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Pang, J., Hao, M., Shi, Y., Li, Y., Zhu, M., Hu, J., Liu, J., Zhang, Q., and Liu, Z. (2018). "Enhancing the ethanol yield from Salix using a Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum co-culture system," BioRes. 13(3), 5377-5393.


A strategic method that utilizes the co-culture of Clostridium thermocellum ATCC 27405 and Thermoanaerobacterium thermosaccharolyticum DSM 571 was developed to improve the ethanol yield from the thermophilic fermentation of Salix. The co-culture conditions of the two strains were optimized using single factor and response surface experiments to enhance the ethanol yield. An even higher ethanol yield was obtained under the optimum co-culture conditions in fermenter tanks than what was observed in pre-experiments in serum bottles. The maximal ethanol concentration and yield were 0.2 g/L and 11.1%, respectively, and with a 26.4% cellulose degradation ratio and 13.8% hemicellulose degradation ratio when the pH was kept stable at 7.0 in fermenter tanks.

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Enhancing the Ethanol Yield from Salix Using a Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum Co-Culture System

Jian Pang,a Min Hao,a Yali Shi,a Yongli Li,a Mingda Zhu,a Jianhua Hu,a Jianguo Liu,b Qiancheng Zhang,a and Zhanying Liu a,*

A strategic method that utilizes the co-culture of Clostridium thermocellum ATCC 27405 and Thermoanaerobacterium thermosaccharolyticum DSM 571 was developed to improve the ethanol yield from the thermophilic fermentation of Salix. The co-culture conditions of the two strains were optimized using single factor and response surface experiments to enhance the ethanol yield. An even higher ethanol yield was obtained under the optimum co-culture conditions in fermenter tanks than what was observed in pre-experiments in serum bottles. The maximal ethanol concentration and yield were 0.2 g/L and 11.1%, respectively, and with a 26.4% cellulose degradation ratio and 13.8% hemicellulose degradation ratio when the pH was kept stable at 7.0 in fermenter tanks.

Keywords: Clostridium thermocellum ATCC 27405; Thermoanaerobacterium thermosaccharolyticum DSM 571; Co-culture; Salix; Fermenter tank; Ethanol

Contact information: a: School of Chemical Engineering, Inner Mongolia University of Technology, Hohhot, Inner Mongolia 010051, China; b: College of Energy and Power Engineering, Inner Mongolia University of Technology, Hohhot, Inner Mongolia 010051, China;

* Corresponding author:


Utilization of biomass as feedstock for production of sustainable transportation fuels is of interest (Yamada et al. 2013), with lignocellulosic biomass being of particular interest in this context (Gupta and Verma 2015; Li et al. 2016). The primary obstacle impeding the widespread production of energy from lignocellulosic biomass is the absence of a low-cost technology for overcoming the recalcitrance of these materials (Dias et al. 2011).

Consolidated bioprocessing (CBP), in which the production of cellulolytic enzymes, biomass hydrolysis, and fermentation of resulting sugars to desired products occur in a single process step, represents a potential technological advance that could lead to lower costs and higher efficiency of cellulosic bio-ethanol production (Olson et al. 2012; Schuster and Chinn 2012; Yamada et al. 2013; Horisawa et al. 2015). CBP-enabling microorganism(s) must be able to effectively solubilize lignocellulosic biomass substrates and produce the desired products at high yields and titers under industrial conditions. C. thermocellum can rapidly solubilize cellulose, and it has often been considered for use in a CBP process configuration. However, C. thermocellum does not utilize the pentoses derived from hemicellulose as carbon source, which means that it cannot utilize 30% to 40% of the total carbohydrates in plant biomass. As a result, improving the utilization of pentoses is a key factor for making C. thermocellum CBP commercially viable.

