Enhanced Biodegradation of Sugar Cane Bagasse by Coculture of

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Enhanced biodegradation of sugarcane bagasse by co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense supplemented with CaCO3 Jie Bu, Qing-Qing Tian, and Ming-Jun Zhu Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b01362 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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Enhanced biodegradation of sugarcane bagasse by co-culture of Clostridium thermocellum and Thermoanaerobacterium aotearoense supplemented with CaCO3 †

†

Jie Bu1 , Qing-Qing Tian1 , Ming-Jun Zhu1, 2, * 1

School of Bioscience and Bioengineering, South China University of Technology,

382 East Outer Loop Rd, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of China 2

School of Life and Geographical Sciences, Kashi University, 29 Xueyuan Road,

Kashi 844006, Xinjiang Uygur Autonomous Region, People’s Republic of China

∗

Corresponding author, E-mail address: [email protected]; Tel: +8620 39380623;

Fax: +8620 39380601 †

Jie Bu and Qing-Qing Tian contributed equally to this work and should be considered

co-first authors.

1

ACS Paragon Plus Environment

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Abstract The pretreated sugarcane bagasse (SCB) could be directly degraded by a designed co-culture system with Clostridium thermocellum and Thermoanaerobacterium aotearoense for biological hydrogen and ethanol production, and the production was remarkably improved by CaCO3 supplementation. Here, the effects of CaCO3 concentration (10~100 mM) on the production of hydrogen and ethanol were investigated. Under the optimal CaCO3 concentration of 40 mM, the hydrogen production reached 87.56±4.08 mmol/L from 2% pretreated SCB with a yield of 4.38 mmol H2/g SCB, an 88.62% increase over the culture without added CaCO3 (46.42±1.22mmol/L, 2.32 mmol H2/g SCB). Additionally, the maximum ethanol concentration reached 10.60±0.81 mM, a 192.82% increase over the control (3.62±0.14 mM). The stimulatory effect of CaCO3 on biodegradation of SCB was primarily ascribed to the buffering capacity of CO32-.This study developed a novel strategy to improve SCB biodegradation for biofuel production. Key words: Biohydrogen; Ethanol; Sugarcane bagasse; Co-culture; CaCO3

2


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1. Introduction Increasing concerns about fossil fuel exhaustion and the greenhouse effect have drawn the attention of researchers and global leaders to biofuels such as biological hydrogen and ethanol 1. Hydrogen is a clean and sustainable energy carrier for the future because of its high efficient conversion to usable power, high energy density and environmental friendliness. Recently, researchers have shown much interest in biological hydrogen production from sustainable biomass by microbial fermentation due to the obvious advantages 2,3. One of the reasons for the wide attention on bioethanol from biomass is attributed to its properties as a liquid transportation fuel. However, the production of bioethanol from food crops such as grains (first generation biofuels) has resulted in an undesirable direct competition with food supply, and a switch to a more abundant inedible lignocellulosic biomass should help to reduce pressure on the food crops 4. Sugarcane bagasse (SCB), a kind of lignocellulosic materials, is a byproduct in the sugar production process 5 and the global annual production of SCB is about 100 million tons 6. Due to the advantages such as high holocellulose content (approximately 60%) and low ash content, SCB is widely employed and studied as raw material in the field of bioenergy 7. The structure of lignocellulosic materials is generally complex and firm and severely hinder and slow down the biodegradation by organisms and enzymes. Therefore, applying pretreatment is usually necessary to break down the intrinsic structure of lignocellulose and improve its degradability 8, 9. SCB pretreated with sodium hydroxide has been confirmed to have significant 3


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advantages over raw SCB in substrate biodegradation and hydrogen production 7. Consolidated bioprocessing (CBP) is an efficient and economical method for valuable product production from renewable lignocellulosic materials and could dramatically reduce the production cost 10. The key of CBP is to employ a robust microbial strain or a synergistic microbial community by screening or genetic modification. C. thermocellum is an efficient bacterium in degrading cellulose and therefore taken into consideration as candidate for CBP 11. However, the yield of hydrogen and ethanol production using mono-culture system is low and the process is not economical. To address these issues, a co-culture system with many advantages over the mono-culture system was developed to increase the hydrogen and ethanol yield. Thermoanaerobacterium aotearoense, a strictly anaerobic, thermoacidophilic and H2-producing bacterium, can utilize fermentable sugars to produce hydrogen and ethanol efficiently 12. In our previous study, the co-culture system with pretreated SCB for hydrogen production was comfirmed to be cost-effective and showed a synergetic advantage in hydrogen production over mono-culture of Clostridium thermocellum or Thermoanaerobacterium aotearoense with untreated SCB. There were two main aspects for the cooperation of two organisms in the co-culture system. Firstly, C.thermocellum could degrade cellulose efficiently and provide T.aotearoense with fermentable sugars. Meanwhile, hemicellulases secreted from both two organisms could degrade hemicellulose synergistically, though pentose could not be utilized by C.thermocellum. Besides, the synergistic interaction between xylanase and cellulase 4


