Dark bio-hydrogen fermentation by an immobilized mixed culture of Bacillus cereus and Brevumdimonas naejangsanensis

Renewable Energy 105 (2017) 458e464

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Renewable Energy journal homepage: www.elsevier.com/locate/renene

Dark bio-hydrogen fermentation by an immobilized mixed culture of Bacillus cereus and Brevumdimonas naejangsanensis Zhihong Ma, Chan Li, Haijia Su* Beijing Key Laboratory of Bioprocess, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 November 2016 Received in revised form 14 December 2016 Accepted 19 December 2016 Available online 20 December 2016

Based on the immobilized mixed culture technology, both the hydrogen production and synergy mechanism between Bacillus cereus A1 and Brevumdimonas naejangsanensis B1 were investigated. Different immobilization carriers were chosen. In terms of hydrogen yield and the multi-cycle use of the carriers in batch fermentation, corn stalk as carrier was found to be a better candidate than fiber material (polyester fiber) and activated carbon (AC). The obtained cumulative hydrogen production was 2205 mL/ L within 180 h, significantly higher than that of the suspended fermentation. The average cumulative hydrogen production and hydrogen yield were 1845 mL/L and 1.50 mol H2/mol glucose for ten cycles of repeated fermentation batches respectively, which was 62.5% higher than that of suspended fermentation. The experimental results also showed that the system could use starch as direct substrate and the tolerance of the immobilized system to the substrate loading was improved. The activities of amylase and hexokinase were respectively 2 to 3 and 2 times higher than in the suspended fermentation due to the synergistic effect of co-immobilization compared with the suspended fermentation. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Hydrogen Dark fermentation Immobilization Mixed culture

1. Introduction In the past decades, the increasing demand of energy and rapid economic development has stimulated the development of alternative energy sources. Hydrogen energy, as one of the most promising energy candidates, attracts increasing attention due to its environmentally-benign and highly-efficient characteristics [1,2]. Many methods to produce hydrogen were examined and applied, but most of them are energy intensive and fossil-fuel based, which makes hydrogen production more costly and unsustainable [3]. On the contrary, biohydrogen production is energysaving, efficient and pollution-free [4]. Current research of biohydrogen production mainly concentrates on suspended-cell systems. However, the disadvantages such as easy washout of cells lead to a low efficiency of hydrogen production. Therefore the investigation of immobilized-cell systems has become a hot topic in the hydrogen production [5]. Since pure bacteria are prone to be contaminated along with the fermentation, sterilization is hence again required, increasing the workload. Besides, pure bacteria cannot be recycled and the use of

* Corresponding author. E-mail address: [email protected] (H. Su). http://dx.doi.org/10.1016/j.renene.2016.12.046 0960-1481/© 2016 Elsevier Ltd. All rights reserved.

pure bacteria in biohydrogen production becomes difficult to be industrially applied. Bao et al. did research about bio-hydrogen fermentation by mixed culture of Bacillus sp. and Brevumdimonas sp. The results showed that the hydrogen yield was doubled in mixed culture compared with pure culture [6,7]. Researches moreover showed that the synergistic effect of mixed cultures could broaden the type of substrate, improve the adaptability of the system to the environment, and enhance the efficiency of fermentation [8]. The substrate is one of the major costs in biohydrogen production, [9]. Previous studies used simple sugars, such as glucose, maltose, and xylose, as substrates for hydrogen production. A yield of 2.31 mol H2/mol xylose was reported by An et al. using Clostridium beijerinckii YA001 [10]. Haroun et al. used glucose as substrate, and the hydrogen yield was 2.27 mol H2/mol glucose [11]. Alvarez et al. used the G088 strain to produce hydrogen with glucose as substrate, and the hydrogen yield was 1.70 mol H2/mol glucose [12]. But, the cost of using simple sugar as substrates is still high, which increases the total production cost of hydrogen. Since cheap polysaccharoses such as starch and cellulose are unable to be degraded directly owing to the lack of hydrolytic enzymes, it is important (i) to look for cheap and suitable substrates that can considerably decrease the fermentation cost, and (ii) to find functional microorganisms that can directly

