The Cglycerol increased with initial glycerol. Glycerol was completely consumed at the 8th cultural hour when the initial glycerol concentration was 5 g/L and 10 g/L. However, glycerol had not been completely consumed at the initial glycerol concentration from 20 to 40 g/L. Qglycerol increased with the initial glycerol concentration. The Qglycerol at the initial glycerol concentration of 40 g/L was obtained as 7.73 g/g DCW h, as shown in Table 4. However, the total amount of glycerol consumption is less due to the lower Cx at the higher initial concentrations of glycerol.
It was shown that the initial glycerol concentration for the optimum CH2 was different from that of the optimum QH2 shown in Fig. 11a and Table 4. It was revealed that EB-06 biohydrogen production metabolism and glycerol metabolism pathway were affected by the initial glycerol concentration in Table 4. Yx/glycerol (yield coefficient of biomass from glycerol, g/mol) and YH2/glycerol (yield coefficient of biohydrogen from glycerol consumed, mol H2/mol) would decrease with the increasing of initial glycerol concentration, while QH2 and Qglycerol would increase with the initial glycerol concentration. The CH2 is the integral value of the product of QH2 and Cx to the fermentation time. Therefore, the optimum CH2 was obtained when the initial glycerin concentration was 10 g/L, as shown in 11A.
The QH2 was related with the bacteria species and substrate type. A large number of hydrogen-producing bacteria have been isolated and screened to obtain efficient hydrogen-producing strains. (Kumar and Das 2000) isolated Enterobacter clocae IIT-BT08 from the tree leaf extract. It was found that the maximum QH2 was 29.63 mmol H2/g DCW h at pH 6.0 and 37 °C; the QH2 was from 25.0 to 28.0 mmol H2/g DCW h at pH 6.0 and 36 °C. (Xing et al. 2006) screened two ethanol-type hydrogen-producing bacteria Ethanoligenens harbinense sp. R3 and Ethanoligenens harbinense sp.Y3 from CSTR reactor. The maximum QH2 was 35.74 mmol H2/g DCW h when glucose was the substrate. It could be seen that the optimum specific hydrogen production rate would reach to 41.47 mmol H2/g DCW h when the initial glycerol concentration was 40 g/L seen from Table 4. It was indicated that EB-06 had a potential in biohydrogen production.
Effect of initial nitrogen source on hydrogen production by EB-06 through glycerol fermentation
It was well known that the growth and metabolism of microorganisms would be affected by the initial nitrogen source content of the medium. In this experiment, the peptone as nitrogen source contained in medium was set as 4.0 g/L, 2.0 g/L, and 0.0 g/L, respectively.
Figure 12 shows that the final CH2, Cx, and QH2 of the EB-06 biohydrogen production process under the different initial nitrogen source concentration. Moreover, both the final CH2 and Cx were gradually decreased with the decrease of initial nitrogen source concentration, as shown in Fig. 12a, b. The QH2 of EB-06 also decreased with the decrease of initial nitrogen source concentration, as shown in Fig. 12c. The results shown that initial nitrogen source played an important role in cell growth and hydrogen generation during biohydrogen production through glycerol fermentation.
Optimization of biohydrogen production by EB-06 of glycerol fermentation through orthogonal design
The growth and the biohydrogen production by EB-06 through glycerol fermentation could be affected by the medium composition and environmental conditions. Orthogonal experiments can be used to optimize the interaction of multiple factors. The initial glycerol concentration, initial pH, and initial C/N ratio were selected as three key factors to investigate their effects on EB-06 growth and biohydrogen production according to the literature (Poleto et al. 2016). Three-factor and three-level orthogonal experiments were adopted to study the optimal medium composition and optimal culture conditions. All the orthogonal experimental design data are shown in Table 5.
The results of the orthogonal experiment are shown in Table 6. The comprehensive score was characterized by YH2 (mol/mol). It shown that the optimum YH2 was obtained as 1.07 mol H2/mol glycerol at 10 g/L initial glycerol, initial pH 5.0, and initial C/N ratio 5/3.
The range analysis method was used to analyze the orthogonal experimental results of biohydrogen production through EB-06 fermented glycerol with the results shown in Table 7. The range R values of the three factors are 0.19 (B), 0.17 (A), and 0.08 (C), respectively. Therefore, it could be concluded that the main factor was initial pH, followed by the initial glycerol concentration and the initial C/N ratio.
