Bioresource Technology 166 (2014) 458–463

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Trehalose enhancing microbial electrolysis cell for hydrogen generation in low temperature (0 °C) Xu Linji a,b,d, Liu Wenzong c,⇑, Wu Yining a, Poheng Lee b,d, Wang Aijie a,c, Li Shuai a a

State Key Laboratory of Urban Water Energy and Environment, Harbin Institute of Technology (SKLUWRE, HIT), Harbin 150090, PR China The Department of Civil and Environmental Engineering, The Hong Kong Polytechnic University, Hong Kong c Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China d Peng Wei Petrochemical Co., LTD, Longqiao Industrial Park, Fuling District, Chongqing 408121, PR China b

h i g h l i g h t s  Volatile fat acids were accumulated after pretreatment with ultrasound and fermentation.  The optimal concentration of trehalose was 50 mmol/L to improve MEC’s efficiency.  Trehalose improved the actions of anode community in terms of size, biodiversity.  Microbacterium and Proteobacteria were main species affecting MEC’s efficiency.

a r t i c l e

i n f o

Article history: Received 4 March 2014 Received in revised form 6 May 2014 Accepted 8 May 2014 Available online 22 May 2014 Keywords: Microbial electrolysis cell Sludge Hydrogen Trehalose Low temperature

a b s t r a c t This work explored the feasibility of a method combining physical (sonication and base) and biological (partial fermentation) processes for sludge treatment and the effects of trehalose on the hydrogen generation of microbial electrolysis cell at 0 °C. The results demonstrated that the above pretreatment method was favorable, which promoted organics decomposing into lower molecular weight matter. The promotion of trehalose for MEC efficiency was obvious and the optimal concentration of trehalose was 50 mmol/L. With this concentration, the highest hydrogen recovery rate was 0.25 m3-H2/-m3-reactor per day. Coulomb efficiency and energy recovery efficiency were 46.4% and 203%, respectively. Further, the consumption order of mixed substances was VFAs > proteins > carbohydrates. For microorganism community, SEM photographs illustrated that the selectivity of environmental temperature for the species of anode bacteria was strong and denaturing gradient gel electrophoresis indicated that Microbacterium and Proteobacteria were the two main species and Proteobacteria may be one of the species that produced electrons. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Waste activated sludge (WAS) is one of the big problems since too much sludge is being produced everyday. For sludge treatment, the main way is landfill, which does not only occupy a large square of land but also a contaminant environmental system (Mills et al., 2014). However, WAS is an important biofuel resource (Jenicek et al., 2012). With increasing pressures on the global challenges of water shortage, energy security, and climate change, various strategies from different fields are urging adaption for these ⇑ Corresponding author at: Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China. Tel.: +86 10 62915515. E-mail address: [email protected] (W. Liu). http://dx.doi.org/10.1016/j.biortech.2014.05.018 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

challenges. In this regard, a new trend considers using the concept of shifting wastes/wastewater treatment from removal to recovery from the prospective of simultaneously becoming organic. Recently, recovering energy from WAS has gradually caught public attractions and some achievements have been reported (Liu et al., 2013; Cavinato et al., 2013; Coma et al., 2013). Microbial electrolysis cell (MEC) is favorable as it generated hydrogen with 0.8 V power, at 4 °C (Lu et al., 2011). What is more, MEC can use complex carbon recourses as biofuel to produce relatively high concentration of H2 (Lu et al., 2010). Compared with water hydrolysis that needs atleast 1.23 V, MEC can save a lot of energy since it only needs more than 0.2 V to generate hydrogen (Kiely et al., 2011; Pant et al., 2010). Even though MEC has many merits, studies on it are limited hence there are many aspects that have to be looked into. For instance, how to shorten reaction time is a problem

