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Characterization of acidogenic phase metabolism in Clostridium acetobutylicum ATCC 824 (pCD07239) under different culture conditions

Applied Biological Chemistry volume 67, Article number: 80 (2024) Cite this article

Abstract

In this study, we investigated the metabolic behavior of the engineered Clostridium acetobutylicum ATCC 824 (pCD07239) strain during the acidogenic phase under varying glucose concentrations and pH conditions. Unlike the wild-type C. acetobutylicum ATCC 824, the engineered strain exhibited negligible butyrate production and simultaneous butanol production during the acidogenic phase under limited glucose condition of 25 g/L. Specifically, batch fermentations of the engineered strain with 25 g/L glucose at a pH of around 5.0 (initially uncontrolled) demonstrated butanol production of 2.99 g/L, while butyrate remained below 0.30 g/L. Separately, in batch fermentations at pH 6.0 with 90 g/L glucose, acetate production nearly doubled compared to fermentations at pH 5.0 with the same glucose concentrations, reaching a maximum concentration of 11.43 g/L, while butyrate production remained relatively low at 4.04 g/L. Under these pH 6.0 and 90 g/L glucose conditions, butanol production reached 9.86 g/L. These findings indicate that C. acetobutylicum ATCC 824 (pCD07239) maintained low butyrate production, even under conditions favoring acidogenesis, and consistently produced butanol. Additionally, the negligible production of acetone at pH 6.0 further indicates that the traditional phase transition was not prominent, suggesting altered regulation mechanisms in the engineered strain. These findings highlight C. acetobutylicum ATCC 824 (pCD07239) strain’s unique metabolic profile and its potential for efficient biobutanol production under diverse conditions.

Introduction

The industrial revolution has led to the widespread utilization of fossil fuels as the primary source of energy consumption worldwide. However, this has also led to significant environmental issues such as global warming [1, 2]. In response, countries worldwide have recognized the significance of developing green and renewable energy alternatives [3,4,5,6]. Biofuels have emerged as a viable alternative to fossil fuels and have been the focus of scientific research for several decades [7, 8]. Biobutanol, in particular, stands out among biofuels due to its stable physical and chemical properties and higher calorific value [9,10,11].

Louis Pasteur first discovered the production of butanol through microbial fermentation in the 19th century, and the process was later industrialized by Chaim Weizmann during World War I using Clostridium acetobutylicum [11]. This process, known as acetone-butanol-ethanol (ABE) fermentation, played a crucial role in the production of solvents [12]. Since then, C. acetobutylicum strains have been extensively studied for their solventogenic capabilities [13].

Solventogenic C. acetobutylicum can generate these solvents through ABE fermentation, which involves two featured phases namely acidogenic phase and solventogenic phase [11, 14, 15]. During the acidogenic phase, cells generate ATP to support cell growth by producing butyrate and acetate, causing a drop in pH to about 4.5 [16]. During the solventogenic phase, butyrate and acetate are re-assimilated, resulting in the generation of ABE, and the pH is restored to about 6.0 [17]. Recent research has focused on improving butanol production through control of this complex metabolic pathway in C. acetobutylicum [9, 16, 18,19,20,21]. In addition, some previous studies have shown that butanol production routes can be switched to butyrate production through redox regulation [22, 23]. Recently, research has also been conducted to produce butanol from gasified carbon sources through the introduction of the Wood-Ljungdahl pathway into C. acetobutylicum [24,25,26].

In our previous study, we engineered C. acetobutylicum ATCC 824 by introducing carbonyl branch genes for the Wood-Ljungdahl pathway from Clostridium difficile, resulting in negligible butyrate production with butanol production during the acidogenic phase when the engineered C. acetobutylicum ATCC 824 (pCD07239) cells were cultivated in clostridial growth medium [26]. This result contrasts with the behavior of wild-type strain C. acetobutylicum ATCC 824, where butyrate production predominates during the acidogenic phase and butanol production is absent. In this study, our objective is to comprehensively characterize the metabolic behavior of C. acetobutylicum ATCC 824 (pCD07239) during acidogenic phase by conducting batch fermentations under different culture conditions.

Materials and methods

Microbial strain

The bacterial strain C. acetobutylicum ATCC 824 (pCD07239) was constructed in our previous study [26]. The stock of this strain was maintained in 15% glycerol at -80 °C.

