Adsorption Characteristics and Mechanisms of Coal-Microorganisms

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Biofuels and Biomass

Adsorption Characteristics and Mechanisms of CoalMicroorganisms in the process of Biogenic Methane Production from High Volatile Bituminous Coal Daping Xia, Huaiwen Zhang, Xianbo Su, Hao Chen, and Dan Li Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.9b01559 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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Adsorption Characteristics and Mechanisms of Coal-Microorganisms in the process of Biogenic Methane Production from High Volatile Bituminous Coal Daping Xia1,2, Huaiwen Zhang1, Xianbo Su1,2*, Hao Chen3, Dan Li1 Author Affiliations: 1

School of Energy Science and Engineering and College of Resource and Environment,

Henan Polytechnic University, Jiaozuo 454000, China; 2

Collaborative Innovation Center of Coalbed Methane and Shale Gas for Central Plains

Economic Region, Jiaozuo 454000, China; 3

PetroChina Research Institute of Petroleum Exploration and Development, Langfang

065007, China; *Corresponding author. Tel: 0391-3987981; Email address:[email protected] Daping Xia: Email address: [email protected] Huaiwen Zhang: Email address: [email protected] Xianbo Su: Email address: [email protected] Hao Chen: Email address: [email protected] Dan Li: Email address:[email protected]

ACS Paragon Plus Environment

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ABSTRACT: To investigate the adsorption characteristics and mechanisms of coalmicroorganisms in the process of biogenic methane production from coal, simulation experiments of biogas production were conducted under suitable environmental conditions (initial pH=7, and constant temperature at 35 °C) using highly volatile bituminous coal from China’s Yima coalfield. The microbial adsorption characteristics, organic liquid products, coal wettability and adsorption heat were measured using scanning election microscopy, gas chromatography-mass spectrometry, contact angle measurement and micro-calorimetry, respectively, to reveal the adsorption characteristics of the coal and its interaction mechanisms with microorganisms. The results show that: (1) the microbial adsorption capacity changed as fermentation time varied and the maximum values of optical density (at 600 nm) and RNA were 0.122 and 1772.73 μg, respectively, and occurred at 9 d. (2) The contact angle of the coal surface was < 90° during the experiments, and the coal wettability reached its maximum on day 9 of heat production. (3) There was correspondence between the exothermic process and production of intermediate liquid products, the content of small organic molecules was more and the heat output was higher. (4) The generation of gas products differed remarkably as adsorption capacity and adsorption heat during the reactions varied. The maximum methane production and adsorption heat values were, respectively, 1.849 mL/g (at 19 d) and 306.031 J/g (at 9 d). The adsorption heat showed a variable trend (first decreasing, then increasing, decreasing again, and finally increasing) but all phases resulted from an exothermic reaction. This research provides a reference for understanding the biogas production pathway, the degradation mechanism and the improvement of biogas-producing efficiency as microorganisms degrade coal, and enhances knowledge about the potential concentrations of target products. The study further enriches the theory of biogenic gas production from coal. Keywords: coal; microorganisms; adsorption heat; biogenic methane; adsorption characteristics


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1. INTRODUCTION

The first step in interactions between microorganisms and coal is the initial adhesion of the microbes to coal. The degree of contact reflects the operational effect of biogas production by anaerobic digestion. The adsorption system between minerals and microorganisms has been widely studied, and research mainly has evaluated the adsorption characteristics of microorganisms with inorganic minerals, soil or heavy metals, and the adsorption capacity, adsorption time and adsorption force between them1-2. Sun et al.1 studied the adsorption characteristics of Cd on the soil minerals, humic acid, bacteria and their mixture. Rong et al.3 studied the adsorption characteristics of Bacillus subtilis and Pseudomonas putida on mineral surfaces, and expounded the effects of cell structure and surface properties on microorganism adsorption. Dunham-Cheatham et al.4 studied the effect of natural organic matter on the adsorption of Hg microbial cell. The adsorption of minerals on cells and biomolecules is a complex process, which was affected by the structure of minerals and cells, surface properties and environmental conditions3. Coal is a mixture that is mainly constituted by a cross-linked network of macromolecules, and its biosorption characteristics are different from common minerals (comprised of a single component). Many studies indicate that the decomposition, transformation and adsorption of organic matter by microorganisms are accompanied with the generation of thermal effects5. Therefore, thermal effects can be used to measure the microbial activity and further explore reactive theory5. Micro-calorimetry is a nonspecific calorimetry that can be used to study the liquid phase reaction, solid state reaction, gas reaction and the complex biological metabolism reaction. This technique can be used to calculate and reflect the apparent growth rate constant of a microbial community and the degree of biochemical reaction by monitoring the heat output in different gasification stages5. The heat adsorption or heat output can be determined by micro-


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calorimetry in the interaction between microorganisms and soil minerals5. Jiang et al.6 studied the thermal metabolic activity and Cu2+ fixation/transformation ratio of microbes in paddy soil and marsh sediment by using microcalorimetric method. Most studies have focused on the regularity of coal degradation by microorganism and its metabolites7-13, and few have addressed the adsorption characteristics and mechanisms of microorganisms on the coal surface during the process of biogas production. In view of this, biogenic methane experiments were conducted using highly volatile bituminous coal from China’s Yima coalfield to study changes in the microbial adsorption capacity, wettability, adsorption and organic liquid products on the coal surface. This research further enriches the biogas production mechanism of coal, and provides a reference for exploration and development of CBM (coal bed methane).

2. MATERIALS AND METHODS

2.1. Experimental Samples. High volatility bituminous coal was selected from the Yima mine in Henan Province, China (Figure 1). The depth and thickness of coal seam is 500 m and 9.25 m, respectively, and the coal seam geothermal temperature is 25 °C. Fresh lump coal samples (>8 cm × 8 cm × 8 cm) were collected by manual operation at the coal mine working face, and then placed in a low-temperature anaerobic tank and sent to the biological laboratory for storage. The quality of the coal sample is 50 kg. Coal proximate and ultimate analyses are shown in Table 114. Before the experiment, the 2 cm external surface of coal samples was stripped off in the anaerobic workstation, and then crushed into coal particles. The particle size of coal samples was 100–200 μm. Fresh coal mine water (containing indigenous bacteria) was collected at the waterspout in the coal face, and sent to the biological laboratory for storage at 4 °C.


