A novel solid state fermentation coupled with gas stripping enhancing the sweet sorghum stalk conversion performance for bioethanol
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 12 November 2013
Accepted: 20 March 2014
Published: 8 April 2014
Bioethanol production from biomass is becoming a hot topic internationally. Traditional static solid state fermentation (TS-SSF) for bioethanol production is similar to the traditional method of intermittent operation. The main problems of its large-scale intensive production are the low efficiency of mass and heat transfer and the high ethanol inhibition effect. In order to achieve continuous production and high conversion efficiency, gas stripping solid state fermentation (GS-SSF) for bioethanol production from sweet sorghum stalk (SSS) was systematically investigated in the present study.
TS-SSF and GS-SSF were conducted and evaluated based on different SSS particle thicknesses under identical conditions. The ethanol yield reached 22.7 g/100 g dry SSS during GS-SSF, which was obviously higher than that during TS-SSF. The optimal initial gas stripping time, gas stripping temperature, fermentation time, and particle thickness of GS-SSF were 10 h, 35°C, 28 h, and 0.15 cm, respectively, and the corresponding ethanol stripping efficiency was 77.5%. The ethanol yield apparently increased by 30% with the particle thickness decreasing from 0.4 cm to 0.05 cm during GS-SSF. Meanwhile, the ethanol yield increased by 6% to 10% during GS-SSF compared with that during TS-SSF under the same particle thickness. The results revealed that gas stripping removed the ethanol inhibition effect and improved the mass and heat transfer efficiency, and hence strongly enhanced the solid state fermentation (SSF) performance of SSS. GS-SSF also eliminated the need for separate reactors and further simplified the bioethanol production process from SSS. As a result, a continuous conversion process of SSS and online separation of bioethanol were achieved by GS-SSF.
SSF coupled with gas stripping meet the requirements of high yield and efficient industrial bioethanol production. It should be a novel bioconversion process for bioethanol production from SSS biomass.
KeywordsGas stripping (GS) Solid state fermentation (SSF) Traditional static solid state fermentation (TS-SSF) Sweet sorghum stalk (SSS) Bioethanol Particle thickness Ethanol stripping efficiency Carbon dioxide weight loss
The production of biofuels (largely bioethanol) from biomass has attracted much interest from governments around the world because of its higher octane number and higher heat of vaporization [1–4]. Agriculture straw biomass is the most abundant renewable resource on earth, and the annual yield is approximately 700 million tons in China [5, 6]. Of the many agriculture straws currently being investigated for energy and industry, sweet sorghum stalk (SSS) is considered as a cost-effective feedstock for bioethanol production due to its higher drought resistant ability, lower production costs, and higher biomass yield (20 to 30 dry tons/ha) compared with other straws [7–9]. SSS, which is a C4 plant, can efficiently convert sunlight into stored chemical energy by photosynthetic fixation of atmospheric CO2 to produce sugars . A large proportion of these sugars are stored either as soluble sugars or plant cell wall polymers, and hence SSS contains plentiful soluble carbohydrates (especially glucose, fructose, and sucrose) and insoluble carbohydrates (cellulose and hemicellulose), which can be converted into biofuels by microorganisms [8, 9]. Meanwhile, sweet sorghum juice accounts for a large part of SSS biomass, which not only contains abundant soluble sugars used directly as a substrate for bioethanol production, but also provides efficient nutrient supplementation for microbe fermentation [8, 10]. Therefore, SSS biomass should be the first competitor among the biological energy agriculture crops, and utilization of SSS for bioethanol production should be an effective way to reduce the process capital cost [11–14].
Summary of literature reports on solid state fermentation (SSF) of sweet sorghum stalk (SSS) for bioethanol production
Moisture content (w/w) (%)
Fermentation temperature (°C)
Fermentation time (h)
Sweet sorghum stalk
2.0 cm long, 0.15 cm thickness
22.7 g ethanol/100 g SSS (DM)
Sweet sorghum stem
1 to 2 mm in diameter, 3 to 50 mm in length
S. cerevisiae TSH1
6.25 g ethanol/100 g drystalk
Li et al. 