Culture conditions

C. acetobutylicum ATCC 824 (pCD07239) was grown anaerobically at 37 ℃ in an anaerobic chamber (COY Laboratory Products Inc., MI, USA), which was filled with a 96% nitrogen and 4% hydrogen gas mixture. The strain was cultivated statically in clostridial growth medium (CGM) composed of (g/L): (NH4)2SO4, 2; yeast extract, 5; asparagine, 2; NaCl, 1; MgSO4·7H2O, 0.7; KH2PO4, 0.75; K2HPO4, 0.75; MnSO4·5H2O, 0.017; FeSO4·7H2O, 0.01; and p-aminobenzoic acid, 0.004 [27,28,29]. Routine culturing was performed in CGM supplemented with 80 g/L glucose in test tubes and flasks.

Batch fermentation using different initial concentrations of glucose

The experiments were conducted in 5 L bioreactors (Biostar, Biocns, Daejeon, Republic of Korea) with 2.5 L of CGM supplemented with various glucose concentrations (25, 50, or 90 g/L). The medium was autoclaved at 121 °C for 15 min, and then a sterilized glucose solution was added. Before inoculation, the medium was purged with nitrogen at a 0.5 L/min flow rate for about 3 h to establish an anaerobic environment [26]. The inoculation was performed using 200 ml of seed culture at an initial OD600 of about 0.1. The pH was initially uncontrolled, and if it dropped below 5.0 due to organic acids production, 28% (v/v) ammonia solution was automatically added to adjust the pH back to around 5.0. The pH control was turned off once the pH exceeded 5.0 due to acid uptake. All fermentations were conducted at 200 rpm and 37 °C under anaerobic conditions in duplicates. Samples were collected every 3 h using a conical tube during the experiments to determine solvents, organic acids, and glucose and monitor pH changes and cell growth.

Batch fermentation under different pH conditions

These experiments were performed as previously mentioned, except the glucose concentration was fixed at about 90 g/L while the pH was varied to be either 5.5, 6.0, or uncontrolled. Other parameters for fermenter operation were the same as those for the glucose experiment described above, and sampling for metabolite analysis was also performed under the same conditions.

Analytical methods

Cell growth was tracked by gauging optical density at 600 nm (OD600) using a UV-VIS spectrophotometer (U-1900, Hitachi, Japan). For solvent estimation, the samples were centrifuged for 10 min at 11,000 rpm, then passed through 0.20-µm filters and analyzed using an Agilent 7890 N gas chromatography system (Agilent Technologies, CA, USA) [30]. The oven program was set at 100 °C for 7 min, followed by a ramp of 30 °C/min until 210 °C, and then held for 5 min at 210 °C. The concentrations of glucose and organic acids were determined using an UltiMate™ 3000 RSLCnano system (Thermo Scientific, CA, USA) equipped with a refractive index detector (RI-101, Shodex, Japan) and a UV detector (Thermo Scientific) [31, 32]. The flow rate was maintained at 500 µL/min using 5 mmol/L sulfuric acid (mobile phase) through a MetaCarb 87 H column (Agilent Technologies, USA) at 25 °C [33].

Results

Effect of different initial glucose concentrations on acidogenic phase of C. acetobutylicum ATCC 824 (pCD07239)

In our previous study, the engineered C. acetobutylicum ATCC 824 (pCD07239) strain demonstrated a remarkable shift in its metabolic behavior during the acidogenic phase, characterized by negligible butyrate production and simultaneous butanol production when cultivated in CGM containing 80 g/L glucose at pH above 5.0 [26]. In this study, we further characterized ATCC 824 (pCD07239) strain during the acidogenic phase through batch fermentations under different culture conditions. We deliberately reduced the initial glucose concentrations from 80 g/L to 25 g/L (or 50 g/L) to emphasize the acidogenic phase behavior in the engineered ATCC 824 (pCD07239) strain (Fig. 1).

Fig. 1
figure 1

Batch fermentation profiles of C. acetobutylicum ATCC 824 (pCD07239) with (a) 25 g/L and (b) 50 g/L initial glucose at a pH of around 5.0 (initially uncontrolled) during the acidogenic phase. Symbols: filled magenta circle, OD600; filled blue diamond, butyrate; empty square, acetate; cross, pH; empty diamond, glucose; filled red square, butanol; empty triangle, ethanol; and filled inverse triangle, acetone