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N Hebi Jiaozuo

Lasa

Yima

Beijing

Urumqi

Xian Nanning

Zhengzhou Zhoukou Nanyang

Shanghai Taipei 0

100 200 km

Location of sample City

Horizon

Age

Coal rank

2-3

Middle jurassic

High volatile bituminous coal

Ro.ran/%

0.56

Figure 1. Location of coal samples Table 1. Proximate and ultimate analysis of coal samples Proximate analysis (%) Mad 10.45

Aad 5.32

Vad 31.15

Ultimate analysis (%) FCad 53.08

Cdaf 76.24

Hdaf 5.29

Ndaf 1.08

Ro.ran (%) (O+S)daf 17.39

0.56

M, moisture; A, ash yield; V, volatile matter; FC, fixed carbon; ad, air-dry basis; daf, dry ash-free basis; C, carbon; H, hydrogen; N, nitrogen; O, oxygen; S, sulfur; Ro.ran, vitrinite random reflectance

2.2. Enrichment Culture of Methanogens. A trace element solution was created by adding 1000 mL deionized water to 1.5 g triglycolamic acid, 0.5 g MnSO4·2H2O, 3.0 g MgSO4·7H2O, 0.1 g FeSO4·7H2O, 1.0 g NaCl, 0.1 g CoCl2·6H2O, 0.1 gCaCl2·2H2O, 0.01 g CuSO4·5H2O, 0.1 g ZnSO4·7H2O, 0.01 g H3BO3, 0.01 g KAl(SO4)2, 0.02 g NiCl2·6H2O, and 0.01 g Na2MoO4. The methanogens were cultured in mine water to which were added (per L) 1.0 g NH4Cl, 0.1 g MgCl2·6H2O, 0.4 g K2HPO4·3H2O, 0.2 g KH2PO4, 1.0 g yeast extract, 0.001 g resazurin, 0.5 g L-cysteine, 0.2 g Na2S, 2.0 g NaHCO3, 2.0 g sodium formate, 2.0 g sodium acetate, 0.1 g tryptone and 10 mL of trace element solution. 2.3. Experimental Protocols. The gas production experiment included three parallel samples, and the final analysis data is an average of the three sets of data. The fermentations were conducted in

1000 mL flasks, and about 100 g of fresh coal particles was also added into the flasks (Figure 2). The cultivation solution of methanogens (enriched for 4 days) was injected into the


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fermentation flask, which was filled with N2 for 2–3 min to create an anaerobic environment. The gas volume was collected by drainage (saturated salt water), and the biogas production reactor was incubated at 35 ± 1 °C for about 20 d after sealing the reactor completely using waxed film. The reactor liquid containing bacteria was collected from the liquid sampling port and placed in a 50 mL centrifuge tube, which was filled with N2 to create an anaerobic environment. The centrifuge tube was sealed using a waxed film and stored at 4 °C in a refrigerator. 4 3 2

5 6

5

7

9 1

8

1-ferment container; 2-rubber plug; 3, 6-steel needle; 4-liquid sampling port; 5-gas sampling port; 7-rubber tube; 8-gas washing bottle; 9-gas collection device

Figure 2. The biogenic gas production simulation device 2.4. Experimental Methods 2.4.1. Determination of gas composition. The gas composition was quantified using a GC4000A gas chromatography instrument equipped with a TCD detector and a stainless steel TDX-01 column, 3 mm in diameter and 2 m long. The column temperature was 80 °C, the temperature of the injector was set at 120 °C and the detector temperature was 100 °C. The carrier gas was helium, and the flow rate was 30 mL/min. An injection needle was used for manual injection, and the injection volume was 1 mL each time. 2.4.2. Determination of RNA in the bacterial liquid. The approximate RNA content reflecting the microbial adsorption capacity was determined using ultraviolet (UV) spectrophotometry. The RNA test solution (1 mL) was diluted 10 times with pure water and


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delivered into separate centrifuge tubes (denoted A and B). Then, 1 mL pure water was added to centrifuge tube A. Likewise, 1 mL precipitant of perchloric acid-ammonium molybdate was added to centrifuge tube B, shaken and then placed in a refrigerator for 30 min. The A and B test tubes were centrifuged (Eppendorf 5804R centrifuge) at 3000 rpm for 30 min, after which 0.5 mL supernatant was collected. The supernatant was transferred into a volumetric flask. Meanwhile, the coal sample was washed 2–3 times with deionized water after centrifugation and the flushing fluid was also transferred into a volumetric flask (constant volume is 50 mL). The A260 (the absorbance of a test solution at wavelength 260 nm) of the A and B test solutions was analyzed using a UV-Vis spectrophotometer (UV-5200) and distilled water was used as a blank control. The approximate RNA content (μg) was calculated according to Equation 1: RNA =

A A260 - B A260 0.022

× VB × 10

(1)

in which AA260 is the optical density of test solution A at wavelength 260 nm; BA260 is the optical density of test solution B at wavelength 260 nm; VB is the total volume of test solution (50 mL); D is the dilution multiple of the test solution (10 times); and 0.022 is the specific extinction coefficient of RNA. 2.4.3. Determination of adsorption heat in the bacterial liquid. The adsorption heat was determined using a C80 microcalorimeter (Setaram Instrumentation, Caluire, France) in the Coal and CBM Engineering Laboratory of Henan Polytechnic University, Jiaozuo, Henan Province, China. The key components of this instrument were the calorimetric system, battery module system and CS control system, and it was equipped with various sample cells. The temperature range of the C80 was room temperature to 300 °C, the heating rate was 0.001– 2 °C/min, the calorimeter precision was ± 0.1%, and the maximum pressure was 100 MPa. The bacterial liquid was centrifuged at 3000 rpm for 30 min and then 50 mL supernatant was collected for storage at 4 °C. The precipitate of coal particles was dried in a vacuum drying oven at 80 °C after being washed. Approximately 0.2 g of solid coal and 2.0 mL of bacterial