Co-culture of different types of microorganisms is one strategy to overcome the limitations of particular microbes, and there are some reports about the co-culture of different kinds of microorganisms as ways to improve hydrogen, ethanol, and butanol production (Chou et al. 2011; Cheng and Zhu 2013; Li et al. 2013). However, the results of these studies varied greatly with different microorganisms in co-culture systems. Cheng and Zhu (2013) established a co-culture of C. thermocellum and T. aotearoense for bio-hydrogen production, using sugarcane bagasse (SCB) that was pretreated under mild alkali conditions, demonstrating the synergy and the economic advantages of the co-culture over monocultures of either C. thermocellum or T. aotearoense. In a progress report from Daniel I. C. Wang, Charles L. Cooney and their colleagues, C. thermocellum and C. thermosaccharolyticum were co-cultured to produce ethanol with real biomass, corn stover as substrate. The ethanol productivity was improved when the corn stover was treated with 1% alkali; however the dry weight of the substrate was lost (Wang et al. 1980). Co-cultures of Candida shehataeand Saccharomyces cerevisiae showed ethanol yields (YP/S) of 0.42 and 0.51 in synthetic medium and in rice hull hydrolysate (RHH), respectively, while pure cultures of C. shehatae produced slightly lower ethanol yields of 0.40 (Hickert et al. 2013). C. beijerinckii and C. tyrobutyricum were co-cultured in free-cell, immobilized-cell fermentation, and continuous immobilized-cell mode, which significantly enhanced butanol production, yield, and volumetric productivity (Gupta and Verma 2015). In nature, the rumen microorganism is a typical example of co-culture. Perhaps unsurprisingly, when cellulolytic bacteria and non-cellulolytic rumen bacteria were co-cultured to observe the changes of cellulose degradation, the results differed greatly for different combinations of microorganisms (Fondevila and Dehority 1996; Shi et al. 1997; Chen and Weimer 2001). Fibrobacter succinogenesS85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7 were co-cultured in the presence or absence of the non-cellulolytic ruminal bacteria Selenomonas ruminantium or Streptococcus bovis, and the relative abundance of the three strains as well as the relative yields of fermentation products were studied. S. ruminantium altered the relative proportions of the cellulolytic species. R. albus and R. flavefaciens were found to produce inhibitors that suppressed growth of R. flavefaciens and F. succinogenes, respectively (Chen and Weimer 2001). The interactions between Fibrobacter succinogenesPrevotella ruminicola, and Ruminococcus flavefaciens in the digestion of cellulose from Forages were also studied. When the non-cellulolytic P. ruminicola was co-cultured with either of the two cellulolytic species (F. succinogenes or R. flavefaciens), forage cellulose digestion numerically increased over that of the cellulolytic species alone. However, when F. succinogenes and R. flavefaciens were co-cultured, cellulose digestion was reduced compared to F. succinogenes alone (Fondevila and Dehority 1996). Complementary action between Butyrivibrio fibrisolvens D1 and either of the two F. succinogenes strains (Fibrobacter succinogenes S85 and BL2) was identified with respect to co-culture growth and carbohydrate utilization. With the addition of B. fibrisolvens, the solubilization of cell walls of both untreated wheat straw and sulfur-dioxide-treated wheat straw did not change, whereas the concentration of cells increased when Fibrobacter succinogenes BL2 was used in the co-cultures (Shi et al. 1997). Past research has also reported the co-culture of C. thermocellum and C5 sugar fermenting thermophilic ethanologenic bacteria of the genera Thermoanaero bacterium and Thermoanaerobacter (Argyros et al. 2011). Co-cultures of cellulolytic C. thermocellum with non-cellulolytic Thermoanaerobacterium strains (X514 and 39E) significantly improved ethanol yield by a striking 194% to 440%. Strain X514 enhanced ethanolic fermentation much more effectively than strain 39E in co-cultivation, with ethanol production in X514 co-cultures being at least 62% higher than in 39E co-cultures (He et al. 2011). In a similar study, a co-culture of C. thermocellum and C. thermohydrosulfuricum actively fermented MN300 cellulose, microcrystalline cellulose, Solka floc, SO2-treated wood, and steam-exploded wood, with a threefold increase in the ethanol production rate compared to a monoculture of C. thermocellum (Ng et al. 1981).

Salix as a type of biomass resource plays an important role in breaking wind and fixing sands, purifying air, and conserving water and resources in Inner Mongolia. However, large numbers of deadwood are produced after the process of stumping rejuvenation ever year, which causes environmental pollution and waste of resources. Therefore, new ideas are proposed to reuse Salix based on the regional features. In this study, a co-culture system of C. thermocellum and T. thermosaccharolyticum was established to increase ethanol yield from abundant Salix in Inner Mongolia. The strains’ co-culture conditions were optimized to enhance the ethanol yield using single-factor and response surface experiments. The results were confirmed by scaling up from 100 mL anaerobic bottles to 1 L fermenter tanks.



