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Article

Continuous Fermentation Coupled with Online Gas Stripping for Effective Biobutanol Production

by
Zhangnan Lin
1,
Hongjuan Liu
2,
Wei Cong
1 and
Jian’an Zhang
2,*
1
State Key Laboratory of Biochemical Engineering, Key Laboratory of Biopharmaceutical Preparation and Delivery, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
2
Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, China
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(11), 942; https://doi.org/10.3390/fermentation9110942
Submission received: 15 September 2023 / Revised: 13 October 2023 / Accepted: 18 October 2023 / Published: 30 October 2023
(This article belongs to the Special Issue Clean Energy Generation in Microbial Metabolism)
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Abstract

:
The main problems with the butanol fermentation process include high cost of grain raw materials, low product concentration and low butanol productivity caused by butanol cytotoxicity. In this study, cassava, a cheap crop, was used as the raw material. A symbiotic system TSH06, which possesses the capability to synthesize butanol under non-strict anaerobic conditions, was used as the fermentation strain. The fermentation performance of TSH06 in a cassava system was investigated. In order to eliminate product inhibition and promote the concentration and productivity of butanol, a strategy of continuous fermentation coupled with online gas stripping was developed. By using the strategy of two-stage continuous fermentation using immobilized cells coupled with online gas stripping, the butanol productivity reached 0.9 g/(L·h); at the same time, a high butanol concentration was achieved, and the concentration of butanol obtained in the condensate reached 71.2 g/L.

1. Introduction

As an important platform chemical, butanol is widely used in industries such as medicine and chemicals, mainly as a solvent or chemical synthesis raw material. At the same time, butanol, which can be produced by biotechnological processes out of bioproducts, can be used as an eco-friendly and carbon-neutral fuel instead of gasoline or as a fuel additive, and also its disposal is possible through cost-effective biological and non-biological processes. With the highlighting of global carbon emissions and environmental issues, butanol production by fermentation has received widespread attention [1,2,3,4,5].
ABE fermentation can simultaneously produce butanol, acetone, and ethanol, with a ratio of 6:3:1 for butanol, acetone, and ethanol, respectively. Butanol, acetone, and ethanol are all important bulk chemicals and can be used as gasoline additives. The traditional raw materials for acetone–butanol–ethanol (ABE) fermentation by Clostridium species are starch or sugar-based materials, such as corn. Such raw materials are not only expensive but also contribute to the issue of human food supply [6]. Therefore, finding cheap non-food biomass raw materials with high productivity and low cultivation requirements to replace corn has become an urgent challenge for the ABE fermentation industry. Compared to other starch crops, cassava has the potential to become an excellent raw material for fermentation. It has a strong capacity for photosynthesis and biomass synthesis, as well as drought resistance. Cassava can grow in poor soil and does not compete for land with other starch crops such as corn and wheat [7]. At the same time, in China and Southeast Asian countries, cassava is primarily cultivated as a non-food crop, mainly for starch production and feed processing [8]. Therefore, cassava is a promising material for butanol fermentation that can replace corn.
On the other hand, during the fermentation process, butanol exerts a strong toxic effect on microbial cells (in the field of medicine, n-butanol can be used as a topical disinfectant and dissolving agent). When the butanol concentration reaches a certain concentration (approximately 8 g/L), it inhibits the growth of bacteria, disrupts the fermentation process, and ultimately forces the fermentation to stop [9,10,11]. In general, the concentration of butanol in batch fermentation does not exceed 13 g/L, and the total concentration of ABE does not exceed 20 g/L. Low product concentration results in increased energy consumption and higher costs for subsequent separation and purification.
Through fermentation coupled with online separation technology, it is possible to maintain the product concentration below the inhibitory level during the fermentation process, allowing the microbial cells to continuously grow and synthesize butanol. Common techniques for coupling fermentation with online separation in butanol fermentation include extraction, vacuum fermentation, adsorption, pervaporation and gas stripping [12,13]. Among them, fermentation coupled with online gas stripping can produce butanol with simple equipment, and the fermentation process is easy to control [10,14]. This makes it an ideal choice in the fermentation-coupled online separation technology [15]. The method of gas stripping is to introduce gas (usually nitrogen or carbon dioxide) into the fermentation broth; this gas is used to produce bubbles in the fermentation broth, which in turn captures the ABE. The captured ABE is subsequently collected in a condenser. In general, the concentration of butanol in the condensed recovery liquid can reach 70 g/L or higher, and the total concentration of ABE can reach 100 g/L or higher [16].
Continuous fermentation is another method that can solve the issue of product inhibition. This is achieved by continuously introducing fresh medium and allowing it to flow out of the fermentation liquid [17,18]. This process helps dilute the concentration of products in the fermentation liquid, thereby eliminating product inhibition and improving productivity [19,20]. Cell immobilization technology can enhance cell density, fermentation stability, and stress resistance of bacteria, thereby improving the productivity [21]. It is a commonly used method to further enhance continuous fermentation productivity. However, although the traditional single-stage continuous fermentation process can effectively enhance the productivity, the concentration of the final product is often low, which hinders the subsequent separation and purification process.
In the preliminary work, a symbiotic bacterial system named TSH06 was screened, which possesses the capability to synthesize butanol under non-strict anaerobic conditions. TSH06 was found to consist of C. acetobutylicum TSH1 and B. cereus TSH2 through isolation, cultivation, and genetic identification [22]. Its butanol production is comparable to that of traditional strict anaerobic Clostridium species [22]. In this study, cheap non-food-crop cassava was used as the substrate for ABE fermentation. In addition, the gas stripping was coupled with the continuous fermentation process to develop a fermentation strategy that achieves high productivity and high product concentration simultaneously. This fermentation strategy provides a theoretical foundation for the industrial application of cassava butanol.