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liquid was added to a sample cell in the C80 microcalorimeter. According to the test requirement, input sample numbers and quality, a heating rate of 0.5 k/s was selected and a constant temperature time of 8 h was used. 2.4.4. Determination of volatile organic compounds. The volatile organic compounds were determined using gas chromatography–mass spectrometry (GC-MS). The peak was identified (qualitative analysis) by the matching degree of organic compounds. The fermentation broth was filtered through a 0.45 μm filter membrane, then to the filtrate was added 50 μL of 0.25% concentrated hydrochloric acid and 6 g NaCl while stirring to achieve full dissolution. Dichloromethane was used as the extraction solvent and the extraction time was 20 min at room temperature. The GC-MS consisted of an Agilent 7890B gas chromatograph and a 5977A mass spectrometer, which were equipped with an FFAP chromatography column (30 m × 0.25 μm × 0.5 mm) (Agilent Technologies, Inc., Santa Clara, CA, USA). The injector temperature was 250 °C, the splitless mode was used, the carrier gas was helium, and the column flow rate was 1.0 mL/min. The temperature program comprised four phases: initially, the temperature was set at 50 °C for 1 min, then increased to 120 °C at a rate of 15 °C/min, then increased to 170 °C at a rate of 5 °C/min, then increased to 240 °C at a rate of 15 °C/min where it was held constant for 3 min. 2.4.5. 16S gene library construction and Illumina® MiSeq sequencing of the microorganisms. The samples (bacterial liquid) were delivered to a commercial laboratory (Sangon Biotech, Co., Ltd. Shanghai, China) for constructing a gene library using the universal Illumina® adaptor and index. The amplicons from each reaction mixture were pooled in equimolar ratios based on their concentration. Before sequencing, the DNA concentration of each polymerase chain reaction product was determined using a Qubit 2.0 Green doublestranded DNA assay and it was quality controlled using a bioanalyzer (Agilent 2100, Agilent Technologies). The sequencing was performed using Illumina® MiSeq system (Illumina Inc.,


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San Diego, CA, USA). 2.4.6. Determination of other analysis index. The contact angle between coal and microorganisms was determined using a JC2000B2 contact angle measurement instrument. The microorganism’s adsorption characteristics on the coal surface were observed using a QuantaTM FEG 250 field emission scanning electron microscope (FEI Co., Hillsboro, OR, USA). The maximum beam was 2 × 10-7 Å, the working distance was 10 mm, the eyepiece amplification was 500,000 times and the maximum pressure of the sample chamber was 4 KPa.

3. RESULTS AND DISCUSSION

3.1. Biogas production characteristics. The results presented in Figure 3 show that CH4 and CO2 were the main gas components in the process of biogas production from coal. There was no gas generation or the gas concentration was too low to be detected in the initial stage of the experiment. The generation of CH4 and CO2 started on day 6, however, the gas production (GP) was relatively low (CH4, 0.062 mL/g; CO2, 0.126 mL/g). The maximum GP of CH4 and CO2 was, respectively, 1.849 mL/g (on day 19) and 0.290 mL/g (on day 18). The GP of CH4 and CO2 decreased rapidly from 19 to 21 days, indicating that fermentation might have entered the stage of CO2 reduction, and the activity of methanogens was inhibited to a certain degree. The GP of CO2 was close to 0.000 mL/g, indicating the end of the experiment.


Energy & Fuels

2.4

CH4 CO2

Gas production/(mLg-1)

2.0 1.6 1.2 0.8 0.4 0.0 0

2

4

6

8

10

12

14

16

18

20

22

Time/d

Figure 3. Changes in gas composition and gas production during biogenic gas production 3.2. Adsorption Characteristics of Microorganisms. OD600 is the light absorbance (at 600 nm) of a microbial community, and can be used to indicate the size of the microbial community to a certain extent15-16. RNA, as the carrier of genetic information and material basis of gene expression, is the foundation and key to studying the metabolism process and regulation mechanism of microorganisms17. In this study, the total amount of microbial adsorption on the coal surface was reflected by OD600 and RNA. The results are presented in Figure 4. 0.13

0

3

6

9

12

15

18

OD600

0.12

21

RNA

24 2000 1800 1600 1400

0.11 0.10

1000 800

0.09

RNA/g

1200

OD600

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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600 0.08

400 200

0.07 0

3

6

9

12

15

18

21

0 24

Time/d

Figure 4. Optical density at 600 nm (OD600) and RNA during biogenic gas production Figure 4 shows that the microorganisms were at the growth period in the early stage of fermentation, and the OD600 was relatively low (0.084). The OD600 reached a maximum of 0.122


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on day 9, and remained at 0.097 from days 12 to 21. The microorganisms grew slowly from days 3 to 6, when the RNA value increased by 5.54% (from 1227.27 μg to 1295.46 μg). From days 6 to 9, various members of the microbial community strongly adapted to the growth environment, and the metabolic stage changed from the adjustment phase to the logarithmic phase. The RNA reached a maximum of 1772.73 μg on day 9. From days 12 to 18, the RNA fluctuated around 1371.22 μg. The final RNA (1409.09 μg) was much larger than the initial RNA. These results show that new microbial biomass was generated as the experiment progressed and combined with original microbial biomass to jointly accomplish the process of methane production from coal.