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because MEC requires more time when its substance is complicated than that of a simple substance, such as acetic acid. In some latest reports, the combined pretreatment of ultrasound and strong base was used in sludge treatment (Elliott and Mahmood, 2012; Yang et al., 2012). This method has been proved to be a good approach for MEC generating hydrogen because the rate of hydrogen production with the combined methods was 10 times as much as that of without any treatment (Liu et al., 2012). Additionally, even though anode microorganisms of MEC can survive at low temperature, their biological activities are much lower than those in room temperature. Apart from that, at low temperature, the fluidity of substance solution was affected by temperature (Hoffmann and Bremer, 2011), therefore, adding a certain concentration of auxiliary is a good way to maintain their activities (Selva et al., 2013). Different auxiliaries including inorganic and organic auxiliaries have been applied in different areas depending on human purposes (Chen et al., 2014). For a microorganism, trehalose is an effective biological agent for the activity protection of organism in low-temperature because it has no particular option and widely exists inside the bodies of organisms that live in a cold environment. Trehalose was widely applied in many fields, such as seedling cultivation, vaccine store and so on (Gao et al., 2013). Nowadays, several hypotheses try to explain its mechanism of protection of cell activities (Inouye and Phadtare, 2014). However, the widely accepted hypothesis is that trehalose has nonspecific chemical bonds and these bonds replace hydrogen bonds formed below 0 °C. Consequently, crystal structures in the bodies of organisms are removed so that organisms still maintain the mobility of the cell wall. In other words, cells can undergo exchanges of mass and information freely even when they live at 0 °C (Schlichter et al., 2001). There are many engineering applications of trehalose however it has not yet been applied in hydrogen generation from MEC system under psychrophilic condition. This article explored the influences of trehalose on hydrogen generation and energy recovery from sludge fermentation liquid (SFL) that was achieved by the pretreatment of ultrasound and strong base and part fermentation. Finally, this article also studied anode microbial community of MEC system through scan electronic microscope (SEM) and denaturing gradient gel electrophoresis (DGGE). 2. Methods 2.1. WAS pretreatment with ultrasound and strong base Waste activated sludge used in this experiment was from the secondary sedimentation tank of a municipal WWTP in Harbin, China. It was concentrated and stored in a refrigerator (4 °C, 24 h). The characters of concentrated waste activated sludge are shown in table (Table 1). The concentrated WAS was processed by ultrasound (28 + 40 kHz ultrasound frequency, 0.5 kW/L energy density and 10 min processing time). After that, the pH of WAS was adjusted to 10.0 with NaOH to inhibit the activity of methanogenesis. Finally, WAS was injected into a batch anaerobic jar for further hydrolysis and partial fermentation. The parameters of partial fermentation were 37 °C constant temperature, pH = 10.0, 100 rpm/ min air-bath shaker, 3-day fermentation time. Eventually, WAS

