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Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor
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 x x x ( 2 0 1 7 ) 1 e7
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Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor Han-Quan Wen, Jian Du, De-Feng Xing, Jie Ding, Nan-Qi Ren, Bing-Feng Liu* State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, P.O. Box 2614, 73 Huanghe Road, Harbin 150090, China
article info
abstract
Article history:
To achieve stable and efficient photo-fermentative hydrogen production, this work
Received 19 January 2017
investigated photo-fermentative hydrogen production by forming biofilm on the surface of
Received in revised form
carrier in the biofilm reactor (BR). Results showed the hydrogen production performance
5 March 2017
was greatly improved by formed biofilm. The time of hydrogen production and efficiency of
Accepted 18 April 2017
substrate utilization were enhanced obviously compared to the control reactor (CR). When
Available online xxx
the CR was used, hydrogen production stopped at 7th day and maximum cumulative hydrogen volume and hydrogen yield were 1730 ± 87 mL/L and 1.44 ± 0.07 mol H2/mol
Keywords:
acetate, respectively. However, in the BR hydrogen production volume of 3028 ± 150 mL/L
Biofilm reactor
and hydrogen yield of 2.52 ± 0.13 mol H2/mol acetate were obtained, which were enhanced
Photo-fermentative bacteria
about 75% compared to that of the CR. The time of hydrogen production extended from 7
Biohydrogen
days of CR to 12 days of BR and the substrate conversion efficiency increased from 36% of
Substrate utilization
CR to 63% of BR. It was worth noting at 8th day that substrate was almost utilized completely but hydrogen production still lasted for 4 days. This suggested that the formation of biofilm in BR was favorable to continuous hydrogen production and substrate utilization with high efficiency. Results demonstrated the BR can get a more stable and consistent operating process and it was a proper and potential way to produce hydrogen by photo-fermentative bacteria (PFB). © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Currently, energy shortage and environment pollutions caused by the application of fossil fuels were the great challenges human faced [1]. It is necessary to develop a new, clean and renewable energy to replace traditional fossil fuels. Increased attentions were focused on the hydrogen gas due to its abundance, high energy intensity (142 MJ/kg), burning
clearly with only water produced and so on [2]. Among different ways of hydrogen production, photo-fermentation could combine waste degradation, solar energy utilization and hydrogen production in one-step process without oxygen inhibition, thus this technology was considered as the most potential pathway for hydrogen production [3]. However, an obstacle, bad flocculation ability and low hydrogen yield, limited its pace of industrialization, thus made the hydrogen producer easy to flow away with the discharge, led to low
* Corresponding author. Fax: þ86 451 86282008. E-mail address: [email protected] (B.-F. Liu). http://dx.doi.org/10.1016/j.ijhydene.2017.04.150 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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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 x x x ( 2 0 1 7 ) 1 e7
biomass in the reactor and operated at an inefficient and unstable operation condition [4]. To solve above problems, researchers did a lot of work on bacterial immobilization [5], reactor design [6], strain breeding [7] and so on to increase biomass and hydrogen production performance. Among the immobilizations, there are three main ways: embedding, chemisorption and physisorption. Embedding method is a popular way which uses semipermeable material such as agar that allows the micromolecule matters exchange and fixes the bacteria. By embedding the bacteria, cells could easily be separated from the medium, but it will also bring the transport resistance for the macromolecule and light, limit the space for bacterial growth and inhibit the rate of cell refresh and discharge of dead cells [8]. Chemisorption uses the functional group or bonds to fix cells onto the carrier, such immobilization is stable and makes exchange easily, but harmful to the bacteria for its violent reaction. Physisorption allows the cells attach on the surface of carriers freely and it is beneficial for exchange of light and matters. The operation condition was simply and mildly, and the most important advantages of the physisorption is it could form the biofilm freely so the bacteria could show the special characteristic of biofilm. Therefore, in this study, physisorption was adopted to immobilize photofermentative bacteria to form biofilm for improving hydrogen production performance. Biofilm is considered as one potential and proper way for hydrogen production because it could fix more bacteria to enhance biomass and improve hydrogen production and support bacteria second living strategy thus cells distribute energy and matters more choicely [9]. It has been confirmed that all kinds of bacteria can form biofilms and this may be the preferred mode of bacterial existence in nature [10,11]. Though according to the DLVO (DerjaguineLandaueVerweyeOverbeek) theory [12], the small size and high Zeta potential of PFB make cells hard to aggregate, the PFB still have the ability to form biofilm for the following reasons. Firstly, a study has proved PFB, as most ancient bacteria, which lived in a circumstance filled with hydrogen millions of years ago, mainly existed in biofilm form [13]. Secondly, some strains like Rhodopseudomonas faecalis RLD-53 could secret some extracellular polymeric substances (EPS) which are the base matrix for biofilm [14]. Thus biofilm for enhanced hydrogen production was possible in theory. However, up to now, most reported studies about biofilm were focused on the dark fermentation and only a few researches reported the photo-fermentative hydrogen production by formed biofilm. Among reported literature, almost all work investigated on optimization of process condition [15,16]. In a study, activated carbon fibers were used as the fluidized solid carrier to immobilize photo-fermentative bacteria for hydrogen production and the maximum yield of 3.08 mol H2/mol acetate was obtained [17]. Also, another study showed the addition of clay and silica gel was effective in promoting hydrogen production, resulting in 67.2e50.9% and 37.2e32.5% increases in rate and yield of hydrogen production [18]. However, these studies paid more attention to the hydrogen production of the reactor, and little information was discussed on enhancement of hydrogen production by biofilm from two aspects of biomass change and substrate utilization.