Untreated Salix psammophila was collected in autumn from the suburbs of Hohhot City, P.R. China, and ground to pass through a 0.425 mm screen. The cellulose and hemicellulose contents of the resulting biomass were 38.4% and 12.2%, respectively.

Microorganisms and media

C. thermocellum and T. thermosaccharolyticum were kindly provided by Lee Lynd, Dartmouth College, USA. Seed cultures of C. thermocellum were grown for 24 h at 55 ºC under constant orbital shaking at 180 rpm. T. thermosaccharolyticum was cultured under the same conditions for 30 h. To ensure the consistency of mono- and co-cultures, both C. thermocellum and T. thermosaccharolyticum were grown in 150 mL serum bottles in modified MTC medium prepared as described by Zhang and Lynd (2003), with the exception of the addition of MOPS. Stock solutions comprising 100 g/L MOPS sodium salt was adjusted to different pH values using 72% H2SO4. Solution A contained the respective carbon sources supplemented with an appropriate amount of distilled water. For seed cultures of C. thermocellum, 5 g/L Avicel PH105 was added to the modified MTC medium as the carbon source. For seed cultures of C. thermocellum, 5 g/L cellobiose (T. thermosaccharolyticum, 2.5 g/L xylose and 2.5 g/L cellobiose ) were used as carbon source (Shao et al. 2009). The initial concentration of Avicel PH105 was 5 g/L, and untreated Salix was used at a concentration of 15 g/L. Solutions A, B, C, D, E, and F were mixed, purged with nitrogen gas, and sterilized, according to a published protocol (Shao et al. 2011).


Growth curve of both bacteria in monocultures

Growth curves of C. thermocellum and T. thermosaccharolyticum were inoculated into seed cultures and the cell concentration were determined based on the absorbing value of 600 nm every 2 h.

Co-culture of C. thermocellum and T. thermosaccharolyticum in serum bottles

The co-culture experiments were executed in 100 mL serum bottles (Cang Zhou Ming Jie Leechdom Co., Ltd., China) with an active volume of 30 mL, and N2-gassed glass containers with screw-top sealable metal lids before being autoclaved at 120 ºC for 21 min. To explore the optimal C. thermocellum fermentation time, T. thermosaccharolyticum inoculation time, and C. thermocellum toT. thermosaccharolyticum inoculation ratio for ethanol yield and the experiments using Salix as carbon source, C. thermocellum and T. thermosaccharolyticum were separately cultured in modified MTC medium without resazurin.

The concentration of Salix was 15 g/L, and a 10% (v/v) inoculum of C. thermocellum was inoculated in the liquid culture medium in both mono- and co-cultures. To optimize the C. thermocellumfermentation time, samples were taken at 96, 120, 144, and 168 h, by withdrawing fermentation liquid from the serum bottle using a syringe while shaking. To optimize the inoculation time of T. thermosaccharolyticum, the bacteria were inoculated at -48, -24, 0, 24, or 48 h of C. thermocelluminoculation. T. thermosaccharolyticum cultures grown for 30 h were used along with C. thermocellumto inoculate the liquid medium, at T. thermosaccharolyticum inoculation ratios of 1:1, 5:1, 10:1, 1:5, and 1:10. The optimization criterion was maximum ethanol yield.

Response surface analysis

Culture conditions were optimized via response surface methodology (RSM) based on Box-Behnken designs (Box and Behnken 1960). The variables containing X1 (T. thermosaccharolyticum inoculation time, h), X2 (C. thermocellum to T. thermosaccharolyticum inoculation ratio) and X3 (C. thermocellumsampling time, h) were carried out to grope the optimum condition via single-factor test. Subsequently, a three-factor two-level analysis of the response surface experiments was designed to study the optimal values of the three factors and the interactions between them according to the Box-Behnken central composite principle. The response surface experimental design of variables is shown in Table 1. In this study, the experimental design contained fifteen trials.