Z. Ma et al. / Renewable Energy 105 (2017) 458e464

hydrolyze the starch or cellulose into fermentable reducing sugars [13]. Immobilized-cell systems can be operated continuously at low retention time without suffering from washout of cells. Compared with suspended cells, immobilized cells offer additional advantages such as being more tolerant to environmental perturbation, being more stable in operation, being reusable and promoting a higher biological activity due to the higher cell density [14e16]. Considering the effect of different immobilization methods on the hydrogen production, adsorption and embedding are mostly used [17e19]. Embedding either spreads cells into the porous carrying materials, or embeds cells in a gel-forming polymer, thus yielding immobilized cells. Anjana et al. reported that the hydrogen production rate by immobilized cyanobacterium Lyngbya perelegans in alginate was raised approximately two times compared to the suspended-cell system [20]. The mass transfer resistance using the embedding is however too high, so that the biochemical reaction rate is low. Many researchers hence choose adsorption, which is based on electrostatic fixation of the enzyme or microorganism cells upon a carrier to attain the goal of immobilized cells. It is the simplest method with mild preparation conditions and higher cell activity. The adsorption carriers must be internally porous, have a large specific surface area and high mechanical strength. It is reported that the hydrogen production using a porous glass carrier was two times higher than that of suspended fermentation [21]. Kirli et al. reported that the hydrogen yield with immobilization in plastic scouring sponge pads covered by metal mesh increased to 2.1 mol/mol glucose [22]. There are some common carriers used in dark bio-hydrogen fermentation, such as sodium alginate [20], porous glass [21], sponge [22], agar [23], ceramic [24] etc. In this study, we consider that agricultural waste is biodegradable, renewable, biocompatible and non-toxic, which makes it attractive as adsorption material. At present, applicable materials are corn stalk, sunflower stalk and bagasse. Currently, most of the stalks are burned, which leads to low efficiency and environment pollution. Using agriculture stalk as an immobilized carrier, not only achieves agricultural stalk resource recovery, but also provides a highly efficient, low-cost carrier for immobilized microorganism. Therefore, corn stalk was investigated as carrier in this study, and compared with other carrier materials. In order to comprehend the synergistic mechanism of two bacteria using starch substrate, the immobilized mixed culture system of the two hydrogen production bacteria was investigated. The optimal immobilization carrier was obtained by comparing the hydrogen production rate and reusability of different carriers. The influences of the immobilized mixed strains on pH, on the tolerance to the substrate, towards the substrate utilization rate, and the enzyme activity were studied, and the mechanism of biohydrogen production in the system of anaerobic fermentation was explored. 2. Materials and methods 2.1. Microorganisms In this study, the mixed culture of A1 and B1 was used. They belong to Bacillus cereus A1 (CP015727) and Brevumdimonas naejangsanensis B1 (CP015614), respectively [25,26]. They were screened and separated from a sludge in an anaerobic digestion reactor, detailed in previous literature [6]. 2.2. Immobilization methods As the carrier, polyester fiber material, activated carbon (AC) and corn stalk were studied. A certain quantity of carriers was added in the seed medium before it was sterilized. Then, the seed medium

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was inoculated with the mixed strains (A1/B1 ¼ 1/1), at a ratio determined by previous literature [6]. The microorganisms were grown in the seed medium for 72 h at 37  C to achieve the immobilization. Then the carriers were removed and transferred to the fermentation medium for hydrogen production. The composition of the seed and the fermentation medium were established previously [6]. 2.3. Experimental setup and procedure The experimental setup was same as that of Bao et al. [6]. Each reactor was filled with 0.9 L fermentation medium, and 9 g of carrier that had absorbed the microorganisms (in the immobilized fermentations) or 100 mL seed medium with the mixed strains (in the suspended fermentations). Other procedure consult the previous literature [6]. After one batch fermentation, the carriers were removed and added into a new fermentation medium for the next batch experiment without any additional inoculation. 2.4. Analytical methods In this study, some main parameters such as pH, total sugar concentration, hydrogen production, volatile fatty acids (VFAs), were investigated according to standard analytical procedure [6,7]. 3. Results and discussion 3.1. Comparison of immobilized and suspended fermentation process In this work, fiber material, activated carbon (AC) and corn stalk adsorption materials were chosen as immobilizing carriers, and they are all cheap, accessible, and porous materials. The cumulative H2 production are presented in Fig. 1. The cumulative amount of hydrogen increased with various degrees for three immobilized fermentations compared with suspended fermentation. A cumulative amount of hydrogen of 1011 mL/L and a hydrogen yield of 1.04 mol H2/mol glucose was obtained with the suspended fermentation. Compared with the suspended fermentation mode, the yield was increased by 34.4%, 49.4% and 118.1% for fiber, AC and corn stalk respectively. The cause of this difference is that the adhesion strength between cells and carrier is different for different carriers, and the toxicity to microorganism is also different [25]. In the fiber immobilized fermentation system, the substrate utilization ratio was lower and the same as in the suspended fermentation, being around 60% in all cases, as shown in Fig. 2 b. The variation of the pH (Fig. 2 a) in the fiber tests was the highest of all, and the final pH was the lowest, leading to the lowest cumulative amount of hydrogen. On the contrary, the pH of corn stalk was the most stable and the substrate utilization rate in the corn stalk immobilized fermentation was around 90%. Although the substrate utilization rate in the activated carbon immobilized fermentation also reached 90%, the amount of hydrogen was much lower than that in the corn stalk case, tentatively explained by the fact that the starch was decomposed in the activated carbon immobilized fermentation, but had not been used to produce hydrogen completely, while some sugars were decomposed into some intermediate metabolites. The main end products in the liquid phase of different experiments, were acetic acid and butyric acid (Fig. 3). The acetic acid was the highest in suspended fermentations, being 1.08 g/L or 37.8% of the total acid content. In fiber and activated carbon immobilized fermentation, the change of butyric acid content was not obvious,

