Caproic acid production through fermentation with a thermotolerant Clostridium strain M1NH

doi:10.21203/rs.3.rs-3613998/v2

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Caproic acid production through fermentation with a thermotolerant Clostridium strain M1NH

https://doi.org/10.21203/rs.3.rs-3613998/v2

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Thermotolerant caproic acid-producing bacteria were isolated from sewage sludge and evaluated for caproic acid production with various substrates. The ethanol-acetate was the most efficient substrate for enriched culture, with 2.7 g/L caproic acid and a yield of 47%. Clostridium strain M1NH was isolated from enriched culture and identified as a gram-positive, rod-shaped, spore-forming with 92% similarity of 16S rRNA to Clostridium kluyveri. Clostridium M1NH has an optimal pH and temperature of 6-6.5 and 35–40°C with a growth rate of 0.11–0.14 h^-1, suggesting that the strain was thermotolerant. Clostridium strain M1NH displays the highest caproic acid production of 3.2–3.5 g/L at a pH of 6.5, a temperature of 35–40°C, and an ethanol-acetate molarity ratio of 4:1. Clostridium strain M1NH is tolerant to concentrations of ethanol, acetate, and caproic acid of 5%, 5%, and 2%, respectively. The thermotolerant Clostridium M1NH presents a significant advancement in harnessing sustainable biobased production processes, holding great promise for applications in thermotolerant fermentation.

Applied & Industrial Microbiology

Clostridium

caproic acid

fermentation

sewage sludge

thermotolerant

Caproic acid is a six-carbon saturated fatty acid, colorless, odorless, and tasteless with soluble in water and organic solvents. Caproic acid is found naturally in various animal fats and plant oils. It can also be produced synthetically from petroleum. Caproic acid is also produced by chain elongation in anaerobic fermentation by chain-elongating bacteria, including Clostridium kluyveri [1, 