Therefore, this study explored the effects of biofilm on the hydrogen production, biomass change and substrate utilization in the BR and CR. The results demonstrated hydrogen production was greatly improved by the formed biofilm on the carrier.
Materials and methods Bacterium and medium Rhodopseudomonas sp. nov. strain A7 (Accession No. KJ699180) was from the State Key Laboratory of Urban Water Resource and Environment in Harbin Institute of Technology [19]. Growth medium (GM) contains CH3COONa (1.0 g), sodium succinate (1.0 g), peptone (0.5 g), yeast powder (1.0 g), NH4Cl (1.0 g), KH2PO4 (0.5 g), K2HPO4 (0.5 g), MgSO4 (0.2 g), CaCl2 (0.08 g), FeSO4$7H2O (0.012 g), EDTA (0.2 g), L-cysteine (0.5 g), NaCl (0.1 g), vitamin (0.1 mL) and trace elements solution (0.1 mL) in 1 L distilled water and pH was adjusted to 6.86 ± 0.1 [19]. Hydrogen production medium (HPM) contains CH3COONa (4.1 g), KH2PO4 (0.5 g), K2HPO4 (0.5 g), sodium glutamate (1.69 g), MgSO4 (0.2 g), CaCl2 (0.08 g), FeSO4$7H2O (0.012 g), EDTA (0.2 g), L-cysteine (0.5 g), NaCl (0.1 g), vitamin (0.1 mL) and trace elements solution (0.1 mL) in 1 L distilled water and pH was adjusted to 6.86 ± 0.1 [19].
Reactor and operation condition The anaerobic tube of 25 mL was used as the reactor. The reactors without carriers were set as the CR. The reactors were filled with 10 mL medium and blew by argon for 5 min to maintain anaerobic conditions (Hungates technology [20]). And then reactors were sterilized at 121 C for 15 min and inoculated with 1 mL seed bacteria (inoculation ratio of 10%), which was in the mid-exponential growth phase after 24 h of cultivation in the growth medium. The reactors were placed in the shaker at 120 rpm, 35 C and 150 W/m2 of light intensity on surface of reactors. The biogas volume was measured with a low friction glass injector. The BR was made by introducing the silica gel sheet (1 cm 10 cm, Xiang Tai sealing material company, China) into the CR to act as the carrier for the formation of biofilm.
Surface morphology of the bacteria The biofilm samples were firstly fixed by 2.5% glutaraldehyde at 4 C in the dark for 12 h then washed gently with 0.01 mol/L phosphate buffered saline twice. Then samples were dehydrated successively by 30%, 50%, 70%, 80% and 90% ethanol once for 10 min respectively and 100% twice for 15 min. And the samples was replaced with ratio of 1:1 (v/v) of ethanol and isoamyl acetate and 100% isoamyl acetate for 15 min, respectively. Lastly after being freeze-dried and coated with gold powder completely, the samples were observed under the scanning electron microscope (SEM). The instrument of SEM was the JEM-2100 (HR) transmission electron microscopy (JEOL, Ltd., Tokyo, Japan) operated at an accelerating voltage of 200 kV.
Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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Analysis method
80
Calculations Hydrogen volume was calculated by the formula: VHydrogen;t ¼ VSpare þ VBiogas;t CHydrogen; t VSpare CHydrogen;t1
(1)
Z VHydrogen;total ¼
VHydrogen;t
(2)
where VHydrogen;t and VBiogas;t are the volume of hydrogen (mL/L medium) and biogas (mL/L medium) on the t time, respectively; t is the time (day, t 2); CHydrogen;t and CHydrogen;t1 are the concentration of hydrogen on the t time and t 1 time respectively (%); VHydrogen;total is the total volume of hydrogen production (mL/L medium); VSpare is the spare volume of reactor (1500 mL). Hydrogen yield was calculated by the following formula: CHydrogen yield ¼
VHydrogen;t ac
CR BR
70
Hydrogen concentration (%)
The biomass of free bacteria were extracted by centrifugation at 12,000 rpm for 5 min. For the bacteria on the biofilm, cells were extracted by washing the carrier gently three times with ddH2O and put in the ultrasonic cleaner for 10 min. For both the free bacteria and biofilm bacteria in CR and BR, the dry weight was measured by being dried until unchanged at 105 C. The optical biomass (OD660) was measured by a visible spectrophotometer (Unic 7200, Unic Shanghai Instrument Company, Shanghai, China). Light density was detected by the TENMARS TM-207 solar Power meter with 1.5 M remove sensor radiation energy test (Tenmars Electronics CO., LTD., Taiwan, China). The hydrogen concentration was detected by a gas chromatograph (Agilent 7890A) which column was Agilent 19091J-433: HP-5 5% Phenyl Methyl Siloxane and operation of the oven, column and thermal conductivity detector (TCD) were at 35 C for 4 min, 325 C and 200 C respectively. Substrate concentration was detected by a gas chromatograph (Agilent 7890A) equipped with a Agilent 19095N-123: 30 m 530 mm 1 mm and a flame ionization detector (FID) and operation of the injector, oven, detector and FID were at 250 C, 70 C first then programmed heating in 5 mine170 C and maintained for 1 min and 300 C, respectively. Each isolate was assayed more than three times and the results were presented as the average of the assays.