It was shown that the optimum YH2 of 1.07 mol H2/mol glycerol was obtained by EB-06 in this paper. The results were close to 1.12 mol H2/mol glycerol reported by (Ito et al. 2005), which was the maximum value of YH2 reported so far. And the yield of biohydrogen to glycerol obtained by this paper was higher than that of others shown in Table 8.
A highly efficient biohydrogen-producing strain was isolated, identified, and named as Enterobacter aerogenes EB-06.
The characteristics of the biohydrogen production of Enterobacter aerogenes EB-06 through glycerol fermentation were analyzed in this paper. The paper also studied effects of the key environmental factors, such as oxygen, initial pH, initial glycerol concentration, and nitrogen source on the biohydrogen production process.
Based on the analysis of orthogonal experiment, it was found that the main factor affecting biohydrogen production was initial pH, followed by the initial glycerol concentration, and, finally, the initial C/N ratio. Simultaneously, the optimum YH2 of 1.07 mol H2/mol glycerol was obtained at the initial glycerol concentration 10.00 g/L, the initial pH value 5, and the C/N ratio 5/3.
- C H2 :
amount of hydrogen production per volume of fermentation broth (mmol /L)
- C x :
cell concentration per volume of fermentation broth (g/L)
- OD660 :
the optical density of bacteria at 660 nm
dry cell weight
- C glucose :
residual glucosel concentration per volume of fermentation broth (g/L)
- C glycerol :
residual glycerol concentration per volume of fermentation broth (g/L)
- Q glycerol :
specific glycerol consumption rate [(g glycerol consumed) (g dry cell formed)−1 h−1]
- Q H2 :
specific H2 formation rate [(mmol H2 formed) (g dry cell formed)−1 h−1]
- μ :
specific growth rate of biomass (h−1)
- Y H2/glycerol :
yield coefficient of H2 from glycerol [(mmol H2 formed) (g glycerol consumed)−1]
- Y x/glycerol :
yield coefficient of biomass from glycerol [(g dry cell formed) (g glycerol consumed)−1]
American Type Culture Collection
People’s Republic of China
thermal conductivity detector
refractive index detector
molecular evolutionary genetic analysis
Polymerase Chain Reaction
National Center for Biotechnology Information
nicotinamide adenine dinucleotide
Asadi N, Zilouei H (2017) Optimization of organosolv pretreatment of rice straw for enhanced biohydrogen production using Enterobacter aerogenes. Bioresour Technol 227:335–344
Azman NF, Abdeshahian P, Kadier A, Al-Shorgani NKN, Salih NKM et al (2016) Biohydrogen production from de-oiled rice bran as sustainable feedstock in fermentative process. Int J Hydrogen Energy 41:145–156
Chookaew T, O-Thong S, Prasertsan P (2014) Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions. Int J Hydrogen Energy 39:9580–9587
Chu CY, Lin T, Lin CY (2013) Effect of substrate concentration and pH on biohydrogen production kinetics from food industry wastewater by mixed culture. Int J Hydrogen Energy 38:15849–15855
Dessì P, Porca E, Waters NR, Lakaniemi AM, Collins G et al (2018) Thermophilic versus mesophilic dark fermentation in xylose-fed fluidised bed reactors: biohydrogen production and active microbial community. Int J Hydrogen Energy 43:5473–5485
Faber MO, Ferreira-Leitão VS (2016) Optimization of biohydrogen yield produced by bacterial consortia using residual glycerin from biodiesel production. Bioresour Technol 219:365–370
Fan XH, Burton R, Zhou YC (2010) Glycerol (byproduct of biodiesel production) as a source for fuels and chemicals—mini review. Open Fuels Energy Sci J 3:17–22
Ghimire A, Frunzo L, Pirozzi F, Trably E, Escudie R et al (2015) A review on dark fermentative biohydrogen production from organic biomass: process parameters and use of by-products. Appl Energy 144:73–95
Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N (2005) Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. J Biosci Bioeng 100:260–265
Jitrwung R, Yargeau V (2011) Optimization of media composition for the production of biohydrogen from waste glycerol. Int J Hydrogen Energy 36:9602–9611
Jitrwung R, Verrett J, Yargeau V (2013) Optimization of selected salts concentration for improved biohydrogen production from biodiesel-based glycerol using Enterobacter aerogenes. Renew Energy 50:222–226
Kumar N, Das D (2000) Enhancement of hydrogen production by Enterobacter cloacae IIT-BT 08. Process Biochem 35:589–593