2. Materials and Methods

2.1. Preparation of Cassava Flour

Cassava (purchased from Guangdong province of China) was cut into small cubes of 1 cm in size and dried at 60 °C to a steady weight. The dried cassava was then ground into powder and set aside for further use.

2.2. Cassava Pretreatment

The cassava medium was stirred and heated to 90~95 °C, then cooled to room temperature and stored for further use.

2.3. Culture Medium

Corn medium: 60 g/L corn meal. The corn medium was heated and boiled for 40 min. The corn medium was then autoclaved at 121 °C for 20 min.
P2 medium: glucose 65 g/L, yeast extract 1 g/L, K2HPO4 0.5 g/L, KH2PO4 0.5 g/L, ammonium acetate 2.2 g/L, and a mineral and vitamin solution. The mineral and vitamin solution contained NaCl 1 g/L, MnSO4·H2O 1 g/L, MgSO4·7H2O 20 g/L, FeSO4·7H2O 1 g/L, thiamin 0.1 g/L, biotin 0.001 g/L, and para-aminobenzoic acid 0.1 g/L. The mineral and vitamin solution was sterilized by filtration using a 0.22-μm membrane filter. Then, the solution was added to the seed medium or fermentation medium at a ratio of 1% (v/v).
Cassava medium: cassava flour 50–80 g/L, yeast extract 2 g/L, K2HPO4 0.5 g/L, KH2PO4 0.5 g/L, ammonium acetate 2.2 g/L and a mineral and vitamin solution.

2.4. Analytical Methods

Biomass: Biomass was measured using the UV spectrophotometric method. The fermentation broth was diluted by a certain factor (5–10 times), and the cell concentration was determined by measuring its absorbance at a wavelength of 600 nm (OD600).
Glucose, maltose, acetic acid, butyric acid, and ABE concentrations: Glucose and maltose were products of starch hydrolysis, while acetic acid and butyric acid were intermediate products in ABE fermentation. High-performance liquid chromatography (Shimadzu, LC-20AT) was used to determine the concentrations of these substances. The samples were diluted by a certain factor (10–50 times), centrifuged for 5 min at a centrifuge speed of 12,000 rpm (13,680× g), and then the supernatant was collected for detection. The centrifugation step was at room temperature. The detecting conditions were set as follows: HPX-87H column, mobile phase of 5 mM sulfuric acid solution, column temperature of 35 °C and a flow rate of 0.6 mL/min. A refractive index detector was used.
Starch concentration: An amount of 1 mL of fermentation broth was mixed with 9 mL of 2 mol/L HCl and boiled at 100 °C for 45 min. The complete hydrolysis of starch was tested with iodine solution. The fully hydrolyzed starch solution was neutralized with 6 mol/L NaOH. The glucose concentration in the solution was measured using a biosensor analyzer, and the measured glucose concentration multiplied by 0.9 was the starch concentration.
Yield: product mass formed per gram of substrate consumed, expressed in g/g.
Productivity: product concentration/fermentation time, expressed in g/(L·h).