60 50 40 30 20 10 0 1
2
3
4
5
6
7
8
9
10
11
12
Time (day) Fig. 1 e The change of hydrogen concentration in BR and CR.
concentration. The experimental results showed the average hydrogen concentration of BR was about 10% higher than that of CR (Fig. 1). The biogases and hydrogen production trends were similar (Figs. 2 and 3). The most biogases were produced between 2 and 5 days in the CR, and the maximum biogases production rate and hydrogen production rate was 533 mL/L/ d and 468 mL/L/d at 4th day, respectively. After 5 days, the biogases and hydrogen production rate slowed down and gases production stopped at 7th day. The total cumulative biogases and hydrogen volume was 1767 ± 88 mL/L and 1730 ± 87 mL/L, respectively. Hydrogen yield was 1.44 ± 0.07 mol H2/mol acetate. However, this work found that BR can extend biogases production time from 7 days of CR to 12 days and hydrogen production mainly occurred between 2 and 10 days. Compared to CR, BR increased efficient and stable hydrogen production time. The maximum gases rate and hydrogen production rate was 600 ± 29 mL/L/d and 507 ± 30 mL/L/d at the 6th day. The cumulative biogases and hydrogen volume reached 4300 ± 200 mL/L and 3028 ± 160 mL/ L, respectively. Hydrogen yield was 2.52 ± 0.13 mol H2/mol acetate, which was 63% of the theoretical yield.
(3)
where CHydrogen yield is the hydrogen yield (mol hydrogen/mol acetate); a is the coefficient of molar density at room temperature (24 L/mol); c is the concentration of sodium acetate (50 mol/L medium).
Results and discussion Biogases and hydrogen production in the BR Biogases production was investigated in CR and BR. The composition of biogases in the photo-fermentation was mainly H2 and CO2. The hydrogen volume was calculated by combing the biogases production rate and the hydrogen
Fig. 2 e Continuous biogas production and production rate by A7 affected by biofilm.
Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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The two kinds of reactors showed the similar gas production trend, but BR could broaden the gases production period from 1 day to 7 days of CR to 1 day to 12 days of BR, BR also had a stable and efficient gases and hydrogen production, which enhanced 143% and 75% compared to the CR. It showed that the biofilm fixed on the carrier could give a better gas and hydrogen production mode. The high efficient hydrogen production time was broaden from 2 to 5 days to 1e10 days, cumulative hydrogen production was higher and the operation was more stable in the BR than the CR.
biofilm biomass 0.05 ± 0.01 g/L) after 7 days. Compared to CR, the biomass in the BR increased about 32% and 2.5% biomass was contributed from the formed biofilm. This result indicated that the formed biofilm by introduction of carrier in the GM could promote the bacterial growth and the increasement of biomass. In the HPM, biomass in both reactors was obviously lower than that in the GM. Biomass in the BR was 2.0 ± 0.1 g/L, which was 18.4% higher than 1.69 ± 0.08 g/L in the CR at 7th day. However, the biomass from biofilm of the BR in the HPM achieved a higher value of 0.15 ± 0.01 g/L, which was 200% higher than 0.05 ± 0.01 g/L in the GM (Fig. 4). The increasement of biomass, especially from biofilm, greatly promoted hydrogen production of strain A7 (Fig. 5). Compared to HPM, GM is a better medium for cell growth but no hydrogen was produced. And a little amount of bacteria was attached to carriers for the formation of biofilm in GM, so biofilm biomass was lower. However, bacteria in the bad circumstance (HPM) can form more biofilm for maintaining energy balance and resisting unfavorable condition. This indicated that bacteria by self-regulation could choose the suitable mode for cell growth and hydrogen production. The formation of biofilm changed the bacterial living condition and affected suspended bacteria activity. Especially, this may help to enhance key enzyme activity of hydrogen production. Therefore, hydrogen production performance was promoted markedly. There was limited information about the effects of biomass from biofilm on the biohydrogen production, and most researches are focused on hydrogen production process.
The effect of formed biofilm on biomass
Substrate utilization in the BR and CR
The biomass change also was detected in different reactors (Fig. 4). In this study, cells were divided into two parts: free bacteria and biofilm bacteria. In the GM, the final biomass could achieve 1.94 ± 0.10 g/L in the CR and 2.55 ± 0.12 g/L in the BR (of which the free biomass occupied 2.53 ± 0.12 g/L and the
The substrate in the medium was mainly used for bacterial growth and hydrogen production, and their utilization efficiency determined biosynthesis of biomass and hydrogen production ability. The initial substrate concentration was 50 mmol/L in CR and BR. The results showed the substrate
3500
CR
Cumulative H2
H2 production rate
BR
Cumulative H2
H2 produciton rate
600
2500
400
2000 1500 200 1000
H2 production rate (mL/L/d)
Cumulative H2(mL/L)
3000
500 0
1
2
3
4
5
6
7
8
9
10
11
12
0
Time (day)
Fig. 3 e Continuous hydrogen production and production rate by A7 affected by biofilm.