was filtrated and the residual part of the sludge was thrown away and the yellow transparent liquid was kept. 2.2. Microbial electrolysis cell installment at low temperature In this article, six single-chamber reactors were made of polycarbonate in Harbin. Each reactor consisted of a cylinder (a 28 ml chamber, 3 cm diameter 4 cm length) and a disposable syringe tube (10 ml). The total volume of each reactor was 38 ml. Anode material was a graphite brush (25 mm diameter, 25 mm length; 0.22 m2 surface area; fiber type: PANEX 33160K, ZOLTEK). Cathode material was a piece of specified carbon cloth (YB-20, YiBang, Taiwan). One side was coated with Pt-C powder (0.5 mg/cm2) catalyst layer, its valid surface area being 7 cm2 and the other side black Polytetrafluoro-ethylene (PTFE) layer. Six reactors were installed in a Low Temp Incubator (MIR-254-PC, SANYO) at 10 °C and they were inoculated with a mixture of fresh WAS and acetate and their ratio of 1:1 (15 ml WAS and 15 ml sodium acetate, 1500 mg/L). Inoculation was repeated 3 times to guarantee that anode was attached a large enough number of anode bacteria on the carbon brush. 0.8 V was applied into each reactor because this value has been proved to be the optimal voltage for MEC producing hydrogen. One to two months later, the Anode biofilm consisted of current-producing bacteria and several co-existing functional species on the anode of MEC became mature. Then the main carbon source was replaced by SFL and the operating conditions were maintained the same until all reactors reached relatively parallel status. After that, the temperatures in these reactors were decreased to 0 °C from 10 °C with every 2 °C gradient decreasing. When all reactors were in stable status at 0 °C, trehalose was added into each reactor with six different gradients (0 mmol/L, 5 mmol/L, 10 mmol/L, 50 mmol/L, 100 mmol/L and 150 mmol/L. Finally, all reactors were run at 0 °C with trehalose from low concentration to high concentration. Eventually, all reactors were run at 0 °C for 5 days for each cycle, and were repeated 2 times with the same operating conditions. 2.3. Detection and calculation Soluble chemical oxygen demand (SCOD) was measured with rapid determination method (COD Digital Reactor Block, 5B-1 (V8); COD Meter, 5B-3C (V8); COD digestion tube, LH-21120-16), digesting at 165 °C, 15 min. Dissolvable carbohydrates were measured by phenol-sulfuric acid method (national standard). Dissolvable proteins were measured by BCA RC DC (Pierce, USA) and current was scanned by a multicenter voltage collection instrument (32-channel data acquisition system, PISO-813, ICP DAS, Co., Ltd, 95 Beijing, China) and the mixed gases were collected by special bags (0.1 L capacity; Cali-5-bond, Calibrated Instruments). The identification and quantification analysis of mixed gas was done by gas chromatography (4890D, J&W Scientific, USA). Chromatographic column type, HP-MoleSieve (30 m  0.53 mm  50 m); carrier gas, high purity nitrogen (6 mL min1, splitting ratio (10:1)); the injection port temperature, column temperature, TCD temperature were 200 °C, 35 °C and 200 °C, respectively. 200 ll gas sample was injected by a microsyringe (Shanghai Anting Scientific Instrument Factory). Residual

Table 1 Characters of waste activated sludge in different phases of treatment. Items

UB-SL SFL

VFAs (mgCOD/L)

SC

HAc

HPr

HBu

HVa

Total

(mgCOD/L)

36.6 1079.8

33.8 1213.2

25.2 428.45

7.7 210.9

103.4 2932.4

86.6 1225.6

SP

SCOD

797.9 1435.8

987.3 5593.9

L. Xu et al. / Bioresource Technology 166 (2014) 458–463

the volume of hydrogen; P stands for atmospheric pressure (101,325 Pa); R, 0.08314 L bar/K mol; T, real temperature, 273 K; MS, the molecular weight of substance; DCOD = SCODin  SCODout, Rt the variation of SCOD, Idt t¼0 Coulombic efficiency (CE), C E ¼ nnCE , nth ¼ 2DMCOD , n (nth, CE ¼ 2F O th

2

theoretical electric quantity; nCE, real electric quantity, I is current, scanned by Current collector; F, Faraday’s constant (96,485 Cmol1). Hydrogen producing rate (Q), 

3

Þr cat ½ð1C=SÞ=Að0:5molH2 =mole Þð86;400s=dÞ v r cat Q ¼ IV ðA=m ¼ 43:2I Fcg Fð96;485C=mole Þc ðmoolH =LÞð103 =m3 Þ g

2

(rcat, hydrogen

reduction rate; cg, the molar density of gas (P/(RT)); Iv, electric current density. WH Energy recovery efficiency (gE), gE ¼ 100  W E2 (W H2 , the total heat value of hydrogen; WE, the invested power). DGGE test was undertaken in biological and chemical laboratory, following the steps of standard handbook. 3. Results and discussion 3.1. The characteristics of WAS and fermentation liquid

