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Article

Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors

by
Thanawat Thanapornsin
,
Lakkana Laopaiboon
and
Pattana Laopaiboon
*
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
*
Author to whom correspondence should be addressed.
Energies 2024, 17(5), 1009; https://doi.org/10.3390/en17051009
Submission received: 12 January 2024 / Revised: 11 February 2024 / Accepted: 18 February 2024 / Published: 21 February 2024
(This article belongs to the Section A4: Bio-Energy)
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Abstract

:
This research aims to study butanol fermentation from sweet sorghum stem juice (SSJ) by immobilized Clostridium beijerinckii TISTR 1461 cells on bamboo chopsticks using Arthrobacter sp. as an efficient bacterium for creating anaerobic conditions in scaled-up bioreactors. For batch culture in a 1-L screw-capped bottle, a butanol concentration (PB), butanol productivity (QB), and butanol yield (YB/S) were 12.09 g/L, 0.26 g/L·h and 0.28 g/g, respectively. These values were ~8 to 14% higher than those of a single culture using oxygen-free nitrogen (OFN) gas to generate anaerobic conditions. When butanol fermentation by the co-culture was scaled-up to 5-L and 30-L stirred-tank fermenters, the butanol production efficiency was not different from that using the 1-L bottles. Additionally, repeated-batch butanol fermentation in the 1-L bottles by the co-culture was successfully operated for four successive cycles with high butanol production. All results clearly indicate that Arthrobacter sp. is promising for creation of anaerobic conditions for butanol production by immobilized Clostridium in large scale bioreactors.

1. Introduction

Industrialization is leading to increased worldwide energy consumption. Non-renewable energy resources such as fossil fuels are extensively applied to increase living standards and development. However, conventional energy resources, i.e., fossil fuels, cause significant environmental issues, including climate change and carbon dioxide emissions [1]. Developing renewable biofuels is critical to replacing conventional energy resources. Ethanol is currently the most common renewable liquid biofuel. However, it has drawbacks such as its lower energy content compared to gasoline, instability in gasoline blends, promotion of corrosion, and inadequate infrastructure for its transport. Another fuel, butanol, is a renewable liquid biofuel with a lower vapor pressure, less flammability, better safety during transport, and more stability when used in internal combustion engines than ethanol [2]. Butanol has a high energy density of 29.2 MJ/L and can be completely substituted for gasoline (energy density 32 MJ/L) without alteration to current internal combustion engines [3]. Additionally, it is an important chemical precursor for paints, polymers, synthetic rubber, brake fluids and plastics [4,5,6].
Biobutanol can be generated via an acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia, e.g., Clostridium beijerinckii, in two phases. The first is an acidogenesis phase that is coupled with bacterial growth. In this phase, Clostridium produces acetic and butyric acids as well as gasses (CO2 and H2). Thereafter, the acids will be converted into acetone, butanol and ethanol in a solventogenesis phase [6]. ABE fermentations must be operated under strictly anaerobic conditions. Oxygen-free nitrogen (OFN) is normally used to generate anaerobic conditions for laboratory scale fermentations [7,8]. Nevertheless, large-scale bioreactors are not practical owing to their high operating costs and complexity. Therefore, the use of obligate aerobic microorganisms to consume residual oxygen is an alternative approach to create anaerobic conditions for ABE fermentations. Arthrobacter is an obligate aerobic microorganism that metabolizes sugars coupled with oxygen consumption under suitable environments [9,10]. Our previous research revealed that Arthrobacter sp. is a promising bacterium for creating anaerobic conditions in butanol fermentations using the free cells of C. beijerinckii TISTR 1461 in a small-scale laboratory reactor [10].
Substrate costs are high for butanol production in industrial ABE fermentations. Sweet sorghum is a non-competitive and multipurpose crop. It is a short-cycle plant with a high degree of tolerance to biotic and abiotic stresses, which can achieve high biomass yields [11]. The juice from its stalks is rich in fermentable sugars (glucose, fructose and sucrose) and contains many trace elements essential for yeast growth and ethanol production, therefore it does not require pretreatment before use. Moreover, it can be grown at almost all temperatures, requiring less water and is more tolerant of increased soil salinity, alkalinity and drought than sugarcane [5,10,12]. Hence, sweet sorghum, a potential energy crop, is an alternative feedstock for future biofuel production.
Generally, batch fermentation is used for ABE fermentations. Nonetheless, batch fermentations have weaknesses such as the need for a new starter or inoculum at process initiation and its high downtime for cleaning between batches, sterilization and setup for the next batch fermentation. Consequently, repeated-batch fermentation is considered superior to the conventional batch fermentation because it addresses some of these weaknesses. Additionally, repeated-batch fermentations require less time, reuse microbial cells in subsequent fermentation runs and have high cell concentrations during fermentation [13,14].
Cell immobilization efficiently enhances fermentation using the robustness of clostridia to increase butanol production [15,16]. Several materials have been studied for cell immobilization in ABE fermentations such as zeolite [17], corn stalks [18], activated carbon [19], sugarcane bagasse [14,20] and sweet sorghum bagasse [21]. In the current study, bamboo chopsticks, a waste material available in huge quantities in Thailand [22], were selected to be carriers for cell immobilization [23].
This research aims to investigate batch and repeated-batch butanol fermentations from sweet sorghum stem juice (SSJ) by co-culture of immobilized C. beijerinckii TISTR 1461 cells on bamboo chopstick pieces and Arthrobacter sp. (for anaerobic condition creation). This is the first report of butanol production by co-culture in scaled-up bioreactors. The relationship between the morphology of C. beijerinckii cells and butanol production during repeated-batch fermentation, which has never been examined, was also investigated.