Fig. 4 e Different kinds of bacterial biomass in the different medium (a) Biomass change in the GM; (b) Biomass change in the HPM. Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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5
Fig. 5 e SEM images of biofilm formation of photo-fermentative bacteria A7 in the HPM at different time (scale bar, 20 mm).
were mainly applied for bacterial growth and energy storage at 1st day, and about 22 mmol/L of acetate was utilized quickly (Fig. 6). At this time, little hydrogen was produced in both reactors (Fig. 3) and maximum rate of substrate utilization reached about 21.6 mmol/L/d (Fig. 6). Then the rate of substrate utilization slowed down in the following 2e7 days, at 7th day CR no longer produced any hydrogen and little amount (8.7 mmol/L) of residual substrate was detected. However, this work noted an interesting phenomenon that substrate was almost consumed completely in BR at the 8th day but hydrogen production still lasted for 4 days. It implied that the PFB by forming biofilm could tolerate adverse conditions and affect suspended bacterial metabolic activity and store more energy or substrate to produce hydrogen in the BR. In addition, this study speculated that the
Substrate concentration (mmol/L)
Substrate concentration Substrate concentration Substrate utilization rate Substrate utilization rate
CR BR CR BR 40
20
20
10
0
0
2
4
6
8
10
12
Time (day)
Fig. 6 e The change of substrate concentration and substrate utilization rate by A7 affected by biofilm.
0
Substrate utilization rate (mmol/L/d)
30
60
bacteria on the biofilm and suspended bacteria can influence each other by some connection result in the increase of utilization efficiency of substrate. Above results showed that the utilization efficiency of substrate in the BR was 97.5% which was higher than 82.6% in the CR. This is one of the reasons for explaining that the performance of BR was better than that of the CR. Immobilized E. aerogenes cells could utilize substrates more such as lactose from 62 mg/L to 69 mg/L than free cells [21]. It showed there are some regulations on central metabolic pathways to utilize substrate affected by biofilm.
Comparison with other researches There were some other researches studying on the immobilized hydrogen production (Table 1). In a study, activated carbon fibers were used as fluidized solid carrier to immobilize PFB and obtained the hydrogen production volume of 3447 ± 69 mL/ L and hydrogen production improved fold of 30% [17]. Another study used the biofilm photobioreactor to produce hydrogen by the Rhodopseudomonas palustris CQK 01 [22]. The light conversion efficiency was 56% and the whole operation time was only 96 h by the biofilm photobioreactor. However, the very low substrate conversion rate of 6.20% also appeared in this research. Activated carbon, silica gel and clay as the solid carriers to promote the hydrogen production and found the highest substrate conversion rate of 59 ± 5% and hydrogen production improved fold of 37% by the carrier of clay but not silica gel [23]. By comparing present study with the other researches, this study showed the highest hydrogen production improved fold of 75% and the high hydrogen yield of 63% using the silica gel sheet. Although not all the studies showed the best carrier was silica, they also found it is beneficial to form the biofilm in the hydrogen production process. Compared with the other researches, this work had the highest increasement in hydrogen production by immobilization and high hydrogen production rate and hydrogen yield.
Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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Table 1 e Comparison between different reactors with immobilization. Bacterial strains
Substrate
Rhodopseudomonas sp. nov.strain A7 R. faecalis RLD-53
Acetate
Biofilm photobioreactor
63.08 ± 3.15%
Acetate
77 ± 1.54%
Rhodopseudomonas palustris CQK 01 Rhodopseudomonas palustris CQK 01 Rhodopseudomonas palustris WP3-5 R. faecalis RLD-53
Glucose
Sealed photo-bioreactor with cells immobilized on actived carbon fiber Biofilm photobioreactor Photobioreactor with entrapped cells Biofilm photobioreactor with clay as carrier Photo-bioreactor with cells immobilized on fluidized biocarrier Photobioreactor with porous glass as an immobilization matrix
Rhodobacter sphaeroides RV
Glucose Acetate Acetate
Succinate
Reactors
The biofilm reactor could extend the time of hydrogen production from 7 days to 10 days and increase conversion efficiency of substrate from 36% to 63%. It also give a stable and consistent operating process. However, this was not the best data because long-time frozen in refrigerator could harm the hydrogen production activity of strain A7 and this could also help explain the extraordinary improvement for biofilm also accelerated the activation process. Thus it needed the further research to confirm the advantages taken by the biofilm for the diversity in bacteria, substrate, immobilization methods, light sources and reactors.
Conclusion This work investigated photo-fermentative hydrogen production in BR and CR. The experimental results indicated that BR can give a better hydrogen production performance whether on cumulative hydrogen volume, obtained hydrogen yield or substrate utilization efficiency. After the biofilm formed, the cumulative hydrogen volume, hydrogen concentration and substrate utilization efficiency were enhanced 75%, 10% and 18% compared to that of CR, respectively. The increasement of biomass, especially from biofilm, greatly promoted hydrogen production of strain A7. Importantly, substrate almost was utilized completely at 8th day but hydrogen production still lasted for 4 days, this also greatly enhanced the utilization and conversion efficiency of substrate. So, formation of biofilm in the BR was a potential way to improve photo-fermentative hydrogen production and stability of system.
Acknowledgement The authors would like to thank the National Natural Science Foundation of China (No. 51678186 and 51478139), 863 Program (No. 2011AA060905) and the Postdoctoral Scientific Developmental Fund of Heilong Jiang Province (No. LBH-Q16080).