Figure 5. Microbial adsorption characteristics on the surface of high volatility bituminous coal The morphology of microorganisms on the coal surface is presented in Figure 5, which shows that aerogenic bacteria, composed of multiple microbes, were present in mixed microbial communities. Different kinds of microbial communities were adsorbed on the coal surface or in coal pores, some were dispersed and some were concentrated in one location, and most consisted of different forms of bacillus and cocci. In the early stage of fermentation, many


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microorganisms were adsorbed on the high volatility bituminous coal surface (mainly cocci, Figure 5a). The quantity of cocci decreased later in the reaction, but the cocci remained the dominant bacteria (Figure 5d). These results seem to suggest that the cocci may be the dominant bacteria during the entire gas production period. 3.3. Change Characteristics of Coal Wettability. Coal wettability reflects its contact capacity with coal microorganisms. The smaller is the contact angle, the more sufficient is the contact and the more thorough is a reaction18. In this study, the contact angle reflected the contact conditions between coal and microorganisms at different stages (Figure 6). The contact angle was in the range 32.00°–52.75° (average 48.07°), which indicated good wettability of the coal. At the beginning of the reaction, the active side chains (such as methyl and ethyl) and oxygencontaining functional groups (such as carboxyl and hydroxyl) were degraded first by extracellular enzymes. Consequently, the aromatic carbon and aromatic hydrogen contents increased, and the contact angle between coal and bacterial liquid was at its maximum (52.75°). The contact angle began to decrease with increasing reaction time and reached a minimum of 32° on day 9. This result showed that microorganisms and coal interacted more completely at this time than previously, and microorganisms were better able to attach on the coal surface to produce gases. The corresponding heat production also reached a maximum on day 9. The contact angle increased slightly on days 9 to 12. From days 12 to 21, the quantity of available organic matter decreased significantly as coal was degraded, and the coal structure was mainly aromatic and the appetency of microorganisms for coal decreased18,19-20. Therefore, the wettability of coal with bacterial liquid stabilized.


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60

52.75 52.07

51.38

51.25

12

15

Contact angle/

50

49.00 48.07

40

32.00 30 20 10 0

3

6

9

18

21

Time/d

Figure 6. Changes in coal surface wettability 3.4. Adsorption Heat Characteristics. Many chemical reactions occur during the process of coal degradation by microorganisms, and these are usually accompanied by the generation of heat. The heat output during these reactions reflects the contact degree between coal and microorganisms to a certain extent. The adsorption heat results are presented in Figure 7. 350 300

Adsorption heat/(Jg-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

306.031

289.091

250 200 150 100

40.232 50 0

3

6

9

5.938

8.694

12

15

11.450 22.473 18

21

Time/d

Figure 7. Adsorption heat changes during the anaerobic fermentation process The biological conversion of coal to methane is complex and includes several steps with the involvement of different functional microorganisms21. Microbial community activity and coal pore characteristics play an important controlling role in the process of adsorption heat generation. In the anaerobic coal fermentation experiment, the adsorption heat exhibited a


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varying trend in which the heat initially decreased, then increased, and decreased again before finally increasing, all heat production resulted from exothermic reactions. As shown in Figure 5a, massive numbers of bacteria were adsorbed on the high volatility bituminous coal surface at the initial stage of fermentation. The nutrients were abundant in this stage, and most microbial cells were able to secrete extracellular polymeric substances, as a result, the volume of microbial cells increased and their adsorption onto the coal surface was promoted22. Both changes led to the relatively high (289.091 J/g) heat absorption on day 3, however, this decreased to 13.92% of initial heat production on day 6 (40.232 J/g). With the continuous degradation of coal by microorganisms, the microbial adsorption capacity reached its maximum value on day 9, and the cells reached their optimal physiological condition (Figure 4, Figure 5c). Meanwhile, the affinity of microorganisms to coal was strongest (Figure 6). Consequently, the maximum adsorption heat (306.031 J/g) was reached on day 9, and accounted for 44.75% of the total heat production. The availability of substrate was insufficient after the maximum heat production occurred, and the acidity of the bacterial environment and toxic substances began to accumulate gradually. The physiological state of microbial cells was abnormal at this time, and most of the cells began to lose their capsular and glycolalyx which resulted in the decreased adsorption of microbial cells onto the coal23. The adsorption heat decreased to 2.05% of the initial heat production on day 12 (5.938 J/g). At the end of fermentation, the adsorption heat of the coal-microorganisms system slightly increased again to 22.473 J/g on day 21, which was due to the formation of micropores and transition pores and the associated increase in internal surface area24-25. 3.5. Relationship between Liquid Intermediates and Exothermic Reactions. Organism metabolism includes the metabolism of both material and energy. The organic compounds in coal undergo complex biochemical reactions as a result of microbial action in the process of material catabolism, which is accompanied with the formation of intermediate products. The


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formation and transformation characteristics of microbial anaerobic metabolic intermediates is an important feedback for microbial adsorption status and metabolic heat generation. The GCMS chromatogram of organic intermediates in fermentation liquid of the both experimental group and control group is presented in Figure 8 (see attachment). Tables 2 and 3 lists the organic compounds of experimental and control group corresponding to the peak numbers shown in Figure 8, respectively. Table 2. GC-MS qualitative results of organic intermediates of the experimental group at different gas production stages Number

Formula

Compound

Matching degree

Number

Formula

Compound

Matching degree

1

C8H9

1,3-dimethyl-benzene

95

37

C31H64

Hentriacontane

91

2

C8H9N3

2-ethyl-2H-benzotriazole

83

38

C22H46

Docosane

81 89

3

C13H27I

1-iodo-tridecane

81

39

C18H36O2

6-Tetradecanesulfonic acid, butyl ester

4

C8H8

Cyclooctatetraene

89

40

C7H8O

3-methyl-phenol

86

5

C14H30

Tetradecane

98

41

C16H34O

2-hexyl-1-decanol

82

6

C21H44

Heneicosane

82

42

C31H64

11-decyl-heneicosane

83

Dimethylmalonic acid, 4chlorophenyl octadecyl ester

85

7

C21H44

10-methyl-eicosane

84

43

C29H47O6

8

C4H8O2

Isobutyric acid

87

44

C6H30O5

Cyclohexyl-15-crown-5

85 83

9

C4H8O2

Butanoic acid

82

45

C19H22N2O3

3,6-Bis-dimethylaminomethyl2,7-dihydroxy-fluoren-9-one

10

C5H10O2

2-methyl-butanoic acid

83

46

C3H7NO2

N-methoxy-N-methylformamide

94

11

C2H8N2

1,2-dimethyl-hydrazine

83

47

C24H38O4

Bis(2-ethylhexyl) phthalate

97

12

C6H12O2

4-methyl-pentanoic acid

89

48

C16H34O

2-Hexadecanol

80

13

C14H30O2

Hexaethylene glycol

83

49

C12H25I

1-iodo-dotriacontane

87

14

C37H35O9

2-[2-[2-[2-[2-[2-[2-(2hydroxyethoxy)-ethoxyl] ethoxyl] ethoxyl] ethoxyl] ethoxyl] ethoxyl] ethanol