Sweet sorghum stalk
Issatchenkia orientalis IPE 100
25 g ethanol/100 g dry stalk
Kwon et al. 
Dry sweet sorghum stalk
0.9 to 1.6 mm
35 to 40
Angel active dry yeast
Shen and Liu 
Sweet sorghum stalk
7.9 g ethanol/100 g fresh stalk
Yu et al. 
The present study aims to systematically identify the effects of gas stripping on SSF performance of SSS solid substrates for bioethanol production compared with TS-SSF. The effects of gas stripping on the ethanol yield and the heat and mass transfer efficiency during SSF were analyzed. Ethanol stripping efficiency was considered as the key metric for evaluation of the ethanol online separation efficiency and GS-SSF performance. Fermentation conditions that might affect GS-SSF performance (including initial gas stripping time, gas stripping temperature, fermentation time, and particle thickness) were also investigated in the present study. Meanwhile, the relations of the ethanol content distribution between the gas phase by gas stripping and fermentation of the solid substrate residue were established in GS-SSF. To our knowledge, this is the first systematic study on gas stripping enhancing SSF performance from SSS for bioethanol production without physicochemical pretreatment.
Results and discussion
Composition analysis of sweet sorghum stalk (SSS)
Chemical components of sweet sorghum stalk (SSS) based on total matter (TM) and dry matter (DM)
Determination of initial gas stripping time
Optimization of ethanol fermentation time
The capital cost of biomass conversion for ethanol production is closely related to fermentation time , which should also obviously affect the gas stripping efficiency. Figure 1B and Figure 1C show the fermentation dynamics of TS-SSF and GS-SSF, respectively, for ethanol production from SSS. The results indicated that the ethanol content based on the DM of the solid substrate is a function of fermentation time. Ethanol content rapidly increased and sugar content obviously decreased with the fermentation time from 10 h to 24 h for TS-SSF and from 10 h to 28 h for GS-SSF after inoculation, respectively, but ethanol content then hardly increased for both TS-SSF and GS-SSF with the increase of fermentation time. This was due to the fact that the nutrition was affluent and easily obtained and utilized by yeast at the beginning of fermentation, while the available sugar content in the fermentation of solid substrate residue was low at the late stage of fermentation. The results showed that the fermentation time of the achievement of the highest ethanol content during TS-SSF was less than that of GS-SSF. It was also interesting to note that the sugar consumption rate and ethanol production rate were both lower for TS-SSF compared with that for GS-SSF. However, the ethanol content after 24 h of fermentation exhibited an opposite trend. From the fermentation point of view, the logical approach should be the selection of optimal fermentation time, leading to maximal ethanol yield based on economic analysis. To our knowledge, an increase of fermentation time obviously increases the capital cost of the bioethanol production process and the possibility of contaminative microbes. Meanwhile, the extension of fermentation time also increased the gas stripping operation cost during GS-SSF. High ethanol productivity and short fermentation time are needed, and it should be not a contradiction in ethanol production. The short-term fermentation may improve the ethanol yield due to the avoidance of end-product inhibition and ethanol volatilization and the reduction of contamination risk during SSF [22, 36]. It can also improve the utilization efficiency of equipment, and hence reduce the process capital cost. Thus, the optimal ethanol fermentation time was determined as 28 h during SSF of SSS in the present study.
