An excellent anaerobic respiration mode for chitin degradation by Shewanella oneidensis MR-1 in microbial fuel cells

Biochemical Engineering Journal 118 (2017) 20–24

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Short communication

An excellent anaerobic respiration mode for chitin degradation by Shewanella oneidensis MR-1 in microbial fuel cells Shan-Wei Li, Raymond J. Zeng, Guo-Ping Sheng ∗ CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China

a r t i c l e

i n f o

Article history: Received 4 August 2016 Received in revised form 25 October 2016 Accepted 12 November 2016 Available online 14 November 2016 Keywords: Anaerobic respiration Chitin degradation Microbial fuel cell (MFC) Shewanella oneidensis

a b s t r a c t Shewanella oneidensis MR-1, a model electroactive bacterium, can oxidize a variety of fermentative organics and utilize an electrode as an electron acceptor for anaerobic respiration. Chitin as the main component of food wastes (shrimp and crab shells) is hardly degraded for bioenergy generation. This study demonstrated that S. oneidensis MR-1 could degrade chitin in microbial fuel cell (MFC). The metabolites during chitin degradation in MFC were succinate, lactate, acetate, formate, and ethanol. Their concentrations produced in MFC were higher than those in the fermentation system, as well as additional electricity could be recovered in MFC. Furthermore, the degradation of GlcNAc (the intermediate of chitin hydrolysis) by S. oneidensis MR-1 was faster than chitin in MFC and fermentation systems. Moreover, the mechanism of enhanced chitin degradation in MFC was speculated. This work might provide a new insight for biomass treatment and energy recovery by S. oneidensis MR-1 in MFC. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Microbial fuel cell (MFC), as the most widely used bioelectrochemical system, can produce electricity from several organic matters for future sustainable energy and chemicals production, in which microorganisms serve as the biocatalyst for oxidizing organic matters [1,2]. Bacterial species identified in MFC are usually called electroactive bacteria, such as Shewanella putrefaciens, Geobacter sulfurreducens, Rhodoferax ferrireducens, Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli and so on [3]. Recently, S. oneidensis and G. sulfurreducens are the species investigated most detailed among the electroactive bacteria. Electricity can be recycled from wastes such as wastewater, solid wastes, sewage sludge and landfill leachate by MFC [4–6]. Waste biomass is a cheap and relatively abundant source to produce electricity by MFC [7,8]. Chitin is the second most abundant biomass in nature, which is a structural material in many marine invertebrates, such as cuttlefish, shrimp [9,10], crab, and lobster as well as fungi and algae. 10 billion tons of chitin are produced in aquatic environments annually [11]. Chitin is a highly insoluble polymer that consists of N-acetyl-␤-d-glucosamine (GlcNAc) linked by ␤1,4-glycosidic bonds, and can be severed as carbon, nitrogen and energy for a variety of organisms. The survival of some bacteria in

∗ Corresponding author. E-mail address: [email protected] (G.-P. Sheng). http://dx.doi.org/10.1016/j.bej.2016.11.010 1369-703X/© 2016 Elsevier B.V. All rights reserved.

an aquatic environment is associated with their ability to utilize chitin as a carbon source. Its biodegradation is also a key step in nutrient recycling processes. Bacterial chitinases [12] provide environmental organisms the ability to acquire carbon under nutrient limiting conditions. It has been reported that only one chitin hydrolase (chitinase A) is found in S. oneidensis [13]. The hydrolysis products of chitin are chito-oligosaccharides and GlcNAc. S. oneidensis MR-1 is primarily cultivated on lactate [14], but is also known to oxidize GlcNAc as carbon and energy source [15]. S. oneidensis MR-1 could use GlcNAc as a substrate for positive growth in aerobic condition [16,17] and anaerobic growth with fumarate [17]. Yang et al. also investigated the utilization pathway of converting GlcNAc to Fru-6-P by S. oneidensis MR-1 [18]. As a popular mode electroactive bacterium [19], S. oneidensis MR-1 can convert wastes into bioelectricity and chemicals by using microbial fuel cell technique [7]. But whether chitin could be utilized and accompanied with energy production by S. oneidensis MR-1 has not been investigated so far. Thus, the aim of this study was to explore the potential of chitin degradation by S. oneidensis MR-1 in both MFC and fermentation systems, respectively. The degradation mechanism of chitin in MFC was also proposed. This work would be useful for the efficient degradation and sustainable utilization of chitin through the bioelectrochemical technology.