H2 yield
Substrate conversion rate
Improved fold
Refs
2.52 ± 0.13 mol H2/mol acetate 3.08 ± 6.16 mol H2/mol acetate
75% 30%
This study [17]
6.20%
0.2 mol H2/mol glucose
No data
[22]
1.70%
0.74 mol H2/mol glucose
No data
[22]
59 ± 5%
2.36 ± 0.2 mol H2/mol acetate 3.24 mol H2/mol acetate
37%
[23]
30%
[24]
2.8 mol H2/mol succinate
No data
[25]
81%
40%
references
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Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/he
Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor Han-Quan Wen, Jian Du, De-Feng Xing, Jie Ding, Nan-Qi Ren, Bing-Feng Liu* State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, P.O. Box 2614, 73 Huanghe Road, Harbin 150090, China
article info
abstract
Article history:
To achieve stable and efficient photo-fermentative hydrogen production, this work
Received 19 January 2017
investigated photo-fermentative hydrogen production by forming biofilm on the surface of
Received in revised form
carrier in the biofilm reactor (BR). Results showed the hydrogen production performance
5 March 2017
was greatly improved by formed biofilm. The time of hydrogen production and efficiency of
Accepted 18 April 2017
substrate utilization were enhanced obviously compared to the control reactor (CR). When
Available online xxx
the CR was used, hydrogen production stopped at 7th day and maximum cumulative hydrogen volume and hydrogen yield were 1730 ± 87 mL/L and 1.44 ± 0.07 mol H2/mol
Keywords:
acetate, respectively. However, in the BR hydrogen production volume of 3028 ± 150 mL/L
Biofilm reactor
and hydrogen yield of 2.52 ± 0.13 mol H2/mol acetate were obtained, which were enhanced
Photo-fermentative bacteria
about 75% compared to that of the CR. The time of hydrogen production extended from 7
Biohydrogen
days of CR to 12 days of BR and the substrate conversion efficiency increased from 36% of
Substrate utilization
CR to 63% of BR. It was worth noting at 8th day that substrate was almost utilized completely but hydrogen production still lasted for 4 days. This suggested that the formation of biofilm in BR was favorable to continuous hydrogen production and substrate utilization with high efficiency. Results demonstrated the BR can get a more stable and consistent operating process and it was a proper and potential way to produce hydrogen by photo-fermentative bacteria (PFB). © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Currently, energy shortage and environment pollutions caused by the application of fossil fuels were the great challenges human faced [1]. It is necessary to develop a new, clean and renewable energy to replace traditional fossil fuels. Increased attentions were focused on the hydrogen gas due to its abundance, high energy intensity (142 MJ/kg), burning
clearly with only water produced and so on [2]. Among different ways of hydrogen production, photo-fermentation could combine waste degradation, solar energy utilization and hydrogen production in one-step process without oxygen inhibition, thus this technology was considered as the most potential pathway for hydrogen production [3]. However, an obstacle, bad flocculation ability and low hydrogen yield, limited its pace of industrialization, thus made the hydrogen producer easy to flow away with the discharge, led to low
* Corresponding author. Fax: þ86 451 86282008. E-mail address: [email protected] (B.-F. Liu). http://dx.doi.org/10.1016/j.ijhydene.2017.04.150 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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biomass in the reactor and operated at an inefficient and unstable operation condition [4]. To solve above problems, researchers did a lot of work on bacterial immobilization [5], reactor design [6], strain breeding [7] and so on to increase biomass and hydrogen production performance. Among the immobilizations, there are three main ways: embedding, chemisorption and physisorption. Embedding method is a popular way which uses semipermeable material such as agar that allows the micromolecule matters exchange and fixes the bacteria. By embedding the bacteria, cells could easily be separated from the medium, but it will also bring the transport resistance for the macromolecule and light, limit the space for bacterial growth and inhibit the rate of cell refresh and discharge of dead cells [8]. Chemisorption uses the functional group or bonds to fix cells onto the carrier, such immobilization is stable and makes exchange easily, but harmful to the bacteria for its violent reaction. Physisorption allows the cells attach on the surface of carriers freely and it is beneficial for exchange of light and matters. The operation condition was simply and mildly, and the most important advantages of the physisorption is it could form the biofilm freely so the bacteria could show the special characteristic of biofilm. Therefore, in this study, physisorption was adopted to immobilize photofermentative bacteria to form biofilm for improving hydrogen production performance. Biofilm is considered as one potential and proper way for hydrogen production because it could fix more bacteria to enhance biomass and improve hydrogen production and support bacteria second living strategy thus cells distribute energy and matters more choicely [9]. It has been confirmed that all kinds of bacteria can form biofilms and this may be the preferred mode of bacterial existence in nature [10,11]. Though according to the DLVO (DerjaguineLandaueVerweyeOverbeek) theory [12], the small size and high Zeta potential of PFB make cells hard to aggregate, the PFB still have the ability to form biofilm for the following reasons. Firstly, a study has proved PFB, as most ancient bacteria, which lived in a circumstance filled with hydrogen millions of years ago, mainly existed in biofilm form [13]. Secondly, some strains like Rhodopseudomonas faecalis RLD-53 could secret some extracellular polymeric substances (EPS) which are the base matrix for biofilm [14]. Thus biofilm for enhanced hydrogen production was possible in theory. However, up to now, most reported studies about biofilm were focused on the dark fermentation and only a few researches reported the photo-fermentative hydrogen production by formed biofilm. Among reported literature, almost all work investigated on optimization of process condition [15,16]. In a study, activated carbon fibers were used as the fluidized solid carrier to immobilize photo-fermentative bacteria for hydrogen production and the maximum yield of 3.08 mol H2/mol acetate was obtained [17]. Also, another study showed the addition of clay and silica gel was effective in promoting hydrogen production, resulting in 67.2e50.9% and 37.2e32.5% increases in rate and yield of hydrogen production [18]. However, these studies paid more attention to the hydrogen production of the reactor, and little information was discussed on enhancement of hydrogen production by biofilm from two aspects of biomass change and substrate utilization.