2. Materials and Methods

2.1. Microorganisms and Inoculum Preparation

A spore suspension of C. beijerinckii TISTR 1461 (from the Thailand Institute of Scientific and Technological Research (TISTR), Khlong Luang, Pathum Thani, Thailand) was activated by heat shock [23]. The activated spores were grown in cooked meat medium (CMM) at 37 °C for 12 h. Then, 5% (v/v) of vegetative cells in CMM was transferred into tryptone-glucose-yeast extract (TGY) medium and incubated at 37 °C for 4–6 h to obtain inocula for butanol production [23]. Sterile CMM and TGY medium were purged with oxygen free nitrogen (OFN) gas to create strictly anaerobic condition before use.
One loopful of Arthrobacter sp. BCC 72131 (from the Thailand Bioresource Research Center (TBRC), Klong Luang, Pathum Thani, Thailand) was grown in sterile nutrient broth (NB) in a shaking incubator at 30 °C with agitation at 200 rpm for 6 h and used as an inoculum [10].

2.2. Raw Materials

Stalks of sweet sorghum cv. KKU 40 (Khon Kaen University, Thailand) were crushed for juice extraction. Sweet sorghum stem juice (SSJ, ~18 °Bx of total soluble solids) was concentrated to ~68 °Bx, then kept at −20 °C. The main components of the SSJ were previously reported [10]. The concentrated juice was diluted to 60 g/L of total sugars and supplemented with urea (KemAus, Cherrybrook, Australia) (0.64 g/L) and buffers (K2HPO4, 0.5 g/L; KH2PO4, 0.5 g/L and ammonium acetate, 2.2 g/L [23]. The pH of the sterile medium was adjusted to 6.5. For a control treatment, the medium was purged with OFN gas to create anaerobic conditions before use for butanol production.

2.3. Carriers

The carriers were sterile bamboo chopsticks (Siam Makro Public Co., Ltd., Khon Kaen, Thailand) having a size of 0.5 cm (diameter.) × 1.9 cm [23].

2.4. Fermentation Procedures

2.4.1. Batch Butanol Fermentation by Co-Culture of Arthrobacter sp. and Immobilized C. beijerinckii Cells in Screw-Capped Bottles

Sterile carriers were added into 1-L screw-capped bottles containing 750 mL of SSJ medium (Figure 1A). The carrier size was 0.5 mm × 1.9 cm with carrier loading of 1:32 (w/v) [23]. Arthrobacter sp. (5%, v/v) was transferred into the SSJ medium and incubated for 6 h to create anaerobic conditions without agitation [10]. Afterwards, Clostridium cells (5%, v/v) were inoculated into the medium. The butanol fermentation was performed as a batch process for 60 h at 37 °C and agitated at 150 rpm using a magnetic bar.