2.5. Bacterial Cultivation

Bacterial Activation: The symbiotic system TSH06, preserved in a glycerol tube at −80 °C, was inoculated into 15 mL of corn medium for activation cultivation. The activation was carried out at 37 °C through static incubation for 18–24 h.
Seed Cultivation: The activated bacteria were transferred into P2 medium for seed cultivation, with an inoculation volume of 7% (v/v) TSH06. The seed cultivation was incubated at 37 °C with static incubation for 18–24 h.
Batch Fermentation Cultivation: The seed solution was inoculated in a P2 medium or cassava medium for fermentation cultivation, with a 7% (v/v) TSH06 inoculation volume. The fermentation experiments were carried out at 37 °C under non-strict anaerobic conditions. The pH was not controlled during the fermentation process.
Continuous Fermentation Cultivation: The fermentation volume was 0.5 L. Batch fermentation was performed at the initial stage, and continuous cultivation was started when the concentration of butanol in the fermentation medium reached 8 g/L (approximately 36~48 h). The culture medium in the storage tank was pumped into the fermenter at a certain rate (25–125 mL/h), while the fermentation broth was pumped out at the same rate, ensuring that the volume of the fermenter remained constant. The pH was not controlled during the fermentation process. Figure 1 shows the continuous fermentation device.
Single-stage continuous fermentation coupled with online gas stripping: The fermentation volume was 0.5 L. Batch fermentation was performed at the initial stage, and continuous cultivation was started when the concentration of butanol in the fermentation medium reached 8 g/L (approximately 36~48 h). The culture medium in the storage tank was pumped into the fermenter at a certain rate (50 mL/h), while the fermentation broth was pumped out at the same rate, ensuring that the volume of the fermenter remained constant. After achieving stable continuous fermentation, the gas-stripping device was activated. The gas used for gas stripping was the self-produced gas from bacterial fermentation, primarily consisting of H2 and CO2. When the gas stripping began, the gas produced by fermentation was pumped into the fermentation broth. The gas produced by TSH06 was circulated through the bioreactor and the condenser system using a peristaltic pump. Butanol, ethanol, and acetone were condensed and collected in the condensing recovery bottle. Subsequently, the gas was reintroduced into the fermentation broth. The gas flow was 3 vvm, the condensation temperature was −5 °C, and the condensation recovery bottle was placed in a low-temperature water bath at −5 °C. pH was not controlled during the fermentation process. Figure 2 shows the single-stage continuous fermentation coupled with the gas-stripping device.
Two-stage continuous fermentation coupled with online gas stripping: The fermentation volume in the first stage was 0.25 L, and the fermentation volume in the second stage was 0.5 L. Batch fermentation was performed at the initial stage, and continuous cultivation was started when the concentration of butanol in the fermentation medium reached 8 g/L (approximately 36~48 h). The culture medium in the storage tank was pumped into the first-stage fermenter at a certain rate. The fermentation broth in the first-stage fermenter was pumped into the second-stage fermenter at the same rate, while the fermentation broth was pumped out at the same rate, ensuring that the volume of the fermenter remained constant. After achieving stable continuous fermentation, the gas-stripping device was activated. The gas flow was 3 vvm, and the condensation temperature was kept at −5 °C. Figure 3 shows two-stage continuous fermentation coupled with the gas-stripping device.
During the fermentation processes, samples were taken at regular intervals, with each sample having a volume of 1 mL. All the fermentation processes were carried out without nitrogen gas flushing and oxygen removal.

2.6. Preparation of Immobilized Cell Carriers

The cassava peelings were sterilized and stored for further use. The sterilization condition was 121 °C for 20 min. The cassava peelings, ranging in particle size from 10 mesh to 60 mesh, were added to the fermentation medium. The additive amount ranged from 2.5% (w/v) to 10% (w/v).

2.7. Scanning Electron Microscopy (SEM) Sample Preparation

Cells were fixed using 2.5% (w/w) glutaraldehyde for 2 h. Then, the fixed cells were cleaned 3 to 4 times with 0.1 mol/L PBS buffer (each washing cycle lasted 15 min). After that, the cells were dehydrated using a series of dehydrating agents. The dehydrating agents included 50% (w/w) ethanol, 70% (w/w) ethanol, 80% (w/w) ethanol, an ethanol–ethyl acetate mixture with a volume ratio of 2:1, and an ethanol–ethyl acetate mixture with a volume ratio of 1:1. Each dehydration step lasted for 30 min. Finally, the cells were vacuum freeze-dried, ion sputter-coated with gold, and observed under vacuum conditions using SEM.

3. Results and Discussion

3.1. Effect of Cassava Concentrations on Butanol Synthesis by TSH06

In the previous study, it was found that TSH06 has the ability to directly produce butanol from cassava flour, without the pretreatment enzymatic hydrolysis or gelatinization [8]. Considering that the concentration of substrate can affect the fermentation performance, in this work, different concentrations of cassava media were used for butanol fermentation to observe the butanol synthesis by TSH06. The fermentation system had a working volume of 60 mL, and the cassava concentrations were 50 g/L, 60 g/L, 70 g/L, and 80 g/L, respectively. The fermentation was conducted for 72 h, and the fermentation results are shown in Table 1, with glucose media fermentation results used as a control.
Table 1 shows that as the initial cassava flour concentration increased from 50 g/L to 70 g/L, the butanol concentration gradually increased. The butanol concentration increased from 8.5 g/L to 11.8 g/L, and the total ABE concentration increased from 12.9 g/L to 17.8 g/L. However, when the initial concentration of cassava flour was increased from 70 g/L to 80 g/L, there was no further increase in the butanol concentration or the total ABE concentration. No significant difference was observed in butanol yield among the various initial concentrations of cassava flour, with butanol yields ranging from 0.22 g/g to 0.24 g/g. In terms of substrate consumption, when the initial concentrations of cassava flour were 50 g/L and 60 g/L, almost all of the starch and glucose were consumed after 72 h of fermentation. When the initial concentration of cassava flour was 70 g/L, there was no residual starch in the fermentation broth at the end of the fermentation. However, a small amount of glucose remained at a concentration of 4.8 g/L. When the initial concentration of cassava flour was 80 g/L, at the end of the fermentation, the starch concentration was found to be 5.5 g/L, while the glucose concentration was measured at 8.0 g/L. In the glucose media, the butanol concentration was 11.7 g/L, the total ABE concentration was 18.3 g/L, the butanol yield was 0.22 g/g, and the productivity was 0.16 g/(L·h).
The results demonstrated that TSH06 could utilize cassava as a substrate for ABE fermentation and could hydrolyze the starch present in cassava media. When the initial concentration of cassava flour ranged from 50 g/L to 80 g/L, with starch concentrations between 38.0 g/L and 61.6 g/L, complete hydrolysis yields glucose concentrations ranging from 42.2 g/L to 68.4 g/L. Considering the butanol yield and substrate consumption rate, when the concentration of cassava flour was 70 g/L, which corresponds to a starch concentration of 54.2 g/L, it achieved both a high butanol concentration (11.8 g/L) and a high substrate utilization rate (94.3%). The concentration, yield, and productivity were comparable to those achieved with glucose media. When the initial concentration of cassava flour exceeded 70 g/L, the production of butanol no longer increased. Therefore, under batch fermentation conditions, the cassava medium with an initial concentration of 70 g/L was considered optimal.