Therefore, this study explored the effects of biofilm on the hydrogen production, biomass change and substrate utilization in the BR and CR. The results demonstrated hydrogen production was greatly improved by the formed biofilm on the carrier.
Materials and methods Bacterium and medium Rhodopseudomonas sp. nov. strain A7 (Accession No. KJ699180) was from the State Key Laboratory of Urban Water Resource and Environment in Harbin Institute of Technology [19]. Growth medium (GM) contains CH3COONa (1.0 g), sodium succinate (1.0 g), peptone (0.5 g), yeast powder (1.0 g), NH4Cl (1.0 g), KH2PO4 (0.5 g), K2HPO4 (0.5 g), MgSO4 (0.2 g), CaCl2 (0.08 g), FeSO4$7H2O (0.012 g), EDTA (0.2 g), L-cysteine (0.5 g), NaCl (0.1 g), vitamin (0.1 mL) and trace elements solution (0.1 mL) in 1 L distilled water and pH was adjusted to 6.86 ± 0.1 [19]. Hydrogen production medium (HPM) contains CH3COONa (4.1 g), KH2PO4 (0.5 g), K2HPO4 (0.5 g), sodium glutamate (1.69 g), MgSO4 (0.2 g), CaCl2 (0.08 g), FeSO4$7H2O (0.012 g), EDTA (0.2 g), L-cysteine (0.5 g), NaCl (0.1 g), vitamin (0.1 mL) and trace elements solution (0.1 mL) in 1 L distilled water and pH was adjusted to 6.86 ± 0.1 [19].
Reactor and operation condition The anaerobic tube of 25 mL was used as the reactor. The reactors without carriers were set as the CR. The reactors were filled with 10 mL medium and blew by argon for 5 min to maintain anaerobic conditions (Hungates technology [20]). And then reactors were sterilized at 121 C for 15 min and inoculated with 1 mL seed bacteria (inoculation ratio of 10%), which was in the mid-exponential growth phase after 24 h of cultivation in the growth medium. The reactors were placed in the shaker at 120 rpm, 35 C and 150 W/m2 of light intensity on surface of reactors. The biogas volume was measured with a low friction glass injector. The BR was made by introducing the silica gel sheet (1 cm 10 cm, Xiang Tai sealing material company, China) into the CR to act as the carrier for the formation of biofilm.
Surface morphology of the bacteria The biofilm samples were firstly fixed by 2.5% glutaraldehyde at 4 C in the dark for 12 h then washed gently with 0.01 mol/L phosphate buffered saline twice. Then samples were dehydrated successively by 30%, 50%, 70%, 80% and 90% ethanol once for 10 min respectively and 100% twice for 15 min. And the samples was replaced with ratio of 1:1 (v/v) of ethanol and isoamyl acetate and 100% isoamyl acetate for 15 min, respectively. Lastly after being freeze-dried and coated with gold powder completely, the samples were observed under the scanning electron microscope (SEM). The instrument of SEM was the JEM-2100 (HR) transmission electron microscopy (JEOL, Ltd., Tokyo, Japan) operated at an accelerating voltage of 200 kV.
Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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Analysis method
80
Calculations Hydrogen volume was calculated by the formula: VHydrogen;t ¼ VSpare þ VBiogas;t CHydrogen; t VSpare CHydrogen;t1
(1)
Z VHydrogen;total ¼
VHydrogen;t
(2)
where VHydrogen;t and VBiogas;t are the volume of hydrogen (mL/L medium) and biogas (mL/L medium) on the t time, respectively; t is the time (day, t 2); CHydrogen;t and CHydrogen;t1 are the concentration of hydrogen on the t time and t 1 time respectively (%); VHydrogen;total is the total volume of hydrogen production (mL/L medium); VSpare is the spare volume of reactor (1500 mL). Hydrogen yield was calculated by the following formula: CHydrogen yield ¼
VHydrogen;t ac
CR BR
70
Hydrogen concentration (%)
The biomass of free bacteria were extracted by centrifugation at 12,000 rpm for 5 min. For the bacteria on the biofilm, cells were extracted by washing the carrier gently three times with ddH2O and put in the ultrasonic cleaner for 10 min. For both the free bacteria and biofilm bacteria in CR and BR, the dry weight was measured by being dried until unchanged at 105 C. The optical biomass (OD660) was measured by a visible spectrophotometer (Unic 7200, Unic Shanghai Instrument Company, Shanghai, China). Light density was detected by the TENMARS TM-207 solar Power meter with 1.5 M remove sensor radiation energy test (Tenmars Electronics CO., LTD., Taiwan, China). The hydrogen concentration was detected by a gas chromatograph (Agilent 7890A) which column was Agilent 19091J-433: HP-5 5% Phenyl Methyl Siloxane and operation of the oven, column and thermal conductivity detector (TCD) were at 35 C for 4 min, 325 C and 200 C respectively. Substrate concentration was detected by a gas chromatograph (Agilent 7890A) equipped with a Agilent 19095N-123: 30 m 530 mm 1 mm and a flame ionization detector (FID) and operation of the injector, oven, detector and FID were at 250 C, 70 C first then programmed heating in 5 mine170 C and maintained for 1 min and 300 C, respectively. Each isolate was assayed more than three times and the results were presented as the average of the assays.