3.2. Substance consumption and hydrogen generation The consumption of compounds of SFL is illustrated in Fig. 1. The amount of SCOD consumed by MEC increased firstly in the range from 0 mmol/L to 50 mmol/L, then it decreased when the concentration was 50 mmol/L. The average SCOD removal reached 2620.6 mg/L, 524.0 mg/L per day and the highest consuming amount was near 2800.0 mg/L, 600 mg/L per day at 50 mmol/L. To VFAs, its average consumption was 1876.4 mgCOD/L each cycle and the highest consuming amount was about 2205.8 mgCOD/L when trehalose was 50 mmol/L. 1004.2 mgCOD/L, 95% acetic acid, 885.6 mgCOD/L, 73% butyric acid, 214.3 mgCOD/L, 50% propionic acid and 80.1 mgCOD/L, 38% pentanoic acid were used by MEC respectively within 5 days. Then, total protein consumption was 415.9 mgCOD/L within 5 days, which reflected that proteins played an important role in the operation of MEC. Finally, the total consumption of carbohydrates was 178.3 mgCOD/L in every cycle. All data are shown in Fig. 1(a). The above results suggested VFAs were the important substance because the consumption of VFA ranked the top. Further, 93% of acetic acid was consumed, which indicated that hydrogen mainly came from acetic acid. Butyric acid came next. Therefore, the order of consumption was acetic acid > Butyric acid > propionic acid > pentanoic acid. Taking VFAs, protein and carbohydrates together, the order of consumption of complex substances was VFAs > protein > carbohydrates. The order reflected that generally anode bacteria tended to use simple substances and

(a) 3000

VFAs

SCOD

Proteins

Carbohyrates

2500 2000 1500 1000 500 0 0

5

10

50

100

150

Trehalose concentraon (mmol/L)

(b)

70

0.3

Hydrogen

60 50 40

the Rate of Hydrogen Producon (m3-H2/-m3-reator)

The differences of ultrasound and base treating sludge liquid (UB-SL) and sludge fermentation liquid (SFL) were obvious. UB-SL was a black liquid while SFL was a yellow and transparent liquid, which demonstrated that there were a series of chemical reactions in the process of pretreatment. The characters of concentrated raw sludge indicated that the amount of total COD was high, near 24,000 mg/L while the amount of soluble COD was very low, just 976.2 mg/L. The amount of VFAs was too low to fit the requirement of anode bacteria. The characters of SFL pretreated with ultrasound, base, partially fermented, are shown in Table 1. It can be seen that SCOD increased to 5593.9 mg/L, 5 times more than that of raw WAS, 987.3 mg/L. The concentration of accumulated VFAs went up to 2932.4 mgCOD/L from 103.4 mgCOD/L, nearly 30 times higher than that of raw sludge. In case of soluble carbohydrates, it went to 225.6 mgCOD/L from 86.6 mgCOD/L. The total amount of soluble proteins ascended to 1435.8 mgCOD/L from 987.3 mgCOD/L. The comparison indicated that WAS with the pretreatment of ultrasound and base and partially anaerobic fermentation was degraded into simpler organic mass to a large degree. The most evident change occurring in the process of pretreatment was VFAs. To MEC, VFAs were the direct carbon source and their total amount

indicated the pretreatment methods were favorable. Soluble carbohydrates also underwent a great increase since ultrasound broke the cells of bacteria in sludge and partial fermentation promoted the release of an insoluble organic. Even though strong base was added, methane generation was incompletely inhibited since a little amount of gas was produced in the process of fermentation. Consequently, the above results indicated that the combined pretreatment of physical and biological processes is favorable.