Effect of temperature on online ethanol separation by gas stripping
Effect of particle thickness on GS-SSF
Among the several parameters, particle size, which is closely related with microbial growth and activity and the fermentation performance, is a critical factor in SSF [38, 40]. It was interesting to note that the ethanol yield apparently increased from 17% to 22% with particle thicknesses from 0.4 cm to 0.05 cm (Figure 3B). The maximum ethanol yield was obtained at the smallest particle thickness within the present particle thickness range. The reason for this phenomenon was that the particle size obviously affects the specific surface area of the solid substrate . The available surface area for microbes increased with the decrease of particle thickness. In other words, the smaller particle thickness would provide a larger surface area for microbial attack. Due to the fact that a sufficient surface area was available for adequate nutrient diffusion and transfer, the growth rate of S. cerevisiae increased, and hence the ethanol production efficiency was improved. However, the energy consumption of size reduction of biomass is higher for small particle thickness than that for large thickness. In addition, too small particle thickness may result in the adhesion and agglomeration of the solid substrate during SSF, which may reduce the solid substrate porosity and the gas diffusion rate and increase the accumulation of heat. The reduced mass and heat transfer efficiency led to the poor growth of microbes, and hence the low product yield [22, 40]. Therefore, it would be necessary to choose a suitable particle size for the biomass conversion process.
The ethanol stripping efficiency obviously decreased from 83.7% to 57.5% with particle thicknesses from 0.4 cm to 0.05 cm (Figure 3B). The maximum ethanol stripping efficiency (83.7%) was obtained at the largest particle thickness (0.4 cm). The possible reason for this result was that the particle thickness obviously affected the packing density of the biomass solid substrate and gas exchange efficiency during SSF [18, 41]. The packing density which determined the solid substrate porosity and the solid substrate bed thickness decreased with the increase of particle thickness. The smallest packing density and the highest porosity of the SSS solid substrate pile were obtained at 0.4 cm thickness. Within the present particle thickness range, the larger particle thickness provided limited surface for microbial attack compared with the smaller thickness, but it provided larger inter-particle space of the solid substrate pile. The larger inter-particle space was helpful for gas diffusion and exchange during gas stripping, and hence improved the ethanol stripping efficiency.
The results implied that the highest ethanol yield was obtained at the smallest particle thickness, but the highest ethanol stripping efficiency was obtained at the largest particle thickness. High ethanol yield was required for the bioconversion process from biomass, while stripping efficiency was a key standard for the downstream process of product separation due to the fact that the high stripping efficiency reduced the capital cost in the industrial process. As a result, the analysis of the ethanol yield and ethanol stripping efficiency combined with energy consumption of size reduction suggested that the 2.0 cm long and 0.15 cm particle thickness was more suitable for the SSS bioconversion process by GS-SSF.
Traditional static solid state fermentation (TS-SSF) versus gas stripping solid state fermentation (GS-SSF)
The ethanol yields during TS-SSF and GS-SSF at different particle thicknesses were compared in the present study (Figure 4B). The results showed that the ethanol yield was higher during GS-SSF than TS-SSF under the same particle thickness, and the corresponding ethanol yield increased by 6% to 10% for GS-SSF compared with TS-SSF. Previous studies reported that the mass and heat transfer was a major bottle neck for SSF, and especially for large-scale production [17, 18, 22]. During SSF, a large amount of heat was generated and the temperature gradient was formed in the solid substrate, which was directly related to the metabolic activities of the microorganisms. The solid substrate biomass used for SSF has low thermal conductivity, and hence the removal of heat from the inner solid substrate residue could be very slow and inefficient [17, 22]. Worse still, accumulation of heat was often fatal during the fermentation process because the increased temperature could affect the growth of microorganisms and the product formation. Meanwhile, the main mass transfer method in the solid substrate is molecular diffusion during TS-SSF, which obviously affects the nutrition transfer efficiency. To remove the accumulated heat and the temperature gradient in the solid substrate and strengthen the fermentation performance, aeration was introduced into the SSF process in previous studies [21, 42]. The application of gas stripping to separate ethanol under a certain temperature removed the heat generated by microbial metabolism through the strengthened gaseous phase during SSF, which improved the mass and heat transfer efficiency. Meanwhile, the gas stripping operation also provided agitation and loosened the solid substrate bed, which was not only helpful for the growth of microbes but also improved the mass and heat transfer efficiency through strengthening the solid phase during GS-SSF.