2.4.2. Batch Butanol Fermentation by Co-Culture of Arthrobacter sp. and Immobilized C. beijerinckii Cells in Scaled-Up Bioreactors

Batch fermentations were carried out in a 5-L stirred-tank bioreactor or an STR (Biostat B, B Braun, Melsungen, Germany) with 3.5-L of the SSJ medium (Figure 1B) and in a 30-L STR (Biostat C, B Braun, Melsungen, Germany) with 20-L of the SSJ medium as a pilot-scale bioreactor (Figure 1C). Mixing and agitation in both bioreactors were performed using a six-blade disc turbine and baffles. The immobilization and fermentation conditions in the scaled-up bioreactors were performed as previously described (Section 2.4.1). The inoculum size of Arthrobacter sp. and Clostridium cells were 5% (v/v), and the fermentation was carried out in triplicate for 60 h at 37 °C and agitated at 150 rpm.

2.4.3. Repeated-Batch Butanol Fermentation by Co-Culture of Arthrobacter sp. and Immobilized C. beijerinckii Cells

Repeated-batch butanol fermentation by co-culture of Arthrobacter sp. and immobilized Clostridium cells was carried out in screw-capped bottles with working volumes of 750 mL at 37 °C and agitated at 150 rpm. First, anaerobic conditions were created in the SSJ medium by Arthrobacter sp. (5%, v/v) as previously described in Section 2.4.1. Immobilization and fermentation were simultaneously initiated by inoculating the active cells of C. beijerinckii (5%, v/v) in the SSJ medium containing the carriers to start Cycle 1. The Cycle 1 fermentation was operated for 24 h before forespores formed [24]. At this time, all fermentation broth was transferred into new screw-capped bottles, and the fermentation was continued by free cells that remained in the fermentation broth. Simultaneously, fresh anaerobic SSJ medium (created by Arthrobactor sp.) was added into the first screw-capped bottles to begin Cycle 2 with immobilized cells. After operating Cycle 2 for 24 h, the fermentation broth was transferred to new screw-capped bottles. The fermentation continued, as was performed in Cycle 1. Then, fresh anaerobic SSJ medium was added into the first screw-capped bottles containing immobilized cells to start the next cycle. The repeated-batch fermentation was continued until the butanol concentration was reduced to 50% that of Cycle 1.

2.5. Analytical Methods

The samples were centrifuged at 7400× g for 10 min. The pH, total sugars, and free amino nitrogen (FAN) of the supernatant were determined.
The pH values were measured using a pH meter. The total sugars (TS) content was determined using a phenol-sulfuric acid method [25]. Free amino nitrogen (FAN) concentration was measured using a ninhydrin assay [26]. Organic (acetic and butyric) acid and organic solvent (acetone, butanol and ethanol) levels were determined using a gas chromatograph [24]. Cell morphology was observed under light microscopy. Butanol yield (YB/S, g/g) and volumetric butanol productivity (QB, g/L·h) were calculated [24]. The structures of bamboo chopsticks and bacterial morphology on the carriers were observed during repeated-batch fermentation using field emission scanning electron microscopy (FE-SEM) (MIRA, TESCAN, Brno, Czech Republic).

2.6. Statistical Analyses

Data are presented as the mean ± standard deviation (SD) from three independent samples. All results were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) at a 95% confidence level. Statistical significance was determined at p < 0.05.