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First, batch fermentation profiles with 25 g/L initial glucose concentrations were analyzed to observe the metabolic behavior of C. acetobutylicum ATCC 824 (pCD07239) during the acidogenic phase (Fig. 1a). When the initial glucose concentration was 25 g/L, butanol production began after 3 h of incubation (at early log phase) and reached 2.99 g/L by the end of the log phase (Table 1). At this point, the glucose was almost entirely consumed, and the highest OD600 was 8.78 (Fig. 1a; Table 1). Notably, butyrate production was negligible after 3 h of incubation, with a maximum titer of 0.30 g/L, while the maximum titer of acetate was observed at 4.54 g/L (Table 1). These results indicate that ATCC 824 (pCD07239) cells mostly remained in the logarithmic growth phase throughout the glucose consumption period under the fermentations using 25 g/L glucose, thereby emphasizing the acidogenic phase (Fig. 1a). This observation is well supported by the negligible production of other solvents, including acetone and ethanol (Table 1). Consequently, most profiles halted at 12 h when the glucose was depleted (Fig. 1a). Moreover, the fermentation with 25 g/L glucose clearly replicated the characteristics observed with the 90 g/L fermentation (control; Fig. 2), where butanol production commenced in the early log phase, and butyrate production was negligible.

Table 1 Summary of fermentation parameters for C. acetobutylicum ATCC 824 (pCD07239) with various initial glucose concentrations
Full size table
Fig. 2
figure 2

Batch fermentation profiles of C. acetobutylicum ATCC 824 (pCD07239) with 90 g/L initial glucose concentration at a pH of around 5.0 (initially uncontrolled) during the acidogenic phase. These profiles were used as a control for experiments with varying glucose concentrations and pH conditions. Symbols: filled magenta circle, OD600; filled blue diamond, butyrate; empty square, acetate; cross, pH; empty diamond, glucose; filled red square, butanol; empty triangle, ethanol; and filled inverse triangle, acetone

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The fermentation with 50 g/L initial glucose was designed to observe the metabolic behavior of the ATCC 824 (pCD07239) strain under intermediate glucose conditions, bridging the gap between the limited (25 g/L) and sufficient (90 g/L) glucose concentrations (Fig. 1b). In the fermentations with 50 g/L initial glucose, butanol production was also observed at early log phase (3 h), similar to the 25 g/L and 90 g/L conditions (Table 1). However, in the fermentation with 50 g/L initial glucose, the acidogenic phase exhibited some distinct characteristics compared to the 25 g/L condition. The logarithmic growth phase extended to 15 h, with the highest OD600 reaching 15.60, indicating higher cell growth (Fig. 1b; Table 1). Butyrate production remained below 1 g/L during the acidogenic phase but significantly increased to 3.94 g/L as the culture transitioned into the solventogenic phase (Table 1). Acetate production increased during acidogenic phase mainly, with a maximum titer of 6.84 g/L. However, it was observed that the production pattern of butyrate did not largely overlap with that of acetate. While the parent ATCC 824 strain rapidly produced both acids during the early fermentation period [9, 29], the engineered ATCC 824 (pCD07239) strain showed a similar pattern for acetate but produced very little butyrate early on, only starting significant production much later (Fig. 1b). The fermentation profile for 50 g/L glucose was generally similar to that observed with 90 g/L glucose, with only slight variations in the final titers of the produced metabolites dependent on the initial glucose concentration (Figs. 1 and 2; Table 1).

Effect of different pH values on acidogenic phase of C. acetobutylicum ATCC 824 (pCD07239)

It is generally observed that higher pH levels in the fermentations using C. acetobutylicum tend to favor the production of organic acids such as butyrate and acetate over solvents like ABE, thereby inducing acidogenesis without phase transition [17, 22, 23]. To see if the engineered ATCC 824 (pCD07239) strain would continue to exhibit its characteristic low butyrate production during the acidogenic phase, even under conditions that typically favor butyrate production, we conducted batch fermentation at pH 6.0 (Fig. 3).

Fig. 3
figure 3

Batch fermentation profiles of C. acetobutylicum ATCC 824 (pCD07239) with 90 g/L initial glucose concentration at pH 6.0. Symbols: filled magenta circle, OD600; filled blue diamond, butyrate; empty square, acetate; cross, pH; empty diamond, glucose; filled red square, butanol; empty triangle, ethanol; and filled inverse triangle, acetone