3.2. Effect of Dilution Rates on Continuous Fermentation

In contrast to batch fermentation, continuous fermentation can effectively enhance the production efficiency and equipment utilization rate. The dilution rate is an important factor in continuous fermentation. In this study, the effect of dilution rate (0.05 h−1, 0.10 h−1, 0.15 h−1, 0.20 h−1, and 0.25 h−1) on the continuous fermentation was investigated. The fermentation system had a volume of 0.5 L, and the initial concentration of glucose was 60 g/L. The results are shown in Figure 4.
According to Figure 4, the product concentrations decreased gradually with the increase in dilution rate. At a dilution rate of 0.05 h−1, the concentration of butanol in the fermentation broth was 9.1 g/L, and the concentration of total ABE was 13.6 g/L. However, when the dilution rate was increased to 0.25 h−1, the concentration of butanol decreased to 3.6 g/L, and the concentration of total ethanol–acetone–butanol was 5.5 g/L.
In terms of substrate utilization, as the dilution rate increased, the utilization rate of substrate in the fermentation broth decreased. At a dilution rate of 0.05 h−1, the concentration of glucose in the effluent broth was 18.6 g/L, and the substrate utilization rate was 69.2%. When the dilution rate was increased to 0.25 h−1, the glucose concentration in the effluent broth was 43.5 g/L, and the substrate utilization rate was only 28.0%. These results indicate that both the concentration of the product and the utilization rate of substrate were negatively correlated with the dilution rate. In other words, as the dilution rate increased, the product concentration and substrate utilization rate decreased.
In terms of butanol productivity, when the dilution rate was below 0.20 h−1, the productivity increased as the dilution rate increased. The maximum productivity reached 1.04 g/(L·h) at a dilution rate of 0.20 h−1. When the dilution rate was further increased to 0.25 h−1, the butanol productivity decreased to 0.90 g/(L·h). Nevertheless, at various dilution rates, the butanol yield remained relatively stable at approximately 0.24 g/g.
The dilution rate plays a crucial role in continuous fermentation processes. In continuous fermentation, an increase in the dilution rate results in a shorter residence time of the medium in the fermenter. This leads to less contact time between cells and nutrients, resulting in lower substrate utilization rates. Conversely, a lower dilution rate means a longer residence time of the medium in the fermenter, allowing cells to have more contact time with nutrients, which results in higher substrate utilization rates [23].
Tashiro et al. conducted a study to examine the fermentation capabilities of C. saccharoperbutylacetonicum N1–4 in a continuous fermentation setting. The result showed that when the dilution rate was set at 0.1 h−1, the concentration of butanol was measured to be 6.5 g/L, with a corresponding butanol productivity of 0.72 g/(L·h). When the dilution rate was elevated to 0.2 h−1, the productivity of butanol increased to 1.24 g/(L·h) [24]. Andrade et al. utilized C. acetobutylicum ATCC 824 as the fermentation strain and performed continuous fermentation with glucose and lactate as substrates. They reported that when the dilution rate was 0.05 h−1, the concentration of butanol was 8.6 g/L, and the productivity was 0.42 g/(L·h) [25]. Zheng et al. utilized xylose as the substrate for continuous fermentation with C. saccharoperbutylacetonicum N1–4. They observed that as the dilution rate increased from 0.14 h−1 to 0.26 h−1, the butanol productivity increased from 0.24 g/(L·h) to 0.53 g/(L·h) [26].
In this study, the maximum productivity of butanol was observed to be 1.04 g/(L·h) when the dilution rate was set at 0.2 h−1. The overall productivity of ABE reached 1.58 g/(L·h). At this dilution rate, the concentration of butanol was measured to be 5.2 g/L, while the concentration of total ABE was found to be 7.9 g/L.