3. Results and Discussion

3.1. Batch Butanol Fermentation by Co-Culture of Arthrobacter sp. and Immobilized C. beijerinckii Cells in Screw-Capped Bottles

Figure 2A shows profiles of the butanol fermentation by co-culture of Arthrobacter sp. (to create anaerobic conditions) and immobilized C. beijerinckii (butanol producer) on bamboo chopsticks in a 1-L screw-capped bottle. Six hours after Arthrobacter inoculation, about 3 g/L of sugar was consumed, with no pH change. The pH value declined dramatically after inoculation with C. beijerinckii, corresponding to the production of acetic and butyric acids. These results indicated an acidogenesis phase during this period. Bacterial cells grew well due to ATP generation from acid production. After 12 h of fermentation, pH slightly increased and high levels of solvents, especially butanol were detected, suggesting that solventogenesis was active during this period. Under these conditions, ~75% of the total sugars (TS) were consumed, with 12.09 g/L of butanol (PB) and 18.14 g/L of ABE (PABE) produced (Table 1). A butanol yield (YB/S) and its productivity (QB) were 0.28 g/g and 0.25 g/L·h, respectively. Batch butanol fermentation by a single culture of immobilized Clostridium on bamboo chopsticks using OFN gas to create anaerobic conditions was also performed in a 1-L screw-capped bottle as a control treatment. The fermentation profiles were similar to those of the co-culture (Figure 2B). Under the control treatment, PB, 10.62 g/L; PABE, 15.23 g/L; QB, 0.22 g/L·h and YB/S, 0.26 g/g values were obtained.
The results indicate that Arthrobacter sp. can consume oxygen in a broth to create an anaerobic environment for butanol production by C. beijerinckii. Interestingly, the PB and PABE of the co-culture were ~14 to 19% higher than those of the single culture (control treatment). During butanol fermentation, cell debris of Arthrobacter sp. was observed under light microscopy. FAN may have been released from the Arthrobacter sp. cell debris, promoting butanol production by C. beijerinckii. To test this hypothesis, the FAN concentration in the broth during fermentation was measured (Figure 3). These results showed that FAN concentrations under a single culture and co-culture were not different at the beginning of butanol fermentation, implying that the SSJ medium composition and environmental parameters of both conditions were similar. After Arthrobacter inoculation, the FAN concentration decreased, suggesting that the bacterium can grow and consume oxygen to create anaerobic conditions in the broth. Unfortunately, the bacterial growth (OD at 600 nm) was not measured in this study due to particulate interference in the SSJ medium. After Clostridium inoculation, FAN in the broth was continuously consumed and butanol production was detected (Figure 2A). After 48 h of fermentation, the FAN concentration slightly increased. This implied that C. beijerinckii did not consume FAN released from Arthrobacter sp., and butanol fermentation ceased. In the single culture fermentation, the FAN concentration continuously decreased and remained unchanged after 48 h (Figure 3), implying that consumed FAN was derived from the SSJ medium, not from cell debris. These findings suggest that the Arthrobacter sp. is capable of creating anaerobic conditions for immobilized Clostridium to produce butanol.

3.2. Batch Butanol Fermentation by Co-Culture in Scaled-Up Bioreactors

Five and 30-L STRs were used to evaluate the feasibility of butanol production by immobilized C. beijerinckii cells employing Arthrobacter sp. to create anaerobic conditions in scaled-up bioreactors. The butanol fermentation profiles in the 5-L and 30-L STRs were similar to those in screw-capped bottles, suggesting that mixing and agitation involving heat and mass transfer to produce butanol were similar. Butanol production efficiency in the scaled-up bioreactors is summarized in Table 1. The PB, PABE, QB and SC values were in the ranges of 11.92–12.22 g/L, 17.80–18.21 g/L, 0.25–0.26 g/L·h and 73.80–74.73%, respectively. These values were not significantly different among the various sized bioreactors, indicating that those using Arthrobacter sp. to create an anaerobic environment and C. beijerinckii as a butanol producer could operate in large-scale bioreactors, at least up to 30-L in size. Sugar was not completely consumed in any of the conditions tested. This may have been due to butanol toxicity [5,23]. Furthermore, the results clearly show that YB/S values were not significantly different in any of the bioreactors tested, suggesting that use of scaled-up bioreactors did not affect the pathway of ABE fermentation by immobilized Clostridium cells on bamboo chopsticks under anaerobic conditions created by Arthrobacter sp. in SSJ medium. Hence, these systems have potential for application in larger scale bioreactors.
Geometric similarity (size and shape) is critical for scaling up bioreactor projects in industries, although achieving it completely is often challenging in practice [27,28]. Typically, during the scale-up of bioreactors, the working volume of the fermentation broth should constitute 70–80% of the bioreactor volume [29]. In this study, the working volumes in the 1-L, 5-L and 30-L bioreactors were slightly different with the values of 75, 70 and 67%, respectively, but they still align closely with recommended working volumes. However, low working volumes, approximately 30% of the total reactor volume (1-L in 3-L and 70-L in 200-L bioreactors), were reported in a scale-up of a bioreactor for successful 2,3-butanediol production [28].