On the other hand, the ethanol content in SSF was much higher than that in SmF [21, 34, 35]. Thus, the ethanol inhibition effect on yeast becomes significant during SSF. Figure 4C shows that the ethanol content in the solid substrate residue during TS-SSF rapidly increased with fermentation time from 10 h to 28 h, and it then reached about 18% and kept at a high level at the later stage of fermentation. However, the ethanol content in the solid substrate was apparently less than 8% at 10 h and decreased from 8% to 5% with fermentation time from 10 h to 42 h after gas stripping during GS-SSF, which was obviously lower than that during TS-SSF. The results also indicated that the ethanol content per 2 h in the gas phase by gas stripping increased with fermentation time from 10 h to 20 h, and then decreased with the progress of fermentation during GS-SSF. The ethanol gas stripping rate was higher than 1.0% (w/w) per 2 h with the fermentation time from 10 h to 28 h. These results revealed that gas stripping significantly carried off the ethanol from fermentation of the solid substrate residue during GS-SSF, which obviously removed the ethanol inhibition effect. The metabolic activities of yeast were improved, leading to the increase of cell density. As a result, the solid substrate was fully utilized by yeast and the ethanol yield was apparently improved during GS-SSF compared with TS-SSF. Meanwhile, gas stripping may also result in an increase in ethanol productivity due to the reduced by-product formation by yeast metabolism during GS-SSF.
Therefore, gas stripping strongly enhanced the SSF performance due to the removal of the ethanol inhibition effect, strengthening of mass and heat transfer, and simplification of the production process compared with TS-SSF. In the present study, SSF coupled with gas stripping (GS-SSF) was a novel SSS bioconversion process for bioethanol production compared with TS-SSF, especially compared with the traditional SmF process (Figure 5).
Our results showed that gas stripping obviously enhanced GS-SSF performance of SSS compared with TS-SSF. Optimal GS-SSF conditions of initial gas stripping time, gas stripping temperature, fermentation time, and particle thickness were 10 h, 35°C, 28 h, and 0.15 cm, respectively. Gas stripping apparently improved the ethanol yield because the ethanol inhibition effect was removed and the mass and heat transfer efficiency improved. Meanwhile, GS-SSF eliminated the need for separate reactors and further simplified the ethanol production process. Therefore, SSF of SSS coupled with gas stripping would be more suitable to improve fermentation ability, and hence reduce the capital cost of the bioconversion process compared with TS-SSF.
Materials and methods
The conversion process diagram and the novel GS-SSF reactor system
The conversion process diagram of a novel SSF coupled with gas stripping for bioethanol production from SSS at different particle thicknesses is given in Figure 5, compared with traditional bioethanol production by SmF.
The novel GS-SSF reactor system is illustrated in Figure 6. It mainly consists of a fermenter, a gas-stripping tank, two carbon adsorption columns, a condensator, a CO2 gas bottle, and a stepping motor.
Raw material preparation
The SSS used in the present study was harvested from the suburb of Beijing, China. For composition analysis, raw material was air-dried to the moisture content of 5% to 10%, and then milled by knife mill (MQF-420, BJZKRF, Beijing, China). The milled raw material was passed through a screen of 2 mm and stored in sealed bags at 4°C. For SSF, raw materials were first cut into 2.0 cm long pieces by knife mill, and then torn into 0.4 cm, 0.3 cm, 0.15 cm, 0.1 cm, and 0.05 cm thicknesses by tearing chopper (Y-S800, BJZKRF), respectively.