60 50 40 30 20 10 0 1
2
3
4
5
6
7
8
9
10
11
12
Time (day) Fig. 1 e The change of hydrogen concentration in BR and CR.
concentration. The experimental results showed the average hydrogen concentration of BR was about 10% higher than that of CR (Fig. 1). The biogases and hydrogen production trends were similar (Figs. 2 and 3). The most biogases were produced between 2 and 5 days in the CR, and the maximum biogases production rate and hydrogen production rate was 533 mL/L/ d and 468 mL/L/d at 4th day, respectively. After 5 days, the biogases and hydrogen production rate slowed down and gases production stopped at 7th day. The total cumulative biogases and hydrogen volume was 1767 ± 88 mL/L and 1730 ± 87 mL/L, respectively. Hydrogen yield was 1.44 ± 0.07 mol H2/mol acetate. However, this work found that BR can extend biogases production time from 7 days of CR to 12 days and hydrogen production mainly occurred between 2 and 10 days. Compared to CR, BR increased efficient and stable hydrogen production time. The maximum gases rate and hydrogen production rate was 600 ± 29 mL/L/d and 507 ± 30 mL/L/d at the 6th day. The cumulative biogases and hydrogen volume reached 4300 ± 200 mL/L and 3028 ± 160 mL/ L, respectively. Hydrogen yield was 2.52 ± 0.13 mol H2/mol acetate, which was 63% of the theoretical yield.
(3)
where CHydrogen yield is the hydrogen yield (mol hydrogen/mol acetate); a is the coefficient of molar density at room temperature (24 L/mol); c is the concentration of sodium acetate (50 mol/L medium).
Results and discussion Biogases and hydrogen production in the BR Biogases production was investigated in CR and BR. The composition of biogases in the photo-fermentation was mainly H2 and CO2. The hydrogen volume was calculated by combing the biogases production rate and the hydrogen
Fig. 2 e Continuous biogas production and production rate by A7 affected by biofilm.
Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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The two kinds of reactors showed the similar gas production trend, but BR could broaden the gases production period from 1 day to 7 days of CR to 1 day to 12 days of BR, BR also had a stable and efficient gases and hydrogen production, which enhanced 143% and 75% compared to the CR. It showed that the biofilm fixed on the carrier could give a better gas and hydrogen production mode. The high efficient hydrogen production time was broaden from 2 to 5 days to 1e10 days, cumulative hydrogen production was higher and the operation was more stable in the BR than the CR.
biofilm biomass 0.05 ± 0.01 g/L) after 7 days. Compared to CR, the biomass in the BR increased about 32% and 2.5% biomass was contributed from the formed biofilm. This result indicated that the formed biofilm by introduction of carrier in the GM could promote the bacterial growth and the increasement of biomass. In the HPM, biomass in both reactors was obviously lower than that in the GM. Biomass in the BR was 2.0 ± 0.1 g/L, which was 18.4% higher than 1.69 ± 0.08 g/L in the CR at 7th day. However, the biomass from biofilm of the BR in the HPM achieved a higher value of 0.15 ± 0.01 g/L, which was 200% higher than 0.05 ± 0.01 g/L in the GM (Fig. 4). The increasement of biomass, especially from biofilm, greatly promoted hydrogen production of strain A7 (Fig. 5). Compared to HPM, GM is a better medium for cell growth but no hydrogen was produced. And a little amount of bacteria was attached to carriers for the formation of biofilm in GM, so biofilm biomass was lower. However, bacteria in the bad circumstance (HPM) can form more biofilm for maintaining energy balance and resisting unfavorable condition. This indicated that bacteria by self-regulation could choose the suitable mode for cell growth and hydrogen production. The formation of biofilm changed the bacterial living condition and affected suspended bacteria activity. Especially, this may help to enhance key enzyme activity of hydrogen production. Therefore, hydrogen production performance was promoted markedly. There was limited information about the effects of biomass from biofilm on the biohydrogen production, and most researches are focused on hydrogen production process.
The effect of formed biofilm on biomass
Substrate utilization in the BR and CR
The biomass change also was detected in different reactors (Fig. 4). In this study, cells were divided into two parts: free bacteria and biofilm bacteria. In the GM, the final biomass could achieve 1.94 ± 0.10 g/L in the CR and 2.55 ± 0.12 g/L in the BR (of which the free biomass occupied 2.53 ± 0.12 g/L and the
The substrate in the medium was mainly used for bacterial growth and hydrogen production, and their utilization efficiency determined biosynthesis of biomass and hydrogen production ability. The initial substrate concentration was 50 mmol/L in CR and BR. The results showed the substrate
3500
CR
Cumulative H2
H2 production rate
BR
Cumulative H2
H2 produciton rate
600
2500
400
2000 1500 200 1000
H2 production rate (mL/L/d)
Cumulative H2(mL/L)
3000
500 0
1
2
3
4
5
6
7
8
9
10
11
12
0
Time (day)
Fig. 3 e Continuous hydrogen production and production rate by A7 affected by biofilm.