93

50

C26H54

Hexacosane

91

15

C14H22O

2,4-di-tert-butylphenol

90

51

C22H46

3-methyl-heneicosane

85

16

C22H46O6

Heptaethylene glycol monododecyl ether

82

52

C30H61Br

1-bromo-triacontane

90

17

C10H20O5

15-crown-5

82

53

C15H30O6

18-propyl-18-crown-6

94

18

C12H24O6

18-crown-6

88

54

C6H12O

2-methyl-3-propyl-oxirane

95 83

19

C12H26

20

C16H22O4

Dodecane

91

55

C6H16O8

2,6-Di-O-methyl-dgalactopyranose

Dibutyl phthalate

96

56

C2H7N

Dimethylamine

94

94

57

C69H138O2

Nonahexacontanoic acid

90

90

58

C18H36

1-octadecene

87

21

C28H58O9

22

C4H10N2O2

Octaethylene glycol monododecyl ether N-Methoxymethyl-Nmethylformamide

23

C7H16

2,3-dimethyl-pentane

84

59

C2HF3O2

trifluoroacetate

86

24

C8H10

o-Xylene

83

60

C30H60O2

Octacosyl acetate

95

25

C10H21I

1-iodo-decane

90

61

C30H62

9-octyl-docosane

87

26

C18H35I

1-iodo-octadecane

80

62

C20H42

2,6,10,14-tetramethyl-hexadecane

89


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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27

C12H26

2-methyl-undecane

82

63

C29H60

Z-14-nonacosane

86

28

C2H4O2

Acetic acid

87

64

C4H8O2

Hexacosyl acetate

97

29

C7H6O

Benzaldehyde

95

65

C14H30

4,6-dimethyl-dodecane

84

30

C4H10O

2-methyl-propanol

91

66

C21H44

31

C16H34

Hexadecane

97

67

C32H36Cl6O8

2,6,10,15-tetramethylheptadecane Bis(6-ethyloct-3-yl) ester-oxalic acid

32

C9H18O2

2-methyl-octanoic acid

95

68

C17H36

Heptadecane

98

33

C5H10O2

Pentanoic acid

83

69

C18H38O

2-(octadecyloxy)-ethanol

90

34

C9H10O

3,4-dimethyl-benzaldehyde

86

70

C26H53

1-methylpentacosane

93

35

C5H10O2

4-methyl-butyrate acid

90

71

C8H16O4

12-crown-4

81

C23H32O2

2,2'-methylenebis[6-(1,1dimethylethyl)-4-methyl- Phenol

98

36

C20H24

Eicosane

93

72

87 86

Table 3. GC-MS qualitative results of organic intermediates of the control group at different gas production stages Number

Formula

Compound

Matching degree

Number

Formula

Compound

Matching degree

1

C16H34

4-ethyl-tetradecane

84

31

C16H34

Hexadecane

93

2

C9H20O2Si

Cycloheyldimethoxymethylsilane

93

32

C18H37I

1-iodo-octadecane

91

3

C14H22

1,3-bis(1,1-dimethylethyl)benzene

93

33

C14H22O

3,5-bis(1,1-dimethylethyl)phenol

94

4

C2H4O2

Acetic acid

84

34

C18H34O2

Trans-13-octadecenoic acid

86

5

C21H44

Heneicosane

80

35

C13H38

Tridecane

81

6

C5H10O2

Isovaleric acid

81

36

C19H40

7-hexyl-tridecane

83

Sulfurous acid, 2-propyl tetradecyl ester

86

Undecanoic acid

94

7

C14H22O

8

C23H38O2

2,4-di-tert-butylphenol Cis-7,10,13,16-docosatetraenoic acid, methyl ester 3,5-Di-tert-butyl-4hydroxybenzaldehyde Cis-5,8,11,14,17eicosapentaenoic acid Cis-5,8,11,14,17eicosapentaenoic acid methyl ester

97

37

C27H36O5S

94

38

C11H22O2

96

39

C15H28O2

89

40

C12H36O6Si6

9-teyradecenoic acid, methyl ester Dodecamethylcyclohexasilo xane

99

41

C12H36O4Si5

Dodecamethylpentasiloxane

Tetradecanoic acid

99

42

C6H12O3

C18H36O2

Octadecanedioic acid

97

43

C14H42O5Si6

14

C6H7N

Benzenamine

84

44

C12H36O4Si5

15

C22H42O4

Oxalic acid, bis(6-ethyloct-3-yl) ester

80

45

C20H40O

Phytol

93

16

C4H8O2

Butanoic acid

91

46

C9H8O

3-phenyl-2-propenal

97

17

C6H11O2

3-methyl-pentanoic acid

85

47

C17H34O2

n-hexadecanoic acid methyl ester

97

18

C24H50

Tetracosane

83

48

C19H38O2

Methyl stearate

97

9

C15H22O2

10

C20H30O2

11

C21H32O2

12

C14H28O2

13

2,2-dimethyl-1,3-dioxolane4-methanol Tetradecamethylhexasiloxane 1,1,1,5,5,5-hexamethyl-3,3bis[(trimethylsily)oxy]trisiloxane