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can significantly improve SCB accessibility. Secondly, xylose and xylooligomers are reported as severe cellulase inhibitors in monoculture of C.thermocellum while could be utilized by T.aotearoense as fast carbon source to produce hydrogen. Therefore, the inhibition was weakened or even eliminated in the co-culture system 7. Ethanol and hydrogen could be generated through complex metabolic pathways. In brief, the cellulose and hemicellulose of SCB were degraded by a series of enzymes from two organisms and converted to fermentable sugars such as glucose, cellobiose, xylose and xylooligomers, etc, which could be utilized by organisms and then entered into glycolytic pathway or pentose-phosphate pathway and generated pyruvate. For C. thermocellum, pyruvate could be metabolized into lactate, formate, acetyl-CoA and CO2, respectively. Acetyl-CoA catabolism led to the production of ethanol or acetate, with NADH reoxidation or ATP generation respectively. Besides, hydrogen could be produced from disposal of reducing equivalents 13. For T. aotearoense, the metabolic pathways of sugars were very similar to C. thermocellum, except for the extra pathway from pentose to pyruvate and the absence of pathways from pyruvate to lactate and formate 12. As byproducts, organic acids decreased the pH of fermentation broth, leading to low utilization of SCB and less hydrogen production 7. Therefore, pH-control strategy is essential to improve the fermentation process. CaCO3 supplementation is considered to be feasible when it comes to effectiveness and cost 14,15

. In the present work, the stimulatory effects of CaCO3 on the hydrogen and

ethanol production by co-culture of C. thermocellum and T. aotearoense were 5


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investigated. The effects of Ca2+ and the carbonate-buffering capacity on the production of hydrogen and ethanol, the growth of C. thermocellum and T. aotearoense, and enzymatic activities of the co-culture system were also evaluated. 2. Materials and methods 2.1 Cellulosic substrates Microcrystalline cellulose, Avicel pH-105, was purchased from FMC (Philadelphia, United States). SCB was donated by Guangzhou Sugarcane Industry Research Institute (Guangzhou, China). Alkaline pretreatment of SCB was carried out according to previous work 7. The following pretreatment parameters were used: concentration of sodium hydroxide 3%, liquid-solid ratio 25 ml/g, temperature 80 oC, and time 3 h. The residul solid was washed with distilled water to neutrality and dried at 50 oC for 24 h. Finally, the dried SCB was ground and sieved through a 200-mesh sieve. The component analysis of pretreated SCB was conducted according to the procedures given by the National Renewable Energy Laboratory 16. The pretreated SCB consisted of cellulose (56.76±1.92%), hemicellulose (21.05±1.22%), Klason lignin (8.30±1.31%), acid soluble lignin (2.68±0.08%) and other components such as ash and benzene. 2.2 Microorganism strains, media and inoculum preparation Clostridium thermocellum ATCC 27405 was a gift of Professor Lee R. Lynd (Dartmouth College, United States). Thermoanaerobacterium aotearoense SCUT27 was isolated from a geothermal spring in the south of China, and its lactate dehydrogenase (ldh) gene was successfully disrupted via homologous recombination 6


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using the constructed vector based on pBLUESCRIPT II SK(+) (Genetimes Technology, Inc, Shanghai, China). Both C. thermocellum and T. aotearoense were grown in modified MTC medium 12. The modified MTC medium used in the study was prepared according to our previous work 7.For C. thermocellum and T. aotearoense, 3 g/L Avicel pH-105 and 5 g/L xylose were added as the carbon source of the two organisms, respectively. The pH of the MTC medium was approximately 6.7 12. C. thermocellum was repeatedly transferred as the seed (over 10 generations continuously) for about 72 h at 55°C with rotary shaking at 150 rpm (C24KC refrigerated incubator shaker, Edison, New Jersey, United States). T. aotearoense was cultured in the same condition until OD600 ~ 0.8 (Thermo Fisher Scientific GENESYS 10, Bremen, Germany), and then transferred to a new liquid seed medium and subcultured until OD600 ~1.0. Additionally, both C. thermocellum and T. aotearoense were anaerobically grown in 120 ml serum bottles for a final working volume of 50 ml. All the bottles were sealed with rubber stopper and aluminum seals, and then were purged and gassed three times with 100% nitrogen. 2.3 Batch fermentation of SCB Batch fermentations of SCB were conducted in 120 mL serum bottles to investigate the production of hydrogen and ethanol. In brief, 20 g/L pretreated SCB and specific concentrations of CaCO3 were loaded into serum bottles and working volume of the bottles was 50 mL. Different concentrations (10, 20, 40, 60, and 100 mM) of CaCO3 were supplemented into MTC medium separately and the production 7