3.3. Effect of Cassava Residue Particle Size and Additive Amount on Fermentation Performance of Immobilized Cells

To enhance the fermentation performance of cassava for butanol production, cell immobilization was utilized for ABE fermentation. From the perspective of fully utilizing cassava raw materials, cassava flour was employed as a substrate for cell growth and fermentation, while cassava peel residue was employed as a carrier for cell immobilization. The impact of particle size and additive amount of cassava peel residue on fermentation performance was investigated.
Figure 5 compares the effects of different particle sizes of cassava peel residue (10 mesh-60 mesh) on ABE fermentation, with a 5% (w/v) additive amount of cassava peel residue. The results of ABE fermentation with free cells were used as a control.
Figure 5 shows that when free cells were used for fermentation, the butanol concentration was 11.8 g/L, and the total ABE concentration was 17.9 g/L. When cassava peel residue with particle sizes of 20 mesh, 40 mesh, and 60 mesh was used as immobilization material, there was no significant difference in butanol production. The butanol production exceeded 13 g/L, and the total ABE concentration was approximately 20 g/L. Compared to free cells, the butanol production increased by 11.0%, and the total ABE production increased by 10.5%. From a product concentration perspective, cassava peel residue with particle sizes of 20 mesh, 40 mesh, and 60 mesh could all be used as immobilization materials for TSH06. Considering the cassava peel residue preparation process, the preparation process for 20-mesh cassava peel residue was relatively simple. Therefore, 20-mesh cassava peel residue was selected as the immobilization material.
The additive amount of immobilization material is another crucial factor in the process of cell immobilization fermentation. Figure 6 compares the effects of different additive amounts of cassava peel residue (2.5% (w/v), 3.3% (w/v), 5% (w/v), 10% (w/v)) on ABE production. The particle size used was 20 mesh. The results of ABE fermentation with free cells were used as control.
After adding cassava peel residue, the production of butanol showed improvement. When the additive amount of cassava peel residue was 2.5% (w/v), the butanol concentration reached 12.5 g/L, and the total ABE concentration amounted to 18.9 g/L. When the additive amount was increased from 2.5% (w/v) to 3.3% (w/v), the butanol concentration further increased from 12.5 g/L to 13.2 g/L. However, when the additive amount was increased from 3.3% (w/v) to 10% (w/v), there was no further increase in butanol production or total ABE production. When the additive amount of cassava peel residue was 3.3% (w/v), compared to free cells, the butanol concentration increased by 11.9%, and the total ABE concentration increased by 10.5%. Based on these results, an additive amount of 3.3% (w/v) of cassava peel residue was the optimal choice for the fermentation process of TSH06.
Figure 7 shows cassava peel residue at different magnifications. The surface of cassava skin residue is rough and porous, which is favorable for bacteria to attach to the surface and grow [27]. Figure 8 shows the SEM images of immobilized cells. A large number of bacteria adhered to the surface of cassava peel residue after 24 h of fermentation. The bacteria primarily existed in the form of rod-shaped cells, and butanol was being synthesized rapidly at this point, indicating the high vitality of the cells. At 48 h, there were still a large number of cells attached to the surface of cassava peel residue.
The above results indicated that using cassava peel residue as a cell immobilization material could enhance butanol production. The butanol concentration increased by 11.9%. However, this was not because cassava peel residue brought in more starch or increased the substrate concentration. There were two reasons for this: (1) The results in Section 3.1 showed that, under batch fermentation conditions, even when the concentration of cassava flour was increased to 80 g/L, the butanol concentration did not exceed 11.8 g/L. (2) No starch was detected on the surface of the cassava peel residue used in this experiment.
Adsorption immobilization is a technique that utilizes the electrostatic attraction between the charged surface of cells and the carrier, allowing the cells to be adsorbed onto the carrier [28]. The adsorption of microorganisms onto the carrier is primarily influenced by van der Waals forces and electrostatic interactions, which include ionic-type hydrogen bonding, between the cell surface and the carrier surface [28]. Cell immobilization technology can effectively increase the cell density in the fermentation broth, enhance fermentation stability, and improve the tolerance of cells to butanol, thereby promoting butanol productivity [29]. The immobilization correlation between the growth rate and dilution rate is split, i.e., even poorly biodegradable compounds can be transformed without wash out effects [30].
Some research has shown that cell immobilization could promote butanol production mainly because of the formation of biofilm on the surface of cell clusters [31]. Biofilm was mainly composed of cells and extracellular matrix. In most cases, the content of extracellular matrix in biofilms could reach over 90% (v/v) [32]. The main components of the extracellular matrix include polysaccharides, proteins, lipids, and nucleic acids. The components of the extracellular matrix were closely related to the formation, protection mechanism, nutrition, metabolites, and signal transmission of biofilms [33,34]. According to Zhuang et al., in the fermentation process, the biofilm formed by cell immobilization could promote the production efficiency and increase the tolerance of cells to butanol. After 24 h of fermentation, 3 g/L butanol was added to both the free cell system and the immobilized cell system. At the end of fermentation, the butanol concentration in the free cell system was 4.8 g/L, whereas the concentration of butanol in the immobilized cell system was 8.6 g/L [35].
The microbial composition of TSH06 and the changes in microbial abundance during the fermentation process were analyzed in previous studies, along with potential interactions among different species. TSH06 was composed of Bacillus cereus and Clostridium acetobutylicum, with the latter being capable of synthesizing butanol while the former cannot [36].During the initial stage of fermentation, Bacillus cereus briefly grew, which led to a rapid decrease in dissolved oxygen in the fermentation broth. Once the dissolved oxygen dropped below 5%, the biomass of Clostridium acetobutylicum gradually increased, becoming the dominant strain. After 12 h of fermentation, over 99% of the bacteria in the fermentation broth were Clostridium acetobutylicum [22]. In this study, the immobilization process promoted butanol production, indicating that it primarily enhanced the growth and fermentation of Clostridium acetobutylicum.