86 90 80 83 86 84

19

C18H38

2-methyl-heptadecane

80

49

C19H34O2

20

C18H36

1-octadecene

96

50

C18H30

9,12-octadecadienoic acid, methyl eater Methyl-8,11,14heptadecatrienoate

21

C8H8O2

Benzeneacetic acid

80

51

C10H21I

1-iodo-2-methyl-nonane

84

22

C14H28Br2

1,14-dibromo-tetradecane

82

52

C21H44

10-methyl-eicosane

93

23

C27H46O

Cholesterol

99

53

C12H25Br

2-bromo-dodecane

89

24

C18H37Cl3Si

Octadecyl trihlorosilane

83

54

C23H48

9-hexyl-heptadecane

89

25

C19H40

3-methyl-octadecane

93

55

C27H56

Heptacosane

90

26

C8H20O2Si

Diethyldiethoxysilane

83

56

C36H74

Hexatriacontane

83


99 87

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Energy & Fuels

27

C13H28

2,4,6-trimethyl-decane

84

57

C26H54

9-butyl-docosane

93 94

28

C11H24

Undecane

90

58

C23H32O2

2,2'-methylenebis[6-(1,1dimethylethyl)-4-methyl]Phenol

29

C16H34

6-propyl-tridecane

86

59

C18H34O2

Oleic acid

89

30

C12H25I

1-iodo-2-methyl-undecane

90

60

C10H15N

Benzenebutanamine

83

The results show that the released heat of fermentation mainly derived from the decomposition of protein-like substances and some organic compounds having a simple molecular structure (such as soluble by-products)26. A comparison of Figures 4, 7 and 8 shows that there was some correspondence among microbial adsorption, microbial thermal characteristics and the formation and distribution of organic compounds. 3.5.1. The relationship between heat output and organic compounds. A comparison of Figure 8, Tables 2 and 3 shows that there were 6 common organic compounds in experimental and control group, including acetic acid, butanoic acid, isovaleric acid, heneicosane, hexadecane and other small organic molecules (SOMs). Meanwhile, the quantity of organic compounds of the experimental group was also significantly more than that control group. This indicates that the generation of organic compounds mainly comes from the degradation of coal. Therefore, in this section, the organic compounds of the experimental group were taken as the main analyzing object. On day 3 of heat production, more organic compounds (concentration>1%), such as 1,3-dimethyl-benzene, tetradecane, 2-methyl-butanoic acid and other small organic molecules, were formed in a specific time range (0–16 min), but the kinds of SOMs were relatively less (Figure 8a, Table 2). On day 9 of heat production, mostly 2,3dimethyl-pentane, o-Xylene, 2-methyl-propanol, benzaldehyde and other SOMs were formed, and the quantity of SOM was also significantly more than that on day 3. Furthermore, the kinds and quantity of macromolecular organic compounds (MOCs) were also larger (Figure 8c, Table 2). The MOCs (concentration > 1%), such as 18-propyl-18-crown-6, were formed in a specific time range (16–28 min) from days 6 to 18 (Figures 8b, 8d, 8e and 8f, Table 2). The adsorption heat increased on day 21, and the SOMs, such as 4,6-dimethyl-dodecane and heptadecane, were


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formed once again in a specific time range (0–16 min), and the kinds and quantity of MOCs decreased. These phenomena occurred because the available nutrient supply was ample and abundant microbial species were present (coexistence of hydrolytic-fermentative bacteria and acid-producing bacteria) at the initial stage of fermentation, the biochemical reaction was complex and the MOCs could be thoroughly degraded. Therefore, the high adsorption heat and large amounts of SOMs were formed. The quantity and kinds of SOMs and the heat output were relatively large on day 9 of heat production, and were consistent with each other. Thus, the integrated analysis indicated that the degradation of coal by microorganisms is a process in which material is transformed from macromolecules to small molecules. This means that the quantity and kinds of SOMs increased and the degradation of MOCs was thorough; furthermore, the biochemical reaction was complicated. In the final period of fermentation, the quantity of available organic compounds decreased, the biochemical reaction was relatively simple and degradability of coal decreased, which led to an accumulation of residual MOCs in the bacterial liquid. 3.5.2. The relationship between heat output and bacterial communities. The heat of adsorption measurements showed that metabolic heat generation of microorganisms had four stages (lag phase, logarithmic phase, resting phase and decline phase) that were similar to the microbial growth process27-29. In the experimental system different types of reactions occurred that involved multiple types of microbes, including hydrolytic-fermentative bacteria, acidproducing bacteria and methanogens, among others. The released heat by exothermic reactions occurred because of the interaction among multiple microorganisms. The heat output was concentrated mainly on days 3 and 9. At these times, the lipid, protein, lignin, cellulose and other macromolecule substances were hydrolyzed, the heat release and the number of bacteria was large, and the microbial diversity was obvious (Figure 9). At the later stage of the reaction, the microbial community consisted mainly of methanogens (the abundance of methanogens


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increased from 44.83% on day 15 to 68.23% on day 21), but the overall abundance and diversity of the microbial community decreased. The main reaction consisted of the degradation and utilization of small molecules by methanogenic bacteria, which decreased the heat release. Acid-prodcuting bacteria Hydrolytic-fermentative bacteria

100

Methanogens Other bacteria

90 80

Relative abundance/%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

70 60 50 40 30 20 10 0

0

3

6

9

12

15

18

21

Time/d

Figure 9. Characteristics of different microbial communities during the anaerobic fermentation process 3.6. Preliminary Adsorption Mechanism. The microbial degradation mechanism of important functional groups of coal was explored from the perspectives of physical adsorption. The larger was the contact area of coal and microorganisms and the more thorough was the interaction and degradation of coal by microorganisms, the better was the gas production. There were more side chains and functional groups (carboxyl, hydroxyl and so on) on the coal surface at the beginning of reaction. The contents of oxygen containing functional groups were also relatively large. For one thing, these were humic acid-active organic substances, and the bacteria mainly secreted extracellular enzymes that acted on proteins and polysaccharides. These two organic substances had an antagonistic effect that decreased the adsorption capacity of microorganisms on coal, and the OD600 and RNA also decreased (Figure 4). For another, these functional groups were easily formed hydrogen bonds with water. Hence, the hydrophilicity of coal was relatively strong, but affinity of bacterial liquid to coal was relatively weak (Figure 6). As the anaerobic


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reaction proceeded, the active organic substances in coal began to be degraded, and the OD600 and RNA significantly increased. At the same time, the contents of oxygen containing functional groups were also decreased, the hydrophilicity of coal and the contact angle began to decrease with increasing reaction time, but the affinity of bacterial liquid to coal began to increase. The heat production reached its maximum value on day 9, when the OD600 and RNA also reached their maxima. Meanwhile, the contact angle reached its minimum value, the affinity to coal was the strongest, the contact degree was the smallest, and the heat production reached its maximum of 306.031 J/g. Subsequently, the heat production gradually decreased, the MOCs (such as benzene-ring compounds) began to dominate, the microbial degradation ability decreased, and the overall growth rate of microorganisms also decreased. There was a serious inconsistency among the peak period of gas production, microbial adsorption and heat output. The lag phase was 9 d. The complete inconsistency among gas production, microbial adsorption and adsorption heat generation indicates that a certain lag phase existed between gas production and microbial adsorption. This lag occurred mainly because the methanogens were the main aerogenic bacteria in the process of methane production; these microorganisms can degrade only acetic acid and other SOMs, and their growth period is relatively long. Therefore, the formation of gas products (CH4, CO2, etc.) was delayed. This analysis reasonably explains the inconsistency between gas production and microbial adsorption on coal during the anaerobic fermentation process.