Consuming Amount (COD mg/L)

volatile fatty acid (VFA) concentration was detected by Agilent gas chromatograph (Agilent, 7890A; J&W Scientific, USA). Flame ionization detector (FID) and an appropriate column (19095N-123 HP-INNOWAX, 30 m  0.530 mm  116 mm, 1.00 lm, J&W Scientific, USA); carrier gas, nitrogen (50 ml/min, splitting ratio (5:1)); the injection port 117 temperature, column temperature, and FID temperature were 250 °C, 300 °C and 300 °C, respectively. Before injecting into GC, VFA samples were filtered with disposable microporous membrane and were acidified with methanoic acid. Microwave plasma torch metal ion spectrometer (Changchun Jilin University Little Swan Instruments 124 Co., Ltd) detected the concentration of different kinds of ions. The conditions were microwave power 110 W, carrier gas 125 flow 1.1 L/min, working gas flow, 0.6 L/min. The genetic map was drawn by MEGA software Calculations as follows: SCOD removal efficiency = (SCODinflow  SCODoutflow/ SCODinflow  100%. SCODinflow, inflowing SCOD of SFL; SCODoutflow, the residual outflowing SCOD of SFL). Hydrogen volume (V H2 ), V H2 ¼ V total  C H2 ; (Vtotal, the total volume of gas; C H2 , the concentration of hydrogen). h i V P=ðRTÞ V PM H H gH2 ¼ D2 COD ¼ RT D2 CODS (V H2 is Hydrogen production (YH2 ), Y H2 gCOD

Volume ( mL)

460

Totoal Volume

0.25

the Rate of Hydrogen Producon

0.2

0.15 30 0.1

20

0.05

10 0

0 0

5

10

50

100

150

Fig. 1. (a) Consumption of different catalogues and hydrogen yield and (b), the total volume and hydrogen volume generated by MEC in a cycle with different trehalose concentrations.

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3.3. Energy efficiency with different trehalose concentrations Fig. 2(a) and (b) describe the real current density–time graph and energy efficiency and coulombic efficiency graph with different trehalose concentrations. When trehalose was 50 mmol/L, the highest current density was 1.7 mA/m2 while the lowest current was 1.0 mA/m2. The highest current density was 2 folds higher than that of the lowest current density (Fig. 2(a)). Coulombic efficiency and energy efficiency were 46.4% and 203% respectively when trehalose was 50 mmol/L while coulombic efficiency and energy efficiency were 12.9% and 81.2% respectively when trehalose was 150 mmol/L. The highest values were 4 folds and 3 folds

(a)

3

0mol/L 10mol/L 100mol/L

Current Density

2.5

5mol/L 50mol/L 150mol/L

2 1.5 1 0.5

118.7

110.7

94.7

102.7

86.7

78.7

70.7

62.7

54.7

46.7

38.7

31.3

23.3

7.3

15.3

0.0

0

Running Time (h)

(b) 250 200

Efficiency ( %)