Microorganism and seed culture preparation
The S. cerevisiae used in this study was obtained from Hubei Angel Yeast Co., Ltd (Hubei, China). S. cerevisiae was pre-cultivated in YPD medium (20 g/L glucose, 10 g/L yeast extract, and 20 g/L peptone) at 30°C and 200 rpm for 15 h. The cells were then inoculated to secondary seed liquid culture medium consisting of 20 g/L glucose, 10 g/L yeast extract, and 20 g/L peptone, and were cultivated at 30°C and 200 rpm for 15 h. The initial optical density (OD) at 600 nm for secondary seed was 0.05.
Five different biomass particles were placed in 1.0 L shake flasks capped with rubber stoppers perforated with a syringe needle for gas release. Next, 0.5 g ammonium sulfate/100 g SSS (DM) and 0.5 g calcium chloride/100 g SSS (DM) were added and mixed evenly. The moisture content of SSS was adjusted to 70% by deionized water, and the initial pH was adjusted to 5.0. The mixture was then sterilized at 121°C for 15 min. After sterilization, 0.5 g yeast/100 g SSS (DM) was added into the mixture and mixed well, and the shake flask was then kept statically in the water bath at 35°C. The fermentation conditions of TS-SSF were based on the experimental optimum of our previous studies.
For GS-SSF, 0.5 g ammonium sulfate/100 g SSS (DM) and 0.5 g calcium chloride/100 g SSS (DM) were added into five different biomass particles and mixed well. The moisture content of SSS was adjusted to 70% by deionized water, and the initial pH was adjusted to 5.0. After heat sterilization at 121°C for 15 min, the mixture was cooled and inoculums of 0.5 g yeast/100 g SSS (DM) were added into the mixture and blended well. As shown in Figure 6, the solid substrate mixtures after sterilization and inoculation were top-loaded into the fermenter through air seal machinery. During ethanol fermentation, the solid substrate mixtures were pushed from the fermenter towards the gas-stripping tank at a predetermined speed by stepping motor. When the solid substrate mixtures reached the gas-stripping tank, ethanol was gas-stripped by CO2 with 10 kg/h at different times. The mixtures of ethanol and CO2 went through an activated carbon adsorption column, and ethanol was absorbed. The absorbed ethanol was desorbed by heating the activated carbon adsorption column and recovered in the receptor by the condensator. The remaining CO2 was recycled, and it was saturated by the humidifier before it was injected into the gas-stripping tank again.
Determination of CO2 weight loss
Carbohydrates were converted to CO2 and ethanol. Production of CO2 overflowed from the shake flask caused a decrease in the weight of the fermented substrate, hence the weight of the shake flask reduced. Weight loss of CO2 in fermentation was measured by an accurate balance (0.01 g). It can be measured based on the total weight loss of the shake flask every 2 hours. The CO2 weight loss rate was defined as CO2 weight loss per 2 h.
Analytical methods and calculations
The composition analysis of SSS was conducted according to the Laboratory Analysis Protocol (LAP) of the National Renewable Energy Laboratory (NREL), Golden, CO, USA [43–45]. The moisture content of SSS was determined using oven drying at 105°C for 24 h. The concentrations of sugar and ethanol were analyzed by HPLC (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA), equipped with a refractive index detector and an Aminex HPX-87H carbohydrate analysis column (Bio-Rad, Hercules, CA, USA) at 35°C with 5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min. Sucrose, glucose, fructose, and ethanol standards used in the experiment were analytical grade and were purchased from Sigma-Aldrich (St Louis, MO, USA).
Gas stripping solid state fermentation
High performance liquid chromatography
Laboratory Analysis Protocol
National Renewable Energy Laboratory
Solid state fermentation
Sweet sorghum stalk
Traditional static solid state fermentation
Yeast extract peptone dextrose.
This work was financially supported by the National Basic Research Program of China (973 Project, 2011CB707401), the National High Technology Research and Development Program (863 Program, 2012AA021302), and the Open Funding Project of the National Key Laboratory of Biochemical Engineering (2013KF-01).
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