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2.3. Chitin degradation experiments The chitin and GlcNAc degradation experiments were operated at 30 ◦ C in MFC and fermentation systems, respectively. The anode and cathode cells equipped with carbon felt electrodes were autoclaved at 120 ◦ C for 20 min before use. The anodic medium containing mineral salt medium and chitin or GlcNAc was sterilized alone in the erlenmeyer flask. The fermentation experiments were performed in 60 mL serum bottles filled with 30 mL mineral salt medium containing chitin or GlcNAc. After that, the bottles were purged with N2 for 15 min to create anaerobic conditions, and then sterilized. The components of mineral salt medium (1 L) contained: 1.044 g KCl, 0.225 g K2 HPO4 , 0.225 g KH2 PO4 , 0.001 g CaCl2 , 0.42 g NaCl, 0.117 g MgSO4 ·7H2 O, 50 mM HEPES, and 10 mL mineral mix (1 L: 1.5 g NTA, 0.1 g MnCl2 ·4H2 O, 0.3 g FeSO4 ·7H2 O, 0.17 g CoCl2 ·6H2 O, 0.1 g ZnCl2 , 0.04 g CuSO4 ·5H2 O, 0.005 g KAl(SO4 )2 ·12H2 O, 0.005 g H3 BO3 , 0.09 g NaMoO4 , 0.12 g NiCl2 , 0.02 g NaWO4 ·2H2 O, and 0.1 g NaSeO4 ). The initial concentrations of chitin and GlcNAc were 2 g/L and 4.5 mM respectively, and the OD600 of S. oneidensis MR-1 was controlled at 0.3. During the degradation experiments, 0.5 mL sample was taken from the anode chambers or serum bottles every few days. The organic metabolites were determined by a high performance liquid chromatography (HPLC1260, Agilent Technologies, USA) with a refractive index detector. The analytical column was Aminex HPX87H (300 mm × 7.8 mm; Bio-Rad, USA), and the mobile phase was 5 mM H2 SO4 . The flow rate was 0.5 mL/min. The morphology of the deposit and the carbon felt electrode in the anode chamber were

614 (C-O)

1659 (C=O) 1557 (CN, NH) 1379 ( CH, C-CH3) 1315 (CN, NH)

3444 (OH)

0.0 4000

3000

2000

1000

Wavenumber ( cm ) -1

Fig. 1. FTIR spectrum and peak assignment of chitin.

Succinate Formate

2.0

Lactate Ethanol

Acetate Current Density

5

a

4

1.5

3

1.0

2 0.5

J (μA/cm2)

The two-chamber MFC was used for chitin or GlcNAc degradation experiments equipped with a 100 mL anode chamber filled with culture medium and a 100 mL cathode chamber filled with 50 mM ferricyanide (pH 7.0 in phosphate buffer). The cation exchange membrane (CMI-7000, Membranes International, Inc., USA) was separated between the anode and cathode chambers, and was immersed in 0.9% NaCl solution for 24 h before use. The carbon felt (3 cm × 3 cm × 3 mm; Beijing Jixing Sheng’an Inc., China) was used as the anode or cathode electrode. A 1000  resistor was connected between the anode and cathode, and the voltages of the resistor were recorded every 10 min. The recovered electrons from chitin or GlcNAc degradation in MFC were calculated through the integration of the output current (I) during the entire experiment.