3.4. Single-Stage Continuous Fermentation Coupled with Online Gas Stripping for Butanol Production

Although the results of continuous fermentation showed that higher productivity could be achieved, the product concentration and substrate utilization were low. With a dilution rate of 0.2 h−1, the concentration of butanol in the fermentation solution was only 5.2 g/L, and the substrate utilization was no more than 50%. The aim of this work was to achieve a high product concentration and enhance substrate utilization, while also maintaining a high productivity. The continuous fermentation process using immobilized cells was coupled with gas stripping for ABE fermentation. A dilution rate of 0.1 h−1 was adopted in this experiment (in order to maintain a relative high butanol concentration and improve the efficiency of online separation). The fermentation system had a volume of 0.5 L, and the results are shown in Figure 9.
In the preliminary work, it was found that the tolerance concentration of TSH06 to butanol was 8 g/L. When the concentration of butanol exceeded 8 g/L, the growth and butanol synthesis of TSH06 were significantly inhibited. Therefore, in the process of fermentation coupled with gas stripping, the concentration of butanol should be controlled so as not to exceed 8 g/L [10]. The first 36 h of fermentation was conducted as a batch fermentation. When the butanol concentration exceeded 8 g/L, continuous fermentation was initiated at a dilution rate of 0.1 h−1. After initiating continuous fermentation, the product concentration exhibited a significant decrease, indicating that the bacteria were in a transitional period from batch fermentation to continuous fermentation. In the previous study, it was found that continuous fermentation reached a steady state within 24–36 h of initiation. Therefore, the gas-stripping device was activated after 36 h of continuous fermentation [8]. After 36 h of continuous fermentation, the concentration of butanol was 8.8 g/L. Gas stripping was then initiated with a recycle gas velocity of 3 vvm. As gas stripping began, a portion of the ABE products was collected in the condensation device, resulting in a significant decrease in butanol concentration in the fermentation broth and a slight increase in productivity, from 0.88 g/(L·h) to 0.90 g/(L·h). From 96 to 144 h, the concentration of butanol remained relatively stable, ranging from 5.5 to 6.3 g/L. After 144 h, the concentration of butanol gradually decreased, while the starch concentration increased. Additionally, the butanol productivity rapidly declined from its peak value of 0.90 g/(L·h) to 0.31 g/(L·h). These observations indicate a significant decrease in the fermentation performance of the cells.
In the single-stage continuous fermentation coupled with online gas stripping, after gas stripping, the concentration of butanol and ABE in the fermentation broth ranged from 6.3–3.1 g/L and 9.2–5.1 g/L, respectively. In this process, the average productivity of butanol was 0.66 g/(L·h), which was more than three times higher than that of batch fermentation. However, the main issue with this system is that it cannot operate steadily for a long period of time. The immobilization system was disrupted due to the gases pumped into the fermentation broth. This hindered proper cell adsorption onto the carrier and impeded normal fermentation. Within 200 h, a noticeable decline in the fermentation performance of the cells was observed, indicating that the immobilization system was not compatible with the gas stripping process in the same fermenter. Therefore, it is necessary to further improve this system.