3.3. Repeated-Batch Butanol Fermentation in Co-Culture

Repeated-batch fermentation was carried out to evaluate its capability of butanol production. It was found that the ABE fermentation could only be operated for four cycles (Table 2). Surprisingly, butanol fermentation efficiencies in terms of PB, PABE and QB in Cycle 2 were higher than those of Cycle 1. This might have been due to a more appropriate environment for butanol fermentation. This hypothesis is supported by morphological observations of bacterial cells in this system. In Cycle 2, PB (12.12 g/L), PABE (17.45 g/L), YB/S (0.28 g/g) and QB (0.25 g/L·h) were relatively high (Table 2). Nonetheless, PB, PABE, SC and QB were reduced by ~39–48% in Cycle 3 and by ~65–67% in Cycle 4 compared to Cycle 1 levels. Therefore, the repeated-batch fermentation was terminated after Cycle 4. Interestingly, YB/S values in all cycles were not different (Table 2), suggesting that the repeated-batch system did not change the pathway of ABE fermentation. Even though PB, PABE, SC and QB in Cycle 4 were reduced by ~65–67% compared to Cycle 1 levels, total butanol productivity in terms of g butanol/h (QP* values) of four cycles (0.12 g/h) was not different from that operated for only one cycle (0.12 g/L) or as a batch fermentation (Table 2). Moreover, the repeated-batch fermentation can reduce the inoculum preparation time (in CMM by 12 h and in TGY by 4 h), reduce the cost of inoculation preparation media for each cycle, resulting in lower operating and labor costs.
The morphology of C. beijerinckii cells attached on bamboo chopsticks was evaluated using FE-SEM. This imagery is shown in Figure 4. The results clearly indicate that C. beijerinckii cells are well attached to bamboo chopsticks (Figure 4B) during the simultaneous immobilization and fermentation technique. Additionally, the results indicate that bacterial cells in Cycle 1 were long, vegetative cells (Figure 4B) whereas Cycle 2 cells were swollen or bulged (Figure 4C), which is the active form, resulting in higher butanol production (Table 2). In Cycle 3, forespores or prespores were clearly observed (Figure 4D). All bacterial cells were completely transformed to spores in Cycle 4 (Figure 4E). These phenomena negatively affected butanol fermentation efficiency (Table 2) [30].
When butanol production by co-culture of Arthrobacter and immobilized Clostridium (this study) in a 5-L stirred-tank bioreactor (PB = 12.22 g/L; QB = 0.26 g/L·h), and co-culture of Arthrobacter and free cells of Clostridium in a 5.6-L stirred-tank bioreactor (PB = 12.59 g/L; QB = 0.26 g/L·h) [30] were compared, product formation by free and immobilized Clostridium were similar. However, for application in repeated-batch fermentation using immobilized cells, all fermented broths can be harvested after finishing each cycle, whereas some portion of the fermented broth (at least 10%) using a free cell system must remain in the bioreactor to be used as an inoculum for the next cycle. Therefore, total butanol productivity or QP* values (g/h) of an immobilized cell system will be higher than QP* values of a free cell system.
All results clearly indicate that the ABE fermentation by co-culture of Arthrobacter sp. and immobilized Clostridium are effective for butanol production in both laboratory and pilot-scale bioreactors. Butanol production by fermentation from a biomass can replace traditional butanol (petro-butanol) production from fossil fuels [31]. Additionally, butanol production by fermentation is more environmentally friendly and energy efficient that fossil-fuel based production.