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of hydrogen and other metabolites were measured during fermentation. After that, CaCl2 or Na2CO3 was used as alternative to study the effect of Ca2+ and CO32- on the production of hydrogen and other metabolites separately. In brief, different concentrations (2.5, 5, 10, 15 and 20 mM) of CaCl2 or Na2CO3 were supplemented into MTC medium and the production of hydrogen and other metabolites were measured. Before inoculating, 100% nitrogen was purged into crimp-sealed serum bottles for three times and then autoclaved at 115oC for 20 min. The cultures with 3 g/L Avicel pH-105 for C. thermocellum and 5 g/L xylose for T. aotearoense were used as inoculum with a volume of 10% (v/v). The seeds of C. thermocellum and T. aotearoense were injected into the bottles at a ratio of 1:1 (v/v), with a total inoculum size of 10% (v/v) for the co-culture processes. The fermentations were conducted in 10 ml serum tubes with a work volume of 5 mL to investigate the effects of CaCO3 on the growth of C. thermocellum and T. aotearoense and the experiments were performed at 55°C with rotary shaking at 150 rpm for 168 h. Samples were withdrawn every 24 h. A gas chromatograph and high performance liquid chromatography (HPLC) were used to measure the production of hydrogen, ethanol and organic acids. All experiments were performed in triplicate and the values were shown as mean ± standard deviation (SD), and the data were analyzed statistically by one-way analysis of variance (ANOVA) with Duncan’s multiple-range test. SPSS for windows (SPSS Inc. Chicago, version 17.0) were used for statistical analysis and a value of P < 0.05 was considered significant. 2.4 Analysis 8


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2.4.1 Hydrogen analysis Hydrogen production was measured with a gas chromatograph (Fuli 9790, China) equipped with a thermal conductivity detector (TCD) and a flame ionization detector (FID) through a TDX-01 column and an AE electric insulating oil analysis column according to the method described by Li et al. 12. 2.4.2 Analysis of ethanol and organic acids Samples were firstly acidified with 10% (w/w) sulphuric acid, centrifuged at 12000 rpm for 10 min (TGL-16H Centrifuge, HEMA Company, Ltd., Zhuhai, China), and then filtered through a 0.22 µm syringe filter (Millipore, Bedford, MA). A Waters 1525 HPLC (Milford, LA, United States) equipped with a refractive index detector (Waters 2414) was used to measure the organic acids and ethanol in supernatant. A Cation H Cartridge Micro-Guard column (Bio-Rad, Hercules CA, United States) and an Aminex HPX-87H column (300×7.8mm) were used, working at 60°C with 5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min 12. 2.4.3 Analysis of SCB components The solid in the fermentation broth was collected by certrifuging and then washed with distilled water for three times. The precipitates were then dried at 50°C for 72 h. Finally, the two-step acid hydrolysis method described in the National Renewable Energy Laboratory was used to measure the contents of cellulose, hemicellulose and lignin in the dried precipitates or raw SCB 16. 2.4.4 Analysis of biomass of C. thermocellum and T. aotearoense A 7500 Fast Real-Time PCR System (Applied Biosystems, Life Technologies, 9


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Grand Isle, New York, United States) was used to measure the biomass of C. thermocellum and T. aotearoense according to the method described by Tang et al. 18. 2.4.5 Analysis of enzymatic activities For the analysis of enzyme activities, fermentation broth was collected and centrifuged at 3000 rpm for 10 min to remove cells and residual substrate. The supernatant was used to measure the enzyme activities. The enzyme activity of carboxymethyl cellulose (CMC) was determined according to International Union of Pure and Applied Chemistry 19. Xylanase activity was determined as described by Lin et al. 20. Briefly, a mixture of 0.5 mL of enzyme solution and 1.5 mL of substrate in 50 mM citrate acid buffer (pH 4.8, 2% CMC for endoglucanase (CMCase) activity and 1% birchwood xylan for xylanase activity) was used for the analysis. After incubation at 55oC for 30 min, the amount of reducing sugar was determined by 3, 5-dinitrosalicylic acid (DNS) method. One unit of CMCase activity (IU) was defined as the amount of enzyme required to produce 1 mg of reducing sugar per 30 min. One unit of xylanase activity (IU) was defined as the amount of enzyme required to produce 1 µm of xylose per minute. β-glucosidase activity was determined by measuring the release of p-nitrophenol (pNP) from p-nitrophenyl β-D-glucoside (pNPG). Briefly, in a 96 well microtiter plate, 10 µL of 100 mM potassium phosphate buffer (pH 6.0) containing 50 mM pNPG was loaded per well, plus the addition of 90 µL enzyme solution. The microtiter plate was then incubated at 55oC for 15 min. Thereafter, the reaction was stopped by adding 100 µL of chilled 1 M Na2CO3, the amount of pNP was determined from the absorbance measurements at 405 nm. One 10