Kurokawa T, Tanisho S (2005) Effects of formate on fermentative hydrogen production by Enterobacter aerogenes. Mar Biotechnol 7:112–118
Leonhartsberger S, Korsa I, Böck A (2002) The molecular biology of formate metabolism in enterobacteria. J Mol Microbiol Biotechnol 4:269–276
Liu F, Fang B (2010) Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae. Biotechnol J 2:374–380
Liu Q, Xiong DW, Hong-Bo HU, Zhang XH (2015) Hydrogen production from glycerol using a genetically engineered Escherichia coli HW2 strain. J Chem Eng Chin Univ 5:1133–1137
Mangayil R, Aho T, Karp M, Santala V (2015) Improved bioconversion of crude glycerol to hydrogen by statistical optimization of media components. Renew Energy 75:583–589
Maru BT, Bielen AAM, Kengen SWM, Constantí M, Medina F (2012) Biohydrogen production from glycerol using Thermotoga spp. Energy Procedia 29:300–307
Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R (2008) Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 74:1124–1135
Nikhil GN, Mohan SV, Swamy YV (2014) Behavior of acidogenesis during biohydrogen production with formate and glucose as carbon source: substrate associated dehydrogenase expression. Int J Hydrogen Energy 39:7486–7495
Niu K, Zhang X, Tan WS, Zhu ML (2011) Effect of culture conditions on producing and uptake hydrogen flux of biohydrogen fermentation by metabolic flux analysis method. Bioresour Technol 102:7294–7300
Pachapur VL, Sarma SJ, Brar SK, Bihan YL, Buelna G et al (2015) Biohydrogen production by co-fermentation of crude glycerol and apple pomace hydrolysate using co-culture of Enterobacter aerogenes and Clostridium butyricum. Bioresour Technol 193:297–306
Poleto L, Souza P, Magrini FE, Beal LL, Torres APR et al (2016) Selection and identification of microorganisms present in the treatment of wastewater and activated sludge to produce biohydrogen from glycerol. Int J Hydrogen Energy 41:4374–4381
Pott RW, Howe CJ, Dennis JS (2014) The purification of crude glycerol derived from biodiesel manufacture and its use as a substrate by Rhodopseudomonas palustris to produce hydrogen. Bioresour Technol 152:464–470
Sarma S, Dubey VK, Moholkar VS (2016) Kinetic and thermodynamic analysis (with statistical optimization) of hydrogen production from crude glycerol using Clostridium pasteurianum. Int J Hydrogen Energy 41:19972–19989
Selembo PA, Perez JM, Lloyd WA, Logan BE (2010) Enhanced hydrogen and 1,3-propanediol production from glycerol by fermentation using mixed cultures. Biotechnol Bioeng 104:1098–1106
Sivaramakrishnan R, Incharoensakdi A (2018) Microalgae as feedstock for biodiesel production under ultrasound treatment—a review. Bioresour Technol 250:877–887
Sørensen B (2011) Hydrogen and fuel cells: emerging technologies and applications, vol 36. Academic Pr Inc, London, p 4356
Trchounian K, Trchounian A (2015) Escherichia coli hydrogen gas production from glycerol: effects of external formate. Renew Energy 83:345–351
Vidal-Limón AM, Tafoya P, Santini BL, Contreras OE, Aguila SA (2017) Electron transfer pathways analysis of oxygen tolerant [NiFe]-hydrogenases for hydrogen production: a quantum mechanics/molecular mechanics—statistical coupled analysis. Int J Hydrogen Energy 42:20494–20502
Xing D, Ren N, Li Q, Lin M, Wang A et al (2006) Ethanoligenens harbinense gen. nov., sp. nov., isolated from molasses wastewater. Int J Syst Evol Microbiol 56:755–760
Zhang W, Sun Z (2008) Random local neighbor joining: a new method for reconstructing phylogenetic trees. Mol Phylogenet Evol 47:117–128
Zhang D, Xiao N, Mahbubani KT, Rio-Chanona EAD, Slater NKH et al (2015) Bioprocess modelling of biohydrogen production by Rhodopseudomonas palustris : model development and effects of operating conditions on hydrogen yield and glycerol conversion efficiency. Chem Eng Sci 130:68–78
WT and XZ conceived and designed the experiments. YL performed the experiments and analyzed the data. YL wrote the paper. YQ helped in manuscript writing. XZ and MZ revised the manuscript. All authors read and approved the final manuscript.
The authors are grateful for the financial support of this research by the Open Project Funding of the State Key Laboratory of Bioreactor Engineering of China.
The authors declare that they have no competing interests.
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