4. Conclusions

Sweet sorghum stem juice can be effectively used as a low-cost substrate for butanol production in ABE fermentations. Co-culture of Arthrobacter sp. and immobilized C. beijerinckii TISTR 1461 on bamboo chopsticks was successful for butanol production in both laboratory and pilot-scale bioreactors. Scaled-up fermentation does not affect the pathways of ABE fermentation and bacterial morphology. Arthrobacter sp. can efficiently create an anaerobic environment for ABE fermentations by immobilized Clostridium in batch and repeated-batch butanol fermentations. The butanol fermentation efficiencies of the co-cultures were ~8 to 14% higher than those of the single culture of immobilized Clostridium using traditional method (OFN gas) to generate anaerobic conditions. Hence, this co-culture approach is promising for use in future industrial-scale butanol bioreactors. Nevertheless, agitation and mixing in large scale production to completely create anaerobic conditions by Arthrobacter sp. is challenging and needs to be further studied.

Author Contributions

Conceptualization, P.L. and L.L.; methodology and formal analysis, P.L. and T.T.; investigation, T.T.; writing: original draft preparation, T.T.; writing: review and editing, P.L. and L.L.; supervision, P.L. and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fundamental Fund of Khon Kaen University, the National Science, Research and Innovation Fund (NSRF), Thailand (Grant. No. 179568), and the Post-Doctoral Training for Frontier Research from Khon Kaen University, Thailand (Grant No. PD2567-04).