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unit of β-glucosidase activity (IU) was defined as the amount of enzyme required to produce 1 µmol of pNP per minute. All the assays were performed in triplicate and the mean was reported with standard deviation. 3. Results and discussion 3.1 Effect of CaCO3 concentration on hydrogen production and metabolites A final hydrogen production of 50.05±1.51 mmol/L and a yield of 1.25 mmol H2/g SCB were achieved with 4% pretreated SCB by the developed co-culture process as previously described by Cheng and Zhu 7. However, the results were unsatisfactory for industrial application. CaCO3 supplementation may be a feasible strategy to further improve the production and yield of hydrogen and ethanol due to the report that it could enhance cell growth and solvent production of Clostridium species when adding into medium 14. Therefore, the effects of CaCO3 on hydrogen and ethanol fermentation by co-culture of C. thermocellum and T. aotearoense were also investigated in the present study. As shown in Fig. 1A, the hydrogen production was remarkably promoted (p5 mM CaCl2 was probably toxic to C. thermocellum and T. aotearoense. There was no doubt that Ca2+, as an essential microelement, played an improtant role in initiating and maintaining the fermentation. While the results showed that CaCl2 supplementation did not help promoting the production of hydrogen and ethanol, suggesting that Ca2+ existed in the MTC medium was enough for the cell growth and metabolism in spite of very low concentration (1.36 mM). Therefore, the stimulatory effects of CaCO3 on the production of hydrogen and ethanol may be attributed to its buffering capacity of CO32-. 3.6 Effect of CO32- on hydrogen and ethanol production 17


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With the aim of determining whether the stimulatory effects of CaCO3 on the production of hydrogen and ethanol were exclusively ascribed to the buffering capacity of CO32-, Na2CO3 was used to investigate its effects on the production of hydrogen and ethanol. Carbonate like MgCO3 has been reported to enhance the utilization of sugars by C. acetobutylicum

22

and other carbonates ((NH4)2CO3,

NH4HCO3, K2CO3, NaHCO3, Na2CO3 and CaCO3) could significantly promoted the total ABE production by C. beijerinckii. 15. As shown in Fig.5, the productions of hydrogen and ethanol increased from 54.82±1.52 mM and 9.38±0.36 to 87.72±1.69 mM and 24.78±1.18 mM, respectively, with the Na2CO3 concentration increased from 2.5 mM to 20 mM. The results were comparable with the production of hydrogen and ethanol from the co-culture system with 40 mM CaCO3 supplementation. The production of formate also increased with the increased concentration of Na2CO3. The results indicated that the buffering capacity of CaCO3 exerted significant influence on fermentation in the co-culture system. It was reported that the presence of CaCO3 increased protein synthesis in Clostridium species shock proteins (GrpE and Dnak)

14

and the levels of different heat

15

, stimulated the changes in the electron transport

system in C. sporogenes BE01 and remarkably reduced the polarization resistance 33. CaCl2 and Na2CO3 were tried to study the effect of Ca2+ and CO32- on the production of hydrogen and ethanol separately. However, different from CaCO3, they were high soluble salts, which may increase the osmotic pressure of media and lead to unfavorable effect due to osmotic pressure, which has been well known to exert profound influence on the growth and metabolism of organisms 34. Thus, to solve 18


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these problems and further enhance the production of hydrogen and ethanol in the co-culture system, more appropriate pH-control strategies should be developed in further study. 4. Conclusions The biodegradation of SCB for biofuel production can be remarkably improved by supplementing CaCO3 into MTC medium in the co-culture system. Not only the biological hydrogen but also the ethanol production can be enhanced by CaCO3 supplementation. The stimulatory effects of CaCO3 on the biodegradation of SCB by the co-culture system are mainly attributed to the carbonate-buffering capacity. Applying a feasible pH-control strategy may be an effective approach for improving the biodegradation of SCB, and this study provides some novel insights into the development of such strategies. Acknowledgments The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China [grant nos. 51478190 & 51278200], Guangdong Provincial Natural Science Foundation Key Project [grant no.2014A030311014], and Guangzhou Science and Technology Program [grant no. 201510010288].