3.5. Two-Stage Continuous Fermentation Coupled with Online Gas Stripping for Butanol Production

In order to prevent the interference to the immobilized system caused by the gas injected into the fermentation broth, a two-stage system was employed for continuous fermentation. The first stage fermenter utilized immobilized cells and had a fermentation volume of 0.25 L, operating at a dilution rate of 0.2 h−1. The second stage fermenter employed free cells and had a fermentation volume of 0.5 L, with a dilution rate of 0.1 h−1. The second-stage fermenter was equipped with online gas stripping, which used the gases produced during fermentation (primarily H2 and CO2) for gas stripping through recycling. The results are shown in Table 2.
According to the results in Table 2, in the two-stage system coupled with online gas stripping, the butanol concentration in the condensation collector reached 71.2 g/L, and the total ABE solvent concentration reached 97.6 g/L. The butanol productivity was 0.9 g/(L·h) with a yield of 0.24 g/g, and the total solvent productivity was found to be 1.33 g/(L·h) with a yield of 0.35 g/g. The utilization of starch was nearly complete, as demonstrated by the 99.3% substrate utilization rate.
Figure 10 shows the fermentation profile. In the first-stage fermenter, continuous fermentation began at 36 h, with the butanol concentration reaching 9.3 g/L and the total ABE concentration reaching 15.2 g/L. After continuous fermentation began, the butanol concentration in the first-stage fermenter remained between 5.1 and 6.8 g/L, while the total ABE concentration remained between 8.3 and 9.5 g/L. In the second-stage fermenter, simultaneous continuous fermentation and online separation were carried out to avoid product inhibition. Between 46 and 96 h, the starch concentration declined from 19.4 g/L to 0 g/L, indicating a rapid increase in substrate utilization. After the system of continuous fermentation coupled with gas stripping entered the steady state (96 h), the average substrate utilization rate was 99.3%; only a small amount of glucose was detected in the fermentation broth, and all starch was consumed. The concentration of butanol in the second-stage fermenter remained between 4.8 and 6.1 g/L, while the total solvent concentration remained between 7.1 and 9.6 g/L. The butanol concentration and total ABE concentration in the condensate were 71.2 g/L and 97.6 g/L, respectively.
Table 3 compares recent studies on multistage continuous fermentation. In recent years, most studies on multistage continuous fermentation for butanol production used glucose as the substrate, while studies using cassava as the fermentation substrate were relatively limited. Compared to glucose, the utilization of cassava as substrate decreased production cost.
From the perspective of product generation, by utilizing continuous fermentation coupled with online gas stripping, the productivity is nearly six times that of the traditional method. This means that the initial construction scale of the fermentation plant can be reduced to one-sixth the size while maintaining the same production capacity. From the perspective of product recovery, it required 36 MJ/kg of butanol for conventional distillation from a solution containing ~10 g/L (w/v) butanol, which was equal to the energy content of butanol itself [16]. For gas stripping, the energy consumption was estimated to be 31 MJ/kg-butanol by Oudshoorn’s study [42] while 29.63 MJ/kg-butanol by Rochón’s study [43]. This energy consumption is estimated based on a butanol concentration of 4–5 g/L in the fermentation broth, which was similar to the concentration of butanol in this study. Although the energy consumption of gas stripping is lower compared to traditional methods (reduced by about 15%), further improvements are still needed to make the process more cost-effective.
In this study, during the continuous fermentation using immobilized cells, the butanol productivity reached 1.26 g/(L·h), but the concentration was only 6.3 g/L. When combined with continuous fermentation and online gas stripping, the butanol productivity was 0.9 g/(L·h), and the concentration of butanol obtained in the condensate reached 71.2 g/L. The results showed that both high productivity and high product concentration could be achieved by this strategy.

4. Conclusions

In this study, we investigated the characteristics of butanol production through cassava fermentation using the symbiotic system TSH06. In addition, a continuous fermentation process coupled with online gas stripping was adopted to enhance productivity and achieve a high concentration of the product. In batch fermentation, the butanol concentration was 11.6 g/L, the butanol yield was 0.24 g/g, and the productivity was 0.16 g/(L·h). In the two-stage continuous fermentation coupled with online gas-stripping system, both high productivity and high product concentration were obtained. The butanol productivity reached 0.9 g/(L·h), the total solvent productivity reached 1.33 g/(L·h), the substrate utilization rate reached 99.3%, and the concentration of butanol and total solvents in the condensate collection solution reached 71.2 g/L and 97.6 g/L, respectively.

Author Contributions

Z.L., H.L. and J.Z. participated in designing the study and Z.L. carried out the experiments as well as drafted the manuscript. W.C. commented on the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 22008246).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available herein.

Acknowledgments

Thanks to Tsinghua University for providing the symbiotic system TSH06. This work was supported by the National Nature Science Foundation of China (No. 22008246).

Conflicts of Interest

The authors declare that they have no competing interests.