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3.5

suspended fibre activated carbon corn stalk

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Acetic and butyric acid concentration (g/L)

cumulative yield of hydrogen (mL)

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Acetic acid Butyric acid

3.0 2.5 2.0 1.5 1.0 0.5 0.0

suspended

time (h) Fig. 1. Variation of cumulative H2 production with time.

7.0

suspended fibre activated carbon corn stalk

6.5 6.0

pH

5.5 5.0 4.5 4.0 3.5 3.0 24

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time (h)

(a) 12

suspended fibre activated carbon corn stalk

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total sugar content (g/L)

activated carbon

corn stalkl

Fig. 3. Variation of liquid end products (VFAs) with time.

but the acetic acid content decreased significantly. In corn stalk immobilized fermentation, the butyric acid concentration was the highest, which increased from 62.2% to 84.2% of the total acids

0

fibre

Immobilized carriers

8

compared with suspended fermentation. Since mostly acetic and butyric acid were produced through immobilized fermentation, the VFAs transformation to ethanol and butanol become more feasible and is currently assessed by transforming appropriate strains through genetic modification. The modified Gompertz equation has been widely used in hydrogen production by batch dark fermentation [2], which is defined as follow:

   Rm  e Pt ¼ Pm exp  exp ðl  tÞ þ 1 Pm Where Pt is the cumulative hydrogen production (mL) at culture time t, Pm is the maximum amount hydrogen production (mL), Rm is the maximum hydrogen production rate (mL/L), were adjusted to measure the hydrogen evolution data, l is the lag time (h) and the value of “e” is 2.71828. As can be seen in Table 1, the lag time (l) of immobilized fermentation was shortened significantly and the H2 production rates were greatly improved in comparison with suspended fermentation, indicating a higher cell density and biological activity in the immobilization process with the immobilized mixed culture adapting rapidly to the environment. Especially in the corn stalk fermentation, the lag time was shortened by 63.7% compared to suspended fermentation and the system ran more steadily and more efficiently. The enzymatic activity will be discussed in section 3.3. 3.2. Batch test of immobilizing carriers In terms of the cumulative amount of hydrogen production, lag time and maximum production rate, the three immobilized fermentations were better than suspended fermentation, although they show a difference. One of the advantages of immobilized

6 4 2

Table 1 Gompertz equation coefficients for three carriers of fermentation compared with the suspended system.

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(b) Fig. 2. Variation of (a) pH, and (b) total sugar with time.

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Fermentation type

l(h)

Rm(mL/h)

R2

suspended fiber activated carbon corn stalk

18.94 14.89 13.12 6.88

16.54 28.03 21.7 25.67

0.989 0.997 0.991 0.983

Z. Ma et al. / Renewable Energy 105 (2017) 458e464

cumulative yield of hydrogen (ml)

2500

2000

1500

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0 suspended -- --fiber-- -- -- activated carbon-- -- -- -- --corn stalk-- -- -- --

Fig. 4. The comparison of immobilized and suspended fermentations. (Different column indicate the cumulative amount of hydrogen production for different batches, including one batch of suspended, 5 batches of fiber as carrier, 7 batches of AC as carrier, 10 batches of corn stalk as carrier).

fermentation is that immobilized cells can be used repeatedly. In order to observe its repeated use, the immobilized carriers were repeatedly used. The cumulative hydrogen production of the three immobilized carriers in batch fermentation was compared in Fig. 4 for repeated

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use of the different materials. The maximum cumulative hydrogen production was obtained in the first batch fermentation, while it showed a trend of decline in subsequent batch fermentations. However, the time of seed development was saved, greatly shortening the fermentation time in immobilized batch fermentation. The fiber carrier system could be repeatedly used in 5 batches of fermentation. An average hydrogen yield of 0.743 mol H2/mol glucose was obtained. However, the cumulative production of hydrogen was rapidly reduced from the second fermentation onwards. Because of the weak adsorption of bacteria, progressive wash-out takes place: fiber as carrier was not good for repeated use. The AC carrier system was repeatedly used in 7 batches of fermentation, and the average hydrogen yield was 1.10 mol H2/mol glucose, which was 19.6% higher than that of the suspended fermentation. Due to the AC's porous structure and its smaller pores, bacteria were more easily adsorbed than using fiber as carrier, thus the AC carrier can be used more repeatedly than the fiber carrier. Corn stalk as carrier, could be repeatedly used in 10 batches of fermentation. The average cumulative production for hydrogen was 1845 mL/L. The average utilization rate of substrate was 90.6%, and the average hydrogen yield was 1.50 mol H2/mol glucose, which was 63.0% higher than that of the suspended fermentation. Corn stalk is mainly composed of cellulose, hemicellulose and lignin, coexisting in plant fiber, forming a porous structure. As shown in Fig. 5, the structure of corn stalk is full of pores, implying that the corn stalk may provide extra surface area for

Fig. 5. Scanning electron microscope view of corn stalk as a support material 200 (a) and 5000 (b); immobilized mixed bacteria on corn stalk 500 (c); immobilized mixed bacteria on corn stalk, 5000 (d).

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Table 2 Comparison of hydrogen yield by different cultures on different carriers. culture

carrier

Operation mode

substrate

H2 yield

Continuous or Batch fermentation

Ref

Clostridium sp. T2 Clostridium sp. T2 Clostridium butyricum Anaerobic sludge Hydrogen produced sludge Sewage sludge Sewage sludge Sewage sludge A1 and B1 A1 and B1 A1 and B1 A1 and B1

mycelia pellets sodium alginate sugarcane bagasse PMMA activated carbon

CSTR CSTR Batch CSTR CSTR

xylose xylose Sugarcane juice sucrose starch

0.414 molH2/mol xylose 0.472 molH2/mol xylose 1.34 molH2/mol hexose 2.0 molH2/mol sucrose 2.74 molH2/mol sucrose

350 h 350 h 5 batches 150 h 240 h

[27] [27] [28] [29] [30]

silicone-immobilized sludge Granular activated carbon Synthetic granular sludge e fiber material activated carbon Corn stalk

CSABR AFBR CSTR Batch Batch Batch Batch

sucrose glucose glucose starch starch starch starch

3.5 molH2/mol sucrose 1.19 molH2/mol glucose e 1.04 molH2/mol glucose 0.743molH2/mol glucose 1.10 molH2/mol glucose 1.50 molH2/mol glucose

HRT ¼ 0.5 HRT ¼ 1 30d 1 batch/180 h 5 batches/900 h 7 batches/1260 h 10 batches/1800 h

[31] [32] [33] [11] This study This study This study

attaching bacteria: its surface is clean before adsorption (Fig. 5 a, b), while scanning electron microscopy (SEM) analysis showed the bacteria distribution within the pore(Fig. 5 c, d)after immobilization. The experimental results of the present study are compared with literature data in Table 2. Compared with other researches, the co-immobilization of two strains ensured their metabolic exchange. Under the synergetic action of both strains, not only simple substrates such as monosaccharide could be used, but also complex substrates such as starch could be used, with a final yield of hydrogen production maintained at a high level. In addition, compared with immobilization on sodium alginate and sludge in the literature, the adsorption method used in this paper was simple and easy to use. And the embedding method isolated the microorganisms from the fermentation broth, which caused a large diffusion resistance between the substrate and the product, declining the speed and efficiency of the fermentation. Compared with the other adsorption materials, corn stalk not only has better physical adsorption ability, but also has better biocompatibility because of the high hydroxyl content. Immobilized microorganisms could be constantly updated, dead cells were removed and replaced by new microbial cells: the activity of the microorganisms could be maintained in the process. Through the comparison, corn stalk was shown to be the optimal adsorption carrier.

3.3. Effect of immobilization on substrate tolerance For suspended fermentation, when the substrate concentration increased from 10 g/L to 15 g/L, the final cumulative H2 production did not increase, as shown in Fig. 6. The reason was that the substrate concentration was too high, the intermediate metabolites were accumulated, resulting in a too fast pH declined, and inhibition of the production of hydrogen. So the optimum fermented substrate concentration was 10 g/L in suspended fermentation. In the corn stalk immobilized fermentation, when the substrate concentration increased from 10 g/L to 15 g/L, the final cumulative H2 production increased from 1845 ml/L to 2385 ml/L, increasing by 29.3%. Seen from the hydrogen yield, it decreased from 1.50 mol H2/mol glucose to 1.29 mol H2/mol glucose, so the optimum was still 10 g/L. Immobilization improved the substrate tolerance of bacteria to a certain degree, as confirmed by literature [34,35]. 3.4. Effect of immobilization on enzyme activity In the fermentation process with starch as substrate, bacteria secrete amylase that will hydrolyze starch into monosaccharide or disaccharide, which was used to produce hydrogen. The amylase activity reflects the ability of bacteria using starch. Hexokinases can catalyze the first committed step of glucose metabolism, i.e. the ATP 35

suspended-10g/L suspended-15g/L immobilized--10g/L immobilized--15g/L

2000

A1,suspended B1,suspended A1/B1=1/1,suspended A1/B1=1/1,immobilized-1 A1/B1=1/1,immobilized-3 A1/B1=1/1,immobilized-10

30

Enzyme activity (U/mL)

cumulative yield of hydrogen (mL)

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25 20 15 10 5

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Time (h) Fig. 6. Variation of cumulative H2 with time under different concentration of starch.

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Time (h) Fig. 7. Variation of the activity of amylase with time.

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HK activity (U/g prot)

National Basic Research Program (973 Program) of China (2014CB745100), the (863) High Technology Project (2013AA020302).

immobilized suspended

20

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15

References

10

5

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Time (h) Fig. 8. Variation of the activity of hexokinase with time.

dependent phosphorylation of glucose (Glu) to yield glucose-6phosphate (G6P). The improvement of hexokinase activity can promote the glycolysis process. So the effect of immobilization on enzyme activity was researched. Fig. 7 showed the variation of the activity of amylase with time in different fermentations. For pure cultures, the amylase activity of A1 is higher than that of B1, illustrating that A1 can hydrolysis starch more effectively. Due to the synergy between two strains, mixed cultures have a higher amylase activity than pure cultures. In the first batch fermentation of immobilized cultures, the activity of amylase was 2 times higher than that of the suspended fermentation, which indicates that mixed cultures quickly adapt to secrete amylase, hydrolyzing starch into low molecular weight sugars. It improved to 3 times in the third and tenth batch fermentation, showing that the stability of the immobilization is better. Therefore, immobilized bacterial fermentation showed great advantages over the suspended fermentation. As clearly shown in Fig. 8, the activity of hexokinase was significantly improved by immobilization. The increased activity of hexokinase prevents the accumulation of the glucose, avoiding the inhibitory effect by high glucose concentration. 4. Conclusions Hydrogen production through anaerobic fermentation by immobilized mixed culture technology has become an important research focus. This study compared three different carriers in the system of co-immobilization, corn stalk offering the best potential as adsorption carrier. The lag period was shortened by immobilization technology. The average cumulative production of hydrogen was 1845 ml/L for 10 batches. The average utilization rate of substrate was 90.6%, and the average hydrogen yield was 1.50 mol H2/ mol glucose, which was 63.0% higher than that of the suspended fermentation. The activity of amylase was 2e3 times higher than that of suspended fermentation, whilst the activity of hexokinase was improved greatly by immobilized mixed culture technology. In addition, the reuse of carrier saved the step of seed development, thus significantly shortening the fermentation time in immobilized mixed culture technology. Acknowledgements The authors express their thanks for the supports from the National Natural Science Foundation of China (21525625), the

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