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List of tables Table 1 Comparison of H2 production from various lignocellulosic biomasses. Table 2 Comparison of enzymatic activities in the co-culture process of C. thermocellum and T. aotearoense with or without added CaCO3. Table 3 Comparison of substrate degradation in the co-culture process of C. thermocellum and T. aotearoense with or without added CaCO3.

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Energy & Fuels

List of figures Fig.1 Effect of different CaCO3 concentrations on hydrogen production and fermentation products in the co-culture process of C. thermocellum and T. aotearoense. Fig.2 Time courses of hydrogen production and fermentation products under the optimal CaCO3 concentration. Fig.3 Time courses of pH values(A) of fermentation broth and biomass of C. thermocellum(B) and T. aotearoense(C). Fig.4 Effect of different CaCl2 concentrations on hydrogen production and fermentation products in the co-culture process of C. thermocellum and T. aotearoense. Fig.5 Effect of different Na2CO3 concentrations on hydrogen production and fermentation products in the co-culture process of C. thermocellum and T. aotearoense.

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Energy & Fuels

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Figures

Fig.1 Effect of different CaCO3 concentrations on hydrogen production and fermentation products in the co-culture process of C. thermocellum and T. aotearoense. (A) Hydrogen production; (B) Ethanol and acetate production.

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Energy & Fuels

Fig.2 Time-profiles of hydrogen production and fermentation products under the optimal CaCO3 concentration. (A) Hydrogen production with added CaCO3(★) and without added CaCO3(△) ; (B) Fermentation products in fermentation broth. Ethanol (○) and acetate (□) production without added CaCO3, ethanol (●) and acetate(■) production supplemented with 40 mM CaCO3. 27


Energy & Fuels

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Fig.3 Time-profiles of pH values (A) of fermentation broth and biomass of C. thermocellum (B) and T. aotearoense (C). pH values(○) and copy number of C. thermocellum(□) and T. aotearoense(△) without added CaCO3; pH values(●) and copy number of C. thermocellum(■) and T. aotearoense(▲) supplemented with 40 mM CaCO3.

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Energy & Fuels

Fig.4 Effects of CaCl2 concentration on hydrogen production and fermentation products in the co-culture process of C. thermocellum and T. aotearoense. (A) Hydrogen production; (B) Fermentation products in fermentation broth.

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Energy & Fuels

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Fig.5 Effect of different Na2CO3 concentrations on hydrogen production and fermentation products in the co-culture process of C. thermocellum and T. aotearoense. (A) Hydrogen production; (B) Fermentation products in fermentation broth.

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Energy & Fuels

Tables Table 1 Comparison of hydrogen production from different lignocellulosic biomasses. Biomass

Inoculum

Temperature Cultivation method (oC)

Highest H2 yield

Reference

(mL/g dry biomass)

Corn stalk

C. thermocellum

55

Batch

61.4

35

Corn stalk

C. thermocellum and T. thermosaccharolyticum

55

Batch

74.9

3

Corn stalk

C. thermocellum and C. thermosaccharolyticum

55

Batch

105.61

36

Rice straw

T. neapolitana

75

Batch

77.1

37

Wheat straw

C. saccharolyticus

70

Batch

44.68

38

Paper sludge

C. thermocellum

60

Batch

50.18

39

Sugarcane bagasse

C. thermocellum and T. aotearoense

55

Batch

56.06

7

Sugarcane bagasse

C. thermocellum and T. aotearoense

55

Batch

98.11

This study

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Table 2 Comparison of enzymatic activities in the co-culture process of C.thermocellum and T.aotearoense with or without added CaCO3. Culture

CMCase (U/mL)

Xylanase (U/mL)

β-Glucosidase (mU/mL)

Co-culture

0.33 ± 0.01

0.14 ± 0.00

0.91 ± 0.29

Co-culture+CaCO3

0.34 ± 0.00

0.18 ± 0.02

2.86 ± 0.10

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Energy & Fuels

Table 3 Comparison of substrate degradation in the co-culture process of C. thermocellum and T. aotearoense with or without added CaCO3. Culture

Degradation ratio of cellulose/%

Degradation ratio of hemicellulose/%

Total degradation ratio/%

Co-culture

31.47±3.49

56.43±0.03

29.74±0.02

Co-culture+CaCO3

48.97±0.40

64.91±1.45

41.46±0.53

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