0.5

1 0.0 0 0

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30

40

50 GlcNAc

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6 6 4 3

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J (μA/cm2)

2.2. MFC installation

1.0

Concentration (mM)

S. oneidensis MR-1 (ATCC® number 700550TM ) was kindly provided by Prof. K. H. Nealson from the University of Southern California [20]. It was cultivated in Luria-Bertani medium, and the dispersed biomass was centrifuged at 5000 g for 5 min. The harvest bacteria were washed twice with 30 mM 4-(2-Hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and resuspended in 30 mM HEPES. The concentration of the bacterial suspension (OD600 ) was determine by a spectrophotometer (UV752N, Jingke Co., China). White or light yellow sheet chitin (reagent grade, Sangon Biotech Inc., China) or N-acetyl-␤-d-glucosamine (GlcNAc, 98%, Aladdin Inc., China) was served as carbon, nitrogen and energy sources. The preparation of suspended chitin was performed as previously described [21]. The contents of C, H, O and N in chitin were analyzed by an elemental analyzer (Vario EL cube, Elementar Co., Germany). The functional groups of chitin were determined by a Fourier Transform Infrared (FTIR) spectrometer (Vertex 70, Bruker Co., Germany).

1.5

Concentration (mM)

2.1. Bacterial culture and chitin analysis

Absorbance (a.u.)

2. Materials and methods

1159 (C-O-C) 1073 (C-O)

S.-W. Li et al. / Biochemical Engineering Journal 118 (2017) 20–24

0 0

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Time (d) Fig. 2. Metabolite concentrations and output current density produced during (a) chitin and (b) GlcNAc degradation by S. oneidensis MR-1 in MFC.

investigated by a scanning electron microscope (SEM) (Sirion200, FEI Co., Netherlands). 3. Results and discussion 3.1. Chitin or GlcNAc degradation in MFC The percentage of C, H, O and N elements in chitin were 42.65%, 6.98%, 45.59% and 6.19%, respectively analyzed by the elemental analyzer. The predicted chemical formula of chitin was C8.07 H15.86 O6.48 N, which was similar to that of GlcNAc (C8 H15 O6 N, the monomer of chitin). FTIR of chitin is shown in Fig. 1 and the characteristic peaks were similar to those of chitin reported by the previous study [22]. Fig. 2a shows that the main products of chitin degradation by S. oneidensis MR-1 in MFC were succinate, lactate, acetate, formate and ethanol with their maximum concentrations of 0.79, 0.22, 1.65, 0.54 and 0.91 mM, respectively. The output current density reached the maximum value of 4.24 ␮A/cm2 at the 2.66th day. This was similar to that of 3.5 ␮A/cm2 generated by the MFC cultivated with anaerobic sludge using 1 g/L chitin as the sub-

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Fig. 3. SEM images of the deposits and the carbon felt electrode in the anode chamber.

strate in previous study, but a substantially longer time of 13.33 day is required [23]. These result implied that chitin could be converted to electricity by S. oneidensis MR-1 in a fast rate. The output current density existed a stable phase from the 5th day to the 23th day, which suggests that chitin as a substrate was sufficient in the first 23 days. In order to verify the degradability of chitin by S. oneidensis MR-1, SEM images of the deposits and the carbon felt electrode after degradation were shown in Fig. 3. After 20 days’ degradation, nanoscale chitin particles were formed, which implied that chitin could be degraded by S. oneidensis MR-1. While after the 23 days’ operation, the output current density started to decrease accompanied with a decrease of succinate, while acetate maintained at a stable concentration. The metabolites of GlcNAc metabolism in MFC were succinate, lactate, acetate, formate and ethanol, and their concentrations increased quickly in one day to 0.35, 0.94, 6.83, 5.47 and 0.09 mM, respectively (Fig. 2b). Simultaneously, GlcNAc was completely consumed, and the output current density reached the maximum value of 6.17 ␮A/cm2 . Then the output current density decreased accompanied with the reduction of metabolites which were exhausted at the 22th day except acetate. These results illustrate that the organic metabolites could be further utilized by S. oneidensis MR-1 in MFC. It has been predicted that succinate, lactate, acetate, formate and ethanol were the main fermentation products of S. oneidensis MR-1 from phosphoenolpyruvate and these organics could also be further utilized by S. oneidensis MR-1 [24]. It has also been reported that S. oneidensis MR-1 could use acetate for aerobic growth and could not utilize acetate for anaerobic growth [17]. So acetate could not be metabolized via anaerobic respiration by S. oneidensis MR-1 in MFC, and it remained in anode chamber after chitin degradation in