bacteria preferentially use those substances that were helpful for them when they were in special conditions. In this experiment, since bacteria were in low temperature, anode bacteria used protein to synthesize enzymes that can protect them from being hurt. The structures of carbohydrates were so complicated that bacteria hardly used them shortly. This was the reason why carbohydrate consumption was the least. In terms of gas produced by MEC as shown in Fig. 1(b), it mainly contains about 90% hydrogen. When trehalose was 50 mmol/L, the total volume and hydrogen volume were 62.1 mL and 55.9 mL respectively, however, when trehalose was 150 mol/L, the total volume and hydrogen volume were 30.8 mL and 27.7 mL, respectively. Further, when trehalose was 150 mmol/L, both the total volume and hydrogen volume were lower than that of control group (trehalose was 0 mmol/L). These data indicated that the protection of biological activities was based on a certain range of trehalose concentration. In this experiment, 50 mmol/L was the optimal concentration. This concentration was lower than the reported concentration, 200 mmol/L for yeast (Elbein et al., 2003) and 500 mmol/L for plants (Tibbett et al., 2002), respectively. Taking substances in SFL and hydrogen volume together, it can be seen that with the optimal concentration, when MEC consumed 2800.0 mg/L SCOD, including VFAs 2205.8 mgCOD/L, proteins 311.9 mgCOD/L and carbohydrates 53.5 mgCOD/L, produced 62.1 mL gas including 55.9 mL hydrogen. Also, the best rate of hydrogen generation reached 0.25 m3-H2/-m3-reactor per day and the control group had 0.13 m3-H2/-m3-reactor per day. However, the lowest rate of hydrogen was 0.12 m3-H2/-m3-reactor per day, which was 0.5 times lower than the highest. In this experiment, 0.25 m3-H2/-m3-reactor per day was achieved when trehalose was 50 mmol/L. This result was 0.03 m3-H2/-m3-reactor per day more than 0.23 m3-H2/-m3-reactor per day, which was achieved with pretreated sludge as carbon source at 4 °C (Lu et al., 2012a,b). From the above results, it can be seen that the utilization of total SCOD for hydrogen was not as high as expected while the utilization of VFAs that only occupied 1/3 SCOD, reached about 70% when MEC was in optimal situation. It indicated that VFAs were mainly carbon sources. Further it can be assumed that a part of VFA was applied into generating hydrogen and the residue was applied in cell mass synthesis. The effects of trehalose on the activities of anode bacteria seemed to be a double-edged sword. The reason was that trehalose helped anode bacteria maintain metabolic activities in cold circumstance within a certain scope so that anode bacteria produced gas. However, with high concentration of trehalose, anode bacteria could not survive since trehalose is a kind of polysaccharide. It deprived water molecules from bacteria cell resulting in the difference in osmotic pressure between inner cell and external cell. Once cells lost water, their activities were inhibited and it even led to cell death (Burdige, 2011; Shivaji and Prakash, 2010). According to the order of complex substance consumption, the activities of bacteria are different and anode bacteria selectively used substances of SFL.

Energy efficiency coulombic efficiency

150

100

50

0 0

5

10

50

100

150

Trehalose Concentraon (mmol/L) Fig. 2. (a) The real time current-time graph, (b) MEC energy recovery efficiency and coulombic efficiency with different trehalose concentrations.

more than the lowest values respectively (Fig. 2(b)). The changing trend of current density and coulombic efficiency indicated that trehalose markedly promoted the efficiency of MEC in terms of increasing the current in a scope from 5 mmol/L to 50 mmol/L. However, the scope was over 50 mmol/L, the inhibition of trehalose became obviously. From above results, we can see that current density in cold temperature was much lower than that at both at 4 °C (Lu et al., 2012a,b) and 25 °C (Linji et al., 2013). One reason was that trehalose increased the inner resistance of MEC (Call and Logan, 2008; Logan et al., 2008) and another reason was that the activities of anode bacteria were low so that they produced fewer electrons. Energy efficiency was calculated according to the thermodynamic value of hydrogen through the real hydrogen produced by MEC. In this article, it can be seen that energy efficiency was positive when trehalose concentration was between 5 mmol/L and 100 mmol/L. Therefore, it was obvious that the higher volume of hydrogen and the higher energy efficiency.

3.4. Morphological features and anode community analysis Morphological features of anode bacteria at different stages can be seen from SEM photographs. In the beginning of cultivation, the density of anode bacteria was heavy, and then it became light gradually with the increase of running time due to the death of some bacteria. When MEC reached a stable situation, biodiversity of anode bacteria decreased dramatically until only few species survived. Further, it was found that the size of single bacteria at 0 °C with trehalose addition was obviously bigger than that at 10 °C in the beginning. Anode bacteria randomly grew on the surface of carbon brush and lived in unit of colony. In order to further clearly understand the constitution of anode species of MEC, DGGE analysis was present in this article. The frame of DGGE compared 16s rRNA bands of anodic bacteria at

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L. Xu et al. / Bioresource Technology 166 (2014) 458–463