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Fermentation 09 00942 g001
Figure 1. Continuous fermentation device.
Figure 1. Continuous fermentation device.
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Fermentation 09 00942 g002
Figure 2. Single-stage continuous fermentation coupled with gas-stripping device.
Figure 2. Single-stage continuous fermentation coupled with gas-stripping device.
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Fermentation 09 00942 g003
Figure 3. Two-stage continuous fermentation coupled with gas-stripping device.
Figure 3. Two-stage continuous fermentation coupled with gas-stripping device.
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Fermentation 09 00942 g004
Figure 4. Effect of dilution rates on continuous fermentation (a): solvent concentration and substrate utilization; (b): butanol productivity and yield.
Figure 4. Effect of dilution rates on continuous fermentation (a): solvent concentration and substrate utilization; (b): butanol productivity and yield.
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Fermentation 09 00942 g005
Figure 5. Effect of different particle sizes of cassava peel residue on ABE concentration.
Figure 5. Effect of different particle sizes of cassava peel residue on ABE concentration.
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Figure 6. Effect of additive amount of cassava peel residue on ABE concentration.
Figure 6. Effect of additive amount of cassava peel residue on ABE concentration.
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Figure 7. Cassava peel residue with different magnifications (A): cassava peel residue; (B): 1000× magnification of cassava peel residue; (C): 2000× magnification of cassava peel residue.
Figure 7. Cassava peel residue with different magnifications (A): cassava peel residue; (B): 1000× magnification of cassava peel residue; (C): 2000× magnification of cassava peel residue.
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Fermentation 09 00942 g008
Figure 8. Microscopic morphology of immobilized cells (a): immobilized cells at 24 h of fermentation; (b): immobilized cells at 48 h of fermentation.
Figure 8. Microscopic morphology of immobilized cells (a): immobilized cells at 24 h of fermentation; (b): immobilized cells at 48 h of fermentation.
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Fermentation 09 00942 g009
Figure 9. Single-stage continuous fermentation coupled online gas stripping. (A) Product concentration and productivity. (B) Substrate concentration.
Figure 9. Single-stage continuous fermentation coupled online gas stripping. (A) Product concentration and productivity. (B) Substrate concentration.
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Figure 10. Two-stage continuous fermentation coupled online gas stripping. (A) Solvent concentration and sugar concentration in fermenter 1 of the two-stage system. (B) Solvent concentration and sugar concentration in fermenter 2 of the two-stage system.
Figure 10. Two-stage continuous fermentation coupled online gas stripping. (A) Solvent concentration and sugar concentration in fermenter 1 of the two-stage system. (B) Solvent concentration and sugar concentration in fermenter 2 of the two-stage system.
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Table 1. Effect of cassava concentrations on butanol synthesis.
Table 1. Effect of cassava concentrations on butanol synthesis.
Fermentation ParameterCassava (g/L)Glucose (g/L)
8070605065
Initial starch concentration (g/L)61.6 ± 1.154.2 ± 0.945.7 ± 1.238.0 ± 1.00
Initial glucose concentration (g/L)000065.3 ± 1.1
Final starch concentration
(g/L)		5.5 ± 1.90000
Final maltose concentration
(g/L)		0.6 ± 0.10.4 ± 0.1000
Final glucose concentration (g/L)8.0 ± 1.24.8 ± 0.90.1 ± 0.10.1 ± 0.111.3 ± 1.4
Butanol(g/L)11.6 ± 0.411.8 ± 0.310.5 ± 0.28.5 ± 0.211.7 ± 0.2
Acetone (g/L)4.3 ± 0.24.4 ± 0.14.0 ± 0.43.2 ± 0.34.9 ± 0.2
Ethanol (g/L)1.6 ± 0.11.6 ± 0.21.4 ± 0.21.2 ± 0.21.7 ± 0.1
Total solvent (g/L)17.5 ± 0.817.8 ± 0.315.9 ± 0.812.9 ± 0.718.3 ± 0.4
Total solvent yield (g/g)0.35 a0.36 a0.35 a0.35 a0.33 b
Butanol yield
(g/g)		0.24 c0.24 c0.23 c0.22 c0.22 d
Butanol productivity
(g/(L·h))		0.160.160.140.120.16
Total solvent
productivity (g/(L·h))		0.240.250.210.180.25
a: Yield of total solvent to starch; b: Yield of total solvent to glucose; c: Yield of butanol to starch; d: Yield of butanol to glucose.
Table 2. Two-stage continuous fermentation coupled with online gas stripping.
Table 2. Two-stage continuous fermentation coupled with online gas stripping.
ParameterButanolABE
Solvent concentration in fermentation broth (g/L)4.8~6.17.1~9.6
Solvent concentration in the condensate collector (g/L)71.297.6
Productivity (g/(L·h))0.91.33
Yield (g/g starch)0.240.35
Substrate utilization %99.3%
Table 3. Multistage continuous fermentation for butanol production.
Table 3. Multistage continuous fermentation for butanol production.
Fermentation StageSubstrateDilution RateStrategyButanol Productivity (g/(L·h))Abe
Productivity (g/(L·h))		Butanol Concentration
(g/L)		Abe
Concentration (g/L)		StrainReference
Two-stageGlucoseD1 = 0.075;
D2 = 0.06		Free cell0.4--5.93--C. acetobutylicum ATCC 824[37]
Two-stageGlucoseD1 = 0.55;
D2 = 0.18		Immobilized cell; Pervaporation1.24--9.3--C. beijerinckii B592[38]
Two-stageGlucoseD1 = 0.25;
D2 = 0.025		Butyric acid addition;
Gas stripping		0.4--4--C. saccharoperbutylacetonicum N1–4 [39]
Two-stageGlucoseD1 = 0.1;
D2 = 0.1		Free cell0.380.589.514.6C. acetobutylicum ATCC 824 [40]
Two-stageGlucoseD1 = 0.1
D2 = 0.04		Stage 1:
continuous
Stage 2:
repeated
fed-batch		0.620.921219.5C. acetobutylicum ATCC 824 [40]
Three-stageCorn stalkD1 = 0.12;
D2 = 0.12
D3 = 0.12		Immobilized cell0.450.6~0.912.419.9C. acetobutylicum ABE 1201 [41]
Two-stageCassavaD1 = 0.2;
D2 = 0.1		Immobilized cell; Gas stripping0.91.3371.297.6TSH06This study
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Lin, Z.; Liu, H.; Cong, W.; Zhang, J. Continuous Fermentation Coupled with Online Gas Stripping for Effective Biobutanol Production. Fermentation 2023, 9, 942. https://doi.org/10.3390/fermentation9110942

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Lin Z, Liu H, Cong W, Zhang J. Continuous Fermentation Coupled with Online Gas Stripping for Effective Biobutanol Production. Fermentation. 2023; 9(11):942. https://doi.org/10.3390/fermentation9110942

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Lin, Zhangnan, Hongjuan Liu, Wei Cong, and Jian’an Zhang. 2023. "Continuous Fermentation Coupled with Online Gas Stripping for Effective Biobutanol Production" Fermentation 9, no. 11: 942. https://doi.org/10.3390/fermentation9110942

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