MFC. Furthermore, the GlcNAc degradation was much faster than that of chitin by S. oneidensis MR-1 in MFC (Fig. 2). 3.2. Chitin or GlcNAc degradation in the fermentation system S. oneidensis MR-1 is a non-fermenting facultative anaerobic ␥-proteobacterium [20]. It is rarely used in the fermentation system for substrate degradation. In order to identify the capacities of chitin degradation by S. oneidensis MR-1, fermentative degradation of chitin and GlcNAc were also investigated. Fig. 4a shows that the metabolites of chitin fermentation were succinate, lactate, acetate and formate, respectively. Fig. 4b shows GlcNAc could be completely consumed by S. oneidensis MR-1, and the metabolites, such as succinate, lactate, acetate, formate and ethanol, reached the maximum concentrations of 0.35, 4.70, 6.17, 4.09 and 0.70 mM, respectively within ten days. These results illustrate that both chitin and GlcNAc could be degraded by S. oneidensis MR-1 in the fermentation system. As the intermediate of chitin hydrolysis, GlcNAc was degraded much faster than the recalcitrant substrate chitin in the fermentation system (Fig. 4). 3.3. Comparison of chitin degradation in MFC and fermentation systems The metabolites of chitin degradation in MFC and fermentation systems were similar, while additional recovered electrons of 212.22 C could be generated in MFC. It has been reported that pyruvate fermentation by S. oneidensis MR-1 cells represented a combination of substrate-level phosphorylation and respiration [25]. It has also been reported that the energetic strategy of S. oneidensis MR-1 is a blend of respiratory and fermentative pathways,

S.-W. Li et al. / Biochemical Engineering Journal 118 (2017) 20–24

Succinate Formate

Lactate Ethanol

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Acetate

Concentration (mM)

0.4

a 0.3

0.2

0.1

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0

8

5

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20 GlcNAc

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6 Fig. 5. Speculative mechanism of chitin degradation in MFC. Reactions 1–12 are listed in Table 1.

4 2 0 0

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Time (d) Fig. 4. Metabolite concentrations and output current density produced during (a) chitin and (b) GlcNAc degradation by S. oneidensis MR-1 in the fermentation system. Table 1 Possible reactions in MFC’s anode chamber. reaction

possible anodic reaction

1 2 3 4 5 6 7 8 9 10 11 12

(C8 H13 NO5 )n + nH2 O → nC8 H15 NO6 C8 H15 NO6 + 2H2 O → 2C4 H6 O4 + NH4 + + 3H+ + 4e− C8 H15 NO6 + 4H2 O → 2C3 H6 O3 + NH4 + + 2CO2 + 7H+ + 8e− C8 H15 NO6 + 4H2 O → 3C2 H4 O2 + NH4 + + 2CO2 + 7H+ + 8e− C8 H15 NO6 + 10H2 O → 8CH2 O2 + NH4 + + 15H+ + 16e− C8 H15 NO6 + 4H2 O → 2C2 H6 O + NH4 + + 4CO2 + 7H+ + 8e− C8 H15 NO6 + 10H2 O → 8CO2 + NH4 + + 31H+ + 32e− C4 H6 O4 + 4H2 O → 4CO2 + 14H+ + 14e− C3 H6 O3 + 3H2 O → 3CO2 + 12H+ + 12e− C2 H4 O2 + 2H2 O → 2CO2 + 8H+ + 8e− CH2 O2 → CO2 + 2H+ + 2e− C2 H6 O + 3H2 O → 2CO2 + 12H+ + 12e−