Full size image

In the fermentations using ATCC 824 (pCD07239) at a constant pH of 6.0, the highest OD600 of 10.32 was observed, which is lower than the cell density observed during fermentation at a pH of around 5.0 (initially uncontrolled) during the acidogenic phase (Table 2). Nonetheless, as expected, acetate production dramatically increased at pH 6.0, with the maximum concentration more than twice that of pH 5.0. The highest acetate concentration observed was about 11.43 g/L at pH 6.0, compared to approximately 5.00 g/L during fermentation at a pH of around 5.0 (Table 2). In contrast, the highest butyrate production reached about 4.04 g/L at pH 6.0, which is actually lower than the approximately 3.94 g/L observed during fermentation controlled at a pH of around 5.0 (Table 2). This result indicates that despite the pH conditions favoring organic acids production (at pH 6.0), butyrate production during the acidogenic phase did not increase dramatically compared to fermentations at a pH of around 5.0. This indicates that while acetate production is highly responsive to external pH changes, butyrate production by the engineered strain ATCC 824 (pCD07239) is largely unaffected by such external pH changes. Additionally, at pH 6.0, acetone production was barely observed, indicating that the traditional phase transition did not occur significantly (Table 2). Nevertheless, interestingly, butanol production reached 9.86 g/L (Table 2). Such a high butanol concentration at pH 6.0 has not been reported previously, suggesting the potential for developing fermentation processes at higher pH using this engineered strain.

Table 2 Summary of fermentation parameters for C. acetobutylicum ATCC 824 (pCD07239) under different pH conditions
Full size table

In the fermentations at pH 5.5 and under uncontrolled pH conditions, acetone and ethanol production exceeded 1 g/L (Fig. 4; Table 2). Butanol production reached a maximum of 12.87 g/L and 6.09 g/L at pH 5.5 and uncontrolled conditions, respectively (Table 2). For butyrate production, although the profiles seem almost acid-crash, the maximum concentration was about 0.54 g/L under uncontrolled pH conditions, which is negligible (Fig. 4b; Table 2). In the fermentation at pH 5.5, butyrate production remained below 1 g/L during the acidogenic phase but increased sharply after the phase transition to the solventogenic phase, reaching a final concentration of 5.84 g/L (Table 2). In contrast, acetate production showed a trend of reaching up to 9.63 g/L even at pH 5.5 (Table 2).

Fig. 4
figure 4

Batch fermentation profiles of C. acetobutylicum ATCC 824 (pCD07239) with 90 g/L initial glucose concentration at (a) pH 5.5 and (b) uncontrolled conditions. Symbols: filled magenta circle, OD600; filled blue diamond, butyrate; empty square, acetate; cross, pH; empty diamond, glucose; filled red square, butanol; empty triangle, ethanol; and filled inverse triangle, acetone

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Discussions

By comparing reported C. acetobutylicum strains, the observed metabolic shifts of ATCC 824 (pCD07239) strain under different culture conditions might be partially understood through the metabolic pathways involved in NADH and ATP production, as well as their relationship with acetate, butyrate, and butanol production (Fig. 5). During the acidogenic phase, typical C. acetobutylicum primarily produces acetate and butyrate, which are associated with ATP generation via substrate-level phosphorylation [34]. This ATP production is crucial for cell growth and maintenance. In the logarithmic cell growth phase, the parent ATCC 824 strain primarily produces acetate and butyrate during the acidogenic phase to compensate for ATP [27, 34]. In the same metabolic process, the butyrate production pathway participates in NAD+ regeneration, also [27, 34]. In contrast, the engineered C. acetobutylicum ATCC 824 (pCD07239) exhibited low butyrate production even under conditions favoring acidogenesis (pH 6.0), suggesting a re-routing of metabolic fluxes (Fig. 5). This indicates that C. acetobutylicum ATCC 824 (pCD07239) has undergone a metabolic shift, favoring alternative pathways for NAD+ regeneration and ATP compensation.

Fig. 5
figure 5

Metabolic pathway of C. acetobutylicum ATCC 824 (pCD07239) during (a) acidogenic and (b) solventogenic phases. Enzymes indicated by abbreviations: PFOR, ferredoxin oxidoreductase; PTA, phosphate transacetylase; AK, acetate kinase; THL, thiolase; BHBD, 3-hydroxybutyryl-CoA dehydrogenase; CRT, crotonase; BCD, butyryl-CoA dehydrogenase; AAD, aldehyde/alcohol dehydrogenase; BdhABC, NADPH-dependent butanol dehydrogenase (or bifunctional aldehyde/alcohol dehydrogenase; AdhE2); and HYD, hydrogenase

Full size image

In this study, for example, ATCC 824 (pCD07239) strain favored acetate formation more than twice its production at pH 6.0 compared to pH 5.0, while producing very little butyrate, relatively. Generally, under such conditions, some reported strains produce both acetate and butyrate well [22, 28, 29]. This butyrate production pathway is well known to participate in NAD+ regeneration and ATP compensation. Therefore, it appears that the ATCC 824 (pCD07239) strain uses alternative pathways for NAD+ regeneration instead of the butyrate production pathway under these conditions (Fig. 5). This is supported by the observation that butanol production reached 9.86 g/L at pH 6.0 (Table 2), which is significantly higher than typical reports for C. acetobutylicum at elevated pH (1.94 g/L) [35].