4. CONCLUSIONS The adsorption of microorganisms onto coal is the first step in producing bio-methane using coal as a substrate. The adsorption capacity of the coal-microorganism system was larger as the contact degree improved. There was a positive correlation between adsorption capacity and adsorption heat, and the trends exhibited by the two metrics over time were similar. The larger


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Energy & Fuels

was the adsorption capacity of coal for microorganisms, the more obvious was the diversity of microorganisms. Furthermore, as the diversity of microorganisms increased, the biochemical reaction was more dramatic, the transformation and degradation of macromolecules was more thorough, the heat output was higher, and the quantity of SOMs was larger. At the later stage of the reaction, the adsorption capacity and adsorption heat tended to stabilize, the types of microbial communities diminished and the types of chemical reactions significantly decreased. Minimal attention (only a preliminary assessment) was given to surface adsorption capacity and adsorption heat. Thus, in future studies it is necessary to further examine the interaction force of coal with microorganisms, as well as the potential changes and heat enthalpy changes, to better reveal the interaction mechanism between coal and microorganisms.

ACKNOWLEDGMENTS This study was funded by the National Science Foundation of China (Grant nos. 41472127, 41472129, and 41502158), Program for Scientific and Technological Research Projects of Henan Province (Grant no. 182102310845). Moreover, the authors are grateful to the editor and anonymous reviewers of this paper.

SUPPORTING INFORMATION The Supporting Information shows the concentration changes of organic intermediates of the both experimental group and control group at different gas production stages. The Figure 8a shows the generation characteristics of organic compounds at day 3, the Figure 8b shows the generation characteristics of organic compounds at day 6 and the rest may be deduced by analogy, and the Figure 8g shows the generation characteristics of organic compounds at day 21. The organic compounds of experimental and control group corresponding to the peak numbers shown in Figure 8 were listed in Tables 2 and 3, respectively.

REFERENCES (1) Sun, Y.; Wu, Q. T.; Cui, L. H.; Wei, Z. B.; Tan, M.; Ma, Q.; Liu, P. Studies on the adsorption


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characteristics of Cd on the soil minerals, humic acid, bacteria and their mixture. Ecology and Environmental Sciences 2016, 25(11), 1813−1821. (2) Jiang, D. H.; Huang, Q. Y.; Cai, P.; Rong, X. M.; Chen, W. L. A method for determination of bacteria adsorption by clay minerals. Acta Pedologica Sinica 2007, 44(4), 656−662. (3) Rong, X. M.; Huang, Q. Y.; Chen, W. L.; Liang, W. Interaction mechanisms of soil minerals with microorganisms and their environmental significance. Acta Ecologica Sinica 2008, 28(1), 376−387. (4) Sarrah, D. C.; Bhoopesh, M.; Satish, M.; Jeremy, B. F. The effect of natural organic matter on the adsorption of mercury to bacteria cells. Geochim. Cosmochim. Acta 2015, 150, 1−10. (5) Rong, X. M. Thermodynamic investigations on the interactions of bacteria with soil clay minerals. Ph.D. Thesis, Huazhong agricultural university, January 2008. (6) Jiang, S. F.; Lan, Y. H.; Xia, Y. H.; Lu, Z. J. Thermal metabolic activity and fixation/transformation of Cu2+ of microbes in wetland soils under Cu2+ stress. Journal of Ecology and Rural Environment 2014, 30(3), 358−363. (7) Hu, B.; Ye, Y. M.; Liu, J. M.; Tai, C. The research progress of coal microbial degradation. Genomics and Applied Biology 2017, 36(11), 4733−4738. (8) Gokcay, C. F.; Kolankaya, N.; Dilek, F. B. Microbial solubilization of lignites. Fuel 2001, 80(10), 1421−1433. (9) Orem, W. H.; Voytek, M. A.; Jones, E. J.; Lerch, H. E.; Bates, A. L.; Corum, M. D.; Warwick, P. D.; Clark, A. C. Organic intermediates in the anaerobic biodegradation of coal to methane under laboratory conditions. Org. Geochem. 2010, 41(9), 997−1000. (10) Gao, L.; Brassell, S. C.; Mastalerz, M.; Schimmelmann, A. Microbial degradation of sedimentary organic matter associated with shale gas and coalbed methane in eastern Illinois Basin (Indiana), USA. Int. J. Coal Geol. 2013, 107, 152−164. (11) Furmann, A.; Schimmelmann, A.; Brassell, S. C.; Mastalerz, M.; Picardal, F. Chemical compound classes supporting microbial methanogenesis in coal. Chem. Geol. 2013, 339, 226−241. (12) Wang, A. K.; Qin, Y.; Lan, F. J. Geochemical characteristics and microbial populations of Neogene brown coal from Zhaotong Basin, China. Environmental Earth Sciences 2013, 68(6), 1539−1544. (13) Chen, L. Y.; Wang, B. Y.; Tai, C.; Guan, J. D.; Zhao, H.; Wang, M. .L.; Han, Z. Y. Composition