Acidovorax soli strain DT17-1 Acidovorax sp. JHL-10 Bacterium amp-w-4 Stenotrophomonas Proteobacteria fluorescens strain TCA3 Proteobacteria fluorescens strain TCA33 Novosphingobium sp. KG Erythrobacter-like sp. V4.BO.03 Mesorhizobium alhagi strain UrPL08 Ochrobactrum sp. B1315 Bacterium 85-L049642-122-014-E11 Arthrobacter nicoanae strain Dc-06 Microbacterium sp. N056 Microbacterium sp. R075N Microbacterium azadirachtae strain T67 Microbacterium phyllosphaerae strain ZSGR33 Lactococcus lacs subsp.strain RC24 Lactococcus sp. 0.02

Fig. 3. Cladogram of anode community through NCBI gene bank on class level.

different stages, with 0.8 V optimal voltage. There were 12 clear bands on each sample and a band represented one kind of species. This frame reflected that the bands of all samples are similar to each other. However, the width of the sample with 50 mmol/L trehalose was bolder and darker than others. It meant that the density of anode bacteria under this condition was heavier than that under other conditions and more bacteria were collected on the anode of MEC. However, it cannot be known to which class they belonged to so that 16s rRNA sequencing was done in this experiment. The cladogram of anode community (Fig. 3) shows the results of the comparison of sequences between experimental and NCBI gene bank. The cladogram reflected the genetic relationship of different species on anode brush to some degree. There were 12 species in total for each band. Among the 12 species, some of their 16s rRNA base sequences matched those base sequences of 16s rRNA of bacteria that have been known, such as Microbacterium sp. N056, Microbacterium sp. R075N, Microbacterium azadirachtae strain T67 and Microbacterium phyllosphaerae strain ZSGR33. The base sequences of 16s rRNA of species had a 98% similarity with those bacteria, which have been known. Preliminarily, Microbacterium was the dominant specie of anodic community. Acidovorax soli strain DT17-1I and Acidovorax sp. JHL-10, Proteobacteria fluorescens strain TCA3 and P. fluorescens strain TCA33, Lactococcus lactis subsp. strain RC24 and Lactococcus sp., all them had over 96% similarity with those bacteria in NCBI gene bank. Even though we cannot identify which species directly produced current, the base sequences of the 16s rRNA of two species match that of Proteobacteria and Microbacterium probably are the current producing bacteria (Call et al., 2009; Lu et al., 2012a,b). These results proved the SEM observation from the perspective of micro level. Additionally, it has been unclear that the detail functions of residual bacteria, but they may be in charge of their own places such as being responsible to the communication of different groups, maintaining the stability of anodic bacteria colony (Fan et al., 2009).

that for MEC using SFL to produce hydrogen in low temperature, this method was favorable. The results achieved in this work indicated that only 33% SCOD was applied to hydrogen production and about 66% of it may be used for cell mass synthesis or still left in the solution. Further, anode bacteria optically used substances in SFL so that almost 90% acetic acid was consumed. Then, the results of hydrogen, energy recovery from SFL illustrated that purer hydrogen (over 80%) has been achieved and energy recovery was considerable. Finally, anodic colony in low temperature situation has been studied in this work. With the optimal trehalose concentration, the highest volume of total gas recovery was 62.1 mL and the best rate of hydrogen production was 0.25 m3-H2/-m3-reactor per day with 203.01%, the best total energy recovery efficiency. Additionally, new discoveries were that the order of VFAs was acetic acid > Butyric acid > propionic acid > pentanoic acid and the order of substance consumption of SFL was VFAs > proteins > carbohydrates. SEM photographs and DGGE analysis illustrated that the population of anode community, the size of anodic bacteria were affected by environmental temperature while the biodiversity was not affected. Finally, Microbacterium was regarded as the dominant species and Proteobacteria and Bacterium were deduced to be current-producing bacteria.

4. Conclusions

References

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Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant Nos. 51078100, 51178140, 51208496), China Postdoctoral Science Foundation (No. 2013T60182), by the National High-tech R&D Program of China (863 Program, Grant No. 2011AA060904), by State Key Laboratory of Urban Water Resource and Environment, HIT (Grant No. HCK201019), and by Heilongjiang Science Foundation for Distinguished Young Scholars (Grant No. JC201003).

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