Table 1. The Response Surface Experimental Design of Variables

Note: X1T. thermosaccharolyticum inoculation time (h); X2C. thermocellum to T. thermosaccharolyticum inoculation ratio; X3C. thermocellum sampling time (h)

Fig. 1. The fermenter tanks were used in this study. a: overall configuration; b: detail of the stirred tanks

Co-culture of C. thermocellum and T. thermosaccharolyticum in fermenter tanks

The INFORS HT Multifors 2 Cell bioreactor (Swiss infors (INFORS) biological technology (Beijing Co., Ltd, China) with an active volume of 1 L was used to extend the co-culture system (Fig. 1). A magnetic stirrer was used continuously to ensure sufficient mixing. An initial load comprising 0.56 L of distilled water with 15 g/L of Salix was added to each fermenter tank and autoclaved at 115 ºC for 30 min. The sterilized fermenter tanks were flushed with nitrogen gas for 2 h prior to adding 72 mL of 100 g/L MOPS buffer pH 7.5, 72 mL yeast extract, 32 mL of solution B, and 16 mL of each of the solutions D, E, and F to each one. Finally, 1 mL of corn oil was added as an antifoaming agent. All chemicals used were molecular biology or analytical grade, unless indicated otherwise. Exponential-phase cultures of C. thermocellum and T. thermosaccharolyticum were used to inoculate the fermenter tanks according to the inoculation times, fermentation time and ratios shown in section 2.4. One fermentation experiment was performed with automated addition of 2M NaOH, in order to keep the pH at 7, and another was performed without active pH regulation.


The solid and liquid phases were separated by centrifugation at 3500 × g for 10 min, after which the solid phase was washed three times with distilled water. The remaining sugar titer and the concentrations of the main fermentation products in the supernatant were determined using a Waters HPLC system (#2695, Milford, MA, USA), equipped with a differential refractometer (e2414, Waters USA) and a Bio-Rad HPX-87H column (Hercules, CA, USA), which was operated with 0.01% (v/v) H2SO4 as mobile phase, and kept at 40 ºC. The degradation of cellulose and hemicellulose was measured using quantitative saccharification, as described by Shao et al. (2009, 2011). Briefly, 1 mL of 72% (w/v) H2SO4 was added to a 28 mL aliquot of the supernatant and autoclaved at 121 ºC for 60 min. After filtering through a centrifugal filter cartridge (0.22 um, Shan Yu Technologies Co. Ltd., China), the product concentrations were determined using the Waters HPLC system. Conversions were calculated as percentages of the originally present solubilized glucan and xylan, based on analysis of residual solids. Glucan and xylan solubilization ratios were calculated according to Eqs. 1 and 2. The degradable components of Salix comprise 38.4% glucan and 12.2% xylan. Salix degradation was calculated via glucan solubilization and xylan solubilization using the method of Jian Pang (Pang et al.2017). The ethanol yield from Salix was calculated based on the ethanol concentration in combination with the cellulose and hemicellulose solubilization values,


where Methanol is the amount of ethanol in the fermentation supernatant (g), and Mcellulose consumed is the amounts of Salix consumed in the fermentation broth (g).


In Eq. 2, a is the percentage of glucan in Salix (38.42%), b is the percentage of xylan in Salix(14.86%), c is glucan solubilization, d is xylan solubilization, Ci is the initial concentration of Salix (15 g/ L), V1 is the broth volume after inoculation (L), and V2 is the initial volume of culture medium (L).

Statistical Analysis

All experiments were conducted in triplicate, and the data are presented as mean values ± standard deviation. An analysis of variance (ANOVA) of the obtained results was conducted with SAS 9.0 software (SAS Institute Inc., Cary, NC, USA).


Co-Culture Experiments in Serum Bottles

Previous studies showed that co-fermentation of ethanol-producing bacteria with cellulolytic bacteria can significantly improve ethanol production (Da Cunha-Pereira et al. 2011). In this study, cheap and abundant Salix waste was used to produce ethanol by co-culture of C. thermocellum and T. thermosaccharolyticum. The growth curves of C. thermocellum and T. thermosaccharolyticum in monocultures were shown in Fig. 2 by measuring optical density at 600 nm. From the Fig. 2, the exponential phase of C. thermocellum and T. thermosaccharolyticum was made and microorganisms’ biomass was quantified by determining the OD value at 600 nm.