Moreover, it was observed that the production pattern of butyrate did not largely overlap with that of acetate under the fermentation with limited glucose condition (25 g/L) at pH 5.0. While the parent ATCC 824 strain rapidly produced both acids during the early fermentation period [13, 16, 27], ATCC 824 (pCD07239) strain showed a similar pattern for acetate but produced very little butyrate (Fig. 1a). These results more clearly demonstrated that the engineered ATCC 824 (pCD07239) strain produces very little butyrate during the acidogenic phase (or early log phase) and consistently produces butanol simultaneously, even under glucose conditions insufficient for solventogenesis. This suggests that the engineered ATCC 824 (pCD07239) strain exhibits a significantly different metabolic profile than the wild-type strain (Fig. 5). For instance, Capilla et al. [36] reported a butanol production of 1.09 g/L from batch fermentation of C. acetobutylicum DSM 792, with a minimum pH of 5.1, using 33 g/L initial glucose, while the ATCC 824 (pCD07239) strain produced 2.99 g/L butanol under similar conditions in this study.

The negligible production of acetone at pH 6.0, while butanol was produced up to 9.86 g/L, suggest that the traditional phase transition was not prominent in C. acetobutylicum ATCC 824 (pCD07239) (Table 2). This could be due to the engineered strain’s altered regulation mechanisms, which prioritize butanol production over other solvents (Fig. 5). The observation of butanol production in the absence of significant acetone production suggests that the pathways involved in butanol production may differ from those involved in the production of the other two solvents. Generally, ABE production in C. acetobutylicum is controlled by the sol operon located on the mega-plasmid pSOL1 containing the adhE1-ctfAB-adc gene cluster [37, 38]. The three genes adhE1, ctfAB, and adc are known to encode for alcohol/aldehyde dehydrogenase, CoA transferase, and acetoacetate decarboxylase, respectively [37, 38]. Therefore, if solvent production is assumed to be driven by the expression of these genes, it is likely that all three solvents would be produced simultaneously. However, the results observed in this study suggest that butanol production in the early log phase may not be due to the activation of the sol operon, as only butanol was observed (Fig. 5). Another alternative pathway could involve alcohol/aldehyde dehydrogenase II, encoded by adhE2, which is not included in the sol operon [39, 40]. Although the exact mechanism is unclear, the early log phase production of butanol alone suggests the possibility of butanol production via the expression of the adhE2 gene.

In conclusion, the engineered C. acetobutylicum ATCC 824 (pCD07239) strain exhibits unique metabolic characteristics that make it a promising candidate for industrial biobutanol production. The ATCC 824 (pCD07239) strain’s ability to maintain low butyrate production and high butanol production under a range of pH conditions, especially at pH 6.0, suggests its adaptability to different fermentation environments. Further research should focus on exploring the omics profiling of the engineered C. acetobutylicum ATCC 824 (pCD07239) in butanol production without butyrate production.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by the Ministry of Science and ICT through the National Research Foundation of Korea (2022R1A2C1006214 and RS-2024-00439872).

Funding

This work was supported by the Ministry of Science and ICT through the National Research Foundation of Korea (2022R1A2C1006214 and RS-2024-00439872).

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Authors and Affiliations

  1. Division of Applied Life Science (BK21 Four), Department of Applied Life Chemistry, Institute of Agriculture & Life Science (IALS), Gyeongsang National University (GNU), Jinju, 52828, Republic of Korea

    Haeng Lim Lee, Selim Ashoor, Zhuang Yao & Yu-Sin Jang

  2. Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Hadayek Shoubra, Cairo, 11241, Egypt

    Selim Ashoor

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  1. Haeng Lim Lee
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  2. Selim Ashoor
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  3. Zhuang Yao
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  4. Yu-Sin Jang
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Contributions

HLL: Investigation, Writing – original draft. ZY: Writing – original draft. SA: Writing – original draft, Data analysis, Writing – review & editing. YSJ: Conceptualization, Supervision, Data analysis, Funding acquisition, Writing – review & editing.

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Correspondence to Yu-Sin Jang.

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The authors declare no competing interests. YSJ is an associate editor of Applied Biological Chemistry. Associate editor status has no bearing on editorial consideration.

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Lee, H.L., Ashoor, S., Yao, Z. et al. Characterization of acidogenic phase metabolism in Clostridium acetobutylicum ATCC 824 (pCD07239) under different culture conditions. Appl Biol Chem 67, 80 (2024). https://doi.org/10.1186/s13765-024-00936-0

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