Data Availability Statement

Data will be made available on request. To limit access and to prevent unauthorized use, the data are not publicly available.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Butanol fermentation in a 1-L screw-capped bottle (A), 5-L (B), and 30-L (C) stirred-tank bioreactors.
Figure 1. Butanol fermentation in a 1-L screw-capped bottle (A), 5-L (B), and 30-L (C) stirred-tank bioreactors.
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Figure 2. Profiles of batch butanol fermentations by co-culture of Arthrobacter sp. and immobilized C. beijerinckii (A) and by a single culture of immobilized C. beijerinckii (B) in 1-L screw-capped bottles showing acetone (♦), butanol (■), ethanol (×), ABE (▲), acetic acid (◊), butyric acid (□), total acid (Δ), pH (○), total sugar (●).
Figure 2. Profiles of batch butanol fermentations by co-culture of Arthrobacter sp. and immobilized C. beijerinckii (A) and by a single culture of immobilized C. beijerinckii (B) in 1-L screw-capped bottles showing acetone (♦), butanol (■), ethanol (×), ABE (▲), acetic acid (◊), butyric acid (□), total acid (Δ), pH (○), total sugar (●).
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Figure 3. Profiles of free amino nitrogen (FAN) concentration during batch butanol fermentation by co-culture of Arthrobactor sp. and immobilized C. beijerinckii (○) and single culture of immobilized C. beijerinckii (●).
Figure 3. Profiles of free amino nitrogen (FAN) concentration during batch butanol fermentation by co-culture of Arthrobactor sp. and immobilized C. beijerinckii (○) and single culture of immobilized C. beijerinckii (●).
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Figure 4. Field emission scanning electron microscopy (FE-SEM) images of bamboo chopsticks (A) and C. beijerinckii immobilized on bamboo chopsticks in Cycle 1 (B), Cycle 2 (C), Cycle 3 (D), and Cycle 4 (E) at 20,000× magnification.
Figure 4. Field emission scanning electron microscopy (FE-SEM) images of bamboo chopsticks (A) and C. beijerinckii immobilized on bamboo chopsticks in Cycle 1 (B), Cycle 2 (C), Cycle 3 (D), and Cycle 4 (E) at 20,000× magnification.
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Table 1. Batch butanol fermentation from SSJ medium by a single culture of immobilized C. beijerinckii and co-culture of Arthrobacter sp. and immobilized C. beijerinckii after 48 h of fermentation.
Table 1. Batch butanol fermentation from SSJ medium by a single culture of immobilized C. beijerinckii and co-culture of Arthrobacter sp. and immobilized C. beijerinckii after 48 h of fermentation.
BioreactorCultureProducts (g/L)SC (%)YB/S (g/g)QB (g/L·h)
AcetoneButanolEthanolABETotal Acids
1-L screw-capped
bottle		Single culture
(control)		4.31 ± 0.13 c10.62 ± 0.20 b0.60 ± 0.02 b15.23 ± 0.33 b4.03 ± 0.23 b,c71.30 ± 1.29 a0.26 ± 0.01 a0.22 ± 0.01 b
Co-culture5.34 ± 0.21 a12.09 ± 0.27 a0.71 ± 0.01 a18.14 ± 0.37 a4.19 ± 0.15 b74.73 ± 2.16 a0.28 ± 0.01 a0.25 ± 0.01 a
5-L stirred-tank
bioreactor		Co-culture5.18 ± 0.05 a,b12.22 ± 0.15 a0.74 ± 0.03 a18.21 ± 0.32 a4.71 ± 0.34 a74.02 ± 1.76 a0.28 ± 0.01 a0.26 ± 0.01 a
30-L stirred-tank
bioreactor		Co-culture4.93 ± 0.29 b11.92 ± 0.19 a0.74 ± 0.02 a17.80 ± 0.34 a3.64 ± 0.24 c73.80 ± 2.08 a0.28 ± 0.01 a0.25 ± 0.01 a
Total acids = acetic and butyric acids; SC = sugar consumption; YB/S = butanol yield and QB = volumetric butanol productivity. a–c Means followed by the same letter within the same column were not significantly different as assessed using Duncan’s multiple range test at a critical value of 0.05.
Table 2. Repeated-batch butanol fermentation efficiency from SSJ medium by co-culture of Arthrobacter sp. and immobilized C. beijerinckii cells in 1-L screw-capped bottle.
Table 2. Repeated-batch butanol fermentation efficiency from SSJ medium by co-culture of Arthrobacter sp. and immobilized C. beijerinckii cells in 1-L screw-capped bottle.
CycleProduct (g/L)SC (%)YB/S (g/g)QB (g/L·h)PB* (g)t* (h)QB* (g/h)
AcetoneButanolEthanolABETotal Acids
13.96 ± 0.37 b11.24 ± 0.22 b0.69 ± 0.04 b15.77 ± 0.11 b3.54 ± 0.34 b71.40 ± 2.11 a0.27 ± 0.01 a0.23 ± 0.01 b8.43700.12
24.68 ± 0.59 a12.12 ± 0.33 a0.75 ± 0.02 a17.45 ± 0.53 a3.26 ± 0.48 b74.83 ± 2.23 a0.28 ± 0.01 a0.25 ± 0.01 a17.521180.15
31.14 ± 0.10 c6.36 ± 0.29 c0.66 ± 0.01 b,c8.17 ± 0.40 c6.84 ± 0.58 a43.47 ± 2.35 b0.27 ± 0.01 a0.13 ± 0.01 c22.291660.13
41.19 ± 0.04 c3.66 ± 0.26 d0.64 ± 0.01 c5.50 ± 0.33 d6.22 ± 0.46 a24.49 ± 2.99 c0.27 ± 0.01 a0.08 ± 0.01 d25.042140.12
Total acids = acetic and butyric acids; SC = sugar consumption; YB/S = butanol yield and QB = volumetric butanol productivity. PB* = total amount of butanol, t* = total fermentation time (48 h per/cycle) including inoculum preparation (in CMM, 12 h and TGY, 4 h) and anaerobic creation (6 h), QB* = total butanol productivity. a–d Means followed by the same letter within the same column were not significantly different as assessed using Duncan’s multiple range test at a critical value of 0.05.
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MDPI and ACS Style

Thanapornsin, T.; Laopaiboon, L.; Laopaiboon, P. Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors. Energies 2024, 17, 1009. https://doi.org/10.3390/en17051009

AMA Style

Thanapornsin T, Laopaiboon L, Laopaiboon P. Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors. Energies. 2024; 17(5):1009. https://doi.org/10.3390/en17051009

Chicago/Turabian Style

Thanapornsin, Thanawat, Lakkana Laopaiboon, and Pattana Laopaiboon. 2024. "Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors" Energies 17, no. 5: 1009. https://doi.org/10.3390/en17051009

APA Style

Thanapornsin, T., Laopaiboon, L., & Laopaiboon, P. (2024). Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors. Energies, 17(5), 1009. https://doi.org/10.3390/en17051009

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