Fig. 4 e Different kinds of bacterial biomass in the different medium (a) Biomass change in the GM; (b) Biomass change in the HPM. Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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5
Fig. 5 e SEM images of biofilm formation of photo-fermentative bacteria A7 in the HPM at different time (scale bar, 20 mm).
were mainly applied for bacterial growth and energy storage at 1st day, and about 22 mmol/L of acetate was utilized quickly (Fig. 6). At this time, little hydrogen was produced in both reactors (Fig. 3) and maximum rate of substrate utilization reached about 21.6 mmol/L/d (Fig. 6). Then the rate of substrate utilization slowed down in the following 2e7 days, at 7th day CR no longer produced any hydrogen and little amount (8.7 mmol/L) of residual substrate was detected. However, this work noted an interesting phenomenon that substrate was almost consumed completely in BR at the 8th day but hydrogen production still lasted for 4 days. It implied that the PFB by forming biofilm could tolerate adverse conditions and affect suspended bacterial metabolic activity and store more energy or substrate to produce hydrogen in the BR. In addition, this study speculated that the
Substrate concentration (mmol/L)
Substrate concentration Substrate concentration Substrate utilization rate Substrate utilization rate
CR BR CR BR 40
20
20
10
0
0
2
4
6
8
10
12
Time (day)
Fig. 6 e The change of substrate concentration and substrate utilization rate by A7 affected by biofilm.
0
Substrate utilization rate (mmol/L/d)
30
60
bacteria on the biofilm and suspended bacteria can influence each other by some connection result in the increase of utilization efficiency of substrate. Above results showed that the utilization efficiency of substrate in the BR was 97.5% which was higher than 82.6% in the CR. This is one of the reasons for explaining that the performance of BR was better than that of the CR. Immobilized E. aerogenes cells could utilize substrates more such as lactose from 62 mg/L to 69 mg/L than free cells [21]. It showed there are some regulations on central metabolic pathways to utilize substrate affected by biofilm.
Comparison with other researches There were some other researches studying on the immobilized hydrogen production (Table 1). In a study, activated carbon fibers were used as fluidized solid carrier to immobilize PFB and obtained the hydrogen production volume of 3447 ± 69 mL/ L and hydrogen production improved fold of 30% [17]. Another study used the biofilm photobioreactor to produce hydrogen by the Rhodopseudomonas palustris CQK 01 [22]. The light conversion efficiency was 56% and the whole operation time was only 96 h by the biofilm photobioreactor. However, the very low substrate conversion rate of 6.20% also appeared in this research. Activated carbon, silica gel and clay as the solid carriers to promote the hydrogen production and found the highest substrate conversion rate of 59 ± 5% and hydrogen production improved fold of 37% by the carrier of clay but not silica gel [23]. By comparing present study with the other researches, this study showed the highest hydrogen production improved fold of 75% and the high hydrogen yield of 63% using the silica gel sheet. Although not all the studies showed the best carrier was silica, they also found it is beneficial to form the biofilm in the hydrogen production process. Compared with the other researches, this work had the highest increasement in hydrogen production by immobilization and high hydrogen production rate and hydrogen yield.
Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150
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Table 1 e Comparison between different reactors with immobilization. Bacterial strains
Substrate
Rhodopseudomonas sp. nov.strain A7 R. faecalis RLD-53
Acetate
Biofilm photobioreactor
63.08 ± 3.15%
Acetate
77 ± 1.54%
Rhodopseudomonas palustris CQK 01 Rhodopseudomonas palustris CQK 01 Rhodopseudomonas palustris WP3-5 R. faecalis RLD-53
Glucose
Sealed photo-bioreactor with cells immobilized on actived carbon fiber Biofilm photobioreactor Photobioreactor with entrapped cells Biofilm photobioreactor with clay as carrier Photo-bioreactor with cells immobilized on fluidized biocarrier Photobioreactor with porous glass as an immobilization matrix
Rhodobacter sphaeroides RV
Glucose Acetate Acetate
Succinate
Reactors
The biofilm reactor could extend the time of hydrogen production from 7 days to 10 days and increase conversion efficiency of substrate from 36% to 63%. It also give a stable and consistent operating process. However, this was not the best data because long-time frozen in refrigerator could harm the hydrogen production activity of strain A7 and this could also help explain the extraordinary improvement for biofilm also accelerated the activation process. Thus it needed the further research to confirm the advantages taken by the biofilm for the diversity in bacteria, substrate, immobilization methods, light sources and reactors.
Conclusion This work investigated photo-fermentative hydrogen production in BR and CR. The experimental results indicated that BR can give a better hydrogen production performance whether on cumulative hydrogen volume, obtained hydrogen yield or substrate utilization efficiency. After the biofilm formed, the cumulative hydrogen volume, hydrogen concentration and substrate utilization efficiency were enhanced 75%, 10% and 18% compared to that of CR, respectively. The increasement of biomass, especially from biofilm, greatly promoted hydrogen production of strain A7. Importantly, substrate almost was utilized completely at 8th day but hydrogen production still lasted for 4 days, this also greatly enhanced the utilization and conversion efficiency of substrate. So, formation of biofilm in the BR was a potential way to improve photo-fermentative hydrogen production and stability of system.
Acknowledgement The authors would like to thank the National Natural Science Foundation of China (No. 51678186 and 51478139), 863 Program (No. 2011AA060905) and the Postdoctoral Scientific Developmental Fund of Heilong Jiang Province (No. LBH-Q16080).
H2 yield
Substrate conversion rate
Improved fold
Refs
2.52 ± 0.13 mol H2/mol acetate 3.08 ± 6.16 mol H2/mol acetate
75% 30%
This study [17]
6.20%
0.2 mol H2/mol glucose
No data
[22]
1.70%
0.74 mol H2/mol glucose
No data
[22]
59 ± 5%
2.36 ± 0.2 mol H2/mol acetate 3.24 mol H2/mol acetate
37%
[23]
30%
[24]
2.8 mol H2/mol succinate
No data
[25]
81%
40%
references
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Please cite this article in press as: Wen H-Q, et al., Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.04.150