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and conversion of intermediate products in the process of anthracite gasification by microorganism. Journal of China Coal Society 2016, 41(9), 2305−2311. (14) Su, X. B., Zhao, W. Z., Xia, D. P. The diversity of hydrogen-producing bacteria and methanogens within an in situ coal seam. Biotechnol. Biofuels 2018, 11(1), 245−262. (15) Lin, H.; Yu, X. L.; Dong, Y. B. Culture and acclimation of mixed bacteria for industrial hypersaline wastewater treatment. CIESC Journal 2010, 61(12), 3272−3278. (16) Long, Y.; Liu, R.; Liang, H. Y.; Liu, T. G. High-throughput screening method for high-yield nisin strain. Acta Microbiological Sinica 2018, 58(7), 1298−1308. (17) Yun, H. The simultaneous recovery and separation of nucleic acid from mining environmental microbes. Masterʹs thesis, Central South University, May 2013. (18) Yang, L. J.; Ouyang, Y. L.; Ke, W. L.; You, Y.; Li, Q. H. Research on the impact factors of coal wettability. Coal 2012, 21(8), 4−5. (19) Guo, H. Y.; Luo, Y.; Ma J. Q.; Xia, D. P.; Ji, C. J.; Su, X. B. Analysis of mechanism and permeability enhancing effect via microbial treatment on different-rank coals. Journal of China Coal Society 2014, 39(9), 1886−1891. (20) Xia, D. P.; Guo, H. Y.; Ma, J. Q.; Si, Q.; Su, X. B. Impact of biogenic methane metabolism on pore structure of coals. Natural Gas Geoscience 2014, 25(7), 1097−1102. (21) Bai, Y. Migration characteristics of methanogenic consortia in coal seam. Masterʹs thesis, Henan Polytechnic University, June 2017. (22) Kim, S. B. Numerical analysis of bacteria transport in saturated porous media. Hydrol. Processes 2006, 20(5), 1177−1186. (23) Geng, J. Research on the transport characteristics of bacillus megaterium in soil and phenanthrenedegrading characteristics. Masterʹs thesis, Beijing University of Chemical Technology, April 2013. (24) Zhou, F. B.; Liu, S. Q.; Pang, Y. Q.; Li, J. L.; Xin, H. H. Effects of coal functional groups on adsorption microheat of coal bed methane. Energy Fuels 2015, 29(3), 1550−1557. (25) Lu, S. Q.; Wang, L.; Qin, L. M. Analysis on adsorption capacity and adsorption thermodynamic characteristics of different metamorphic degree coals. Coal Science and Technology 2014, 42(6), 130−135.


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(26) Wang, J. B.; Yang, L. Z.; Zhang, L. Y.; Xi, B. D. Pig manure and straw ferment produce heat collaboratively and transformation of dissolved organic matter. Research of Environmental Sciences 2013, 26(2), 194−201. (27) Boling, E. A.; Blanchard, G. C.; Russell, W. J. Bacterial identification by microcalorimetry. Nature 1973, 241(5390), 472−473. (28) Beezer, A. E.; Newell, R. D.; Tyrrell, H. J. V. Flow microcalorimetric investigation of yeast growth in a complex medium. Microbios 1978, 22(88), 73−84. (29) Xie, C. L.; Tang, H. K.; Song, Z. H.; Qu, S. S.; Liao, Y. T.; Liu, H. S. Microcalorimetric study of bacterial growth. Thermochim. Acta 1988, 123, 33−41.


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Table 1. Proximate and ultimate analysis of coal samples Table 2. GC-MS qualitative results of organic intermediates of experimental group at different gas production stages Table 3. GC-MS qualitative results of organic intermediates of control group at different gas production stages Figure 1. Location of coal samples Figure 2. The biogenic gas production simulation device Figure 3. Changes in gas composition and gas production during biogenic gas production Figure 4. Optical density at 600 nm (OD600) and RNA during biogenic gas production Figure 5. Microbial adsorption characteristics on the surface of high volatility bituminous coal Figure 6. Changes in coal surface wettability Figure 7. Adsorption heat changes during the anaerobic fermentation process Figure 8. GC-MS chromatogram of organic intermediates at different gas production stages Figure 9. Characteristics of different microbial communities during the anaerobic fermentation process


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2 .4

G a s p r o d u c t i o n / ( m L ⋅g - 1 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

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C H 4 C O 2

2 .0 1 .6 1 .2 0 .8 0 .4 0 .0 0

2

4

6

8

1 0

1 2


T im e /d

1 4

1 6

1 8

2 0

2 2

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3

9

Energy & Fuels

6

1 2

1 5

1 8

2 1

2 4

0 .1 3

2 0 0 0

O D 6 0 0

0 .1 2

R N A

1 8 0 0 1 6 0 0 1 4 0 0

0 .1 1

1 2 0 0 0 .1 0

1 0 0 0 8 0 0

0 .0 9

6 0 0 0 .0 8

4 0 0 2 0 0

0 .0 7 0

0 3

6

9

1 2

1 5


T im e /d

1 8

2 1

2 4

R N A / µg

O D 6 0 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

0

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6 0

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C o n ta c t a n g le /°

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Energy & Fuels

5 2 .7 5

5 2 .0 7

5 1 .3 8

5 1 .2 5

5 0

4 9 .0 0

4 8 .0 7

1 8

2 1

4 0

3 2 .0 0 3 0 2 0 1 0 0 3

6

9

1 2


T im e /d

1 5

3 5 0

A d s o r p t i o n h e a t / ( J ⋅g - 1 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

3 0 0

Energy & Fuels

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3 0 6 .0 3 1

2 8 9 .0 9 1

2 5 0 2 0 0 1 5 0 1 0 0

4 0 .2 3 2 5 0

5 .9 3 8 1 2

8 .6 9 4

1 1 .4 5 0

2 2 .4 7 3

1 5

1 8

2 1

0 3

6

9


T im e /d

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R e la tiv e a b u n d a n c e /%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

1 0 0

A c i d - p r o d c u t i n g Energy b a c& Fuels te ria H y d ro ly tic -fe rm e n ta tiv e b a c te ria

M e th a n o g e n s O th e r b a c te ria

9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0

3

6

9

1 2


T im e /d

1 5

1 8

2 1

Adsorption Characteristics and Mechanisms of Coal-Microorganisms

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