which suggested that electron acceptors used by S. oneidensis MR-1 serves not only to balance internal redox pools but also to prevent the need for less efficient fermentative pathways [15]. So the improvement of chitin degradation in MFC might be resulted by using anode electrode as an electron acceptor for efficient anaerobic respiration by S. oneidensis MR-1. 3.4. Hypothetical mechanisms of chitin degradation in MFC Fig. 5 shows the possible mechanism of chitin degradation by S. oneidensis MR-1 in MFC, which might contain the procedures of hydrolysis, fermentation and anaerobic respiration. It has been reported that chitin should initially be slowly broken down into oligosaccharides, followed by conversion of the oligosaccharides to GlcNAc at a fast rate [26]. The possible anodic reactions are listed in Table 1. In MFC, the excess electrons generated by chitin oxidation dissipated through extracellular electron transfer via outer

membrane c-type cytochromes (OMCs) [27] or flavins (electron shuttles secreted by S. oneidensis MR-1) [28] at the anode. The electron dissipation tended to decrease the NADH/NAD+ ratio, resulting in compensatory cellular regulation to regenerate NADH, which needed continuous organic metabolites generation from chitin and GlcNAc, and thus enhanced the degradation of chitin and GlcNAc by S. oneidensis MR-1. Recently, electrochemically controlling microbial fermentative metabolism with the electrode is defined as ‘electro-fermentation’ [29,30]. An electrode in the fermentation medium can externally induce a shift from balanced to unbalanced fermentation, which can enhance and better control microbial fermentations by increasing the specificity of the metabolic routes and overcoming thermodynamic limits [30]. It has been reported that glycerol can convert into ethanol using an engineered strain of S. oneidensis MR-1 for natively transferring electrons directly to electrodes [31]. So recalcitrant biomass treatment by electroactive bacteria via anaerobic respiration in MFC might be an attractive way for energy recovery. Acknowledgements The authors wish to acknowledge National Natural Science Foundation of China (21377123 and 51322802), the Fundamental Research Funds for the Central Universities, and the Key Research Program of Frontier Sciences, CAS (QYZDB-SSWQDC020) for the partial support of this study. References [1] B.E. Logan, M.J. Wallack, K.Y. Kim, W.H. He, Y.J. Feng, P.E. Saikaly, Assessment of microbial fuel cell configurations and power densities, Environ. Sci. Technol. Lett. 2 (2015) 206–214. [2] M.A. Rosenbaum, A.E. Franks, Microbial catalysis in bioelectrochemical technologies: status quo, challenges and perspectives, Appl. Microbiol. Biotechnol. 98 (2014) 509–518. [3] K. Rabaey, W. Verstraete, Microbial fuel cells: novel biotechnology for energy generation, Trends Biotechnol. 23 (2005) 291–298. [4] M. Sun, L.F. Zhai, W.W. Li, H.Q. Yu, Harvest and utilization of chemical energy in wastes by microbial fuel cells, Chem. Soc. Rev. 45 (2016) 2847–2870. [5] H. Wang, J.D. Park, Z.J. Ren, Practical energy harvesting for microbial fuel cells: a review, Environ. Sci. Technol. 49 (2015) 3267–3277. [6] Z. Ge, J. Li, L. Xiao, Y.R. Tong, Z. He, Recovery of electrical energy in microbial fuel cells, Environ. Sci. Technol. Lett. 1 (2014) 137–141. [7] B.E. Logan, K. Rabaey, Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies, Science 337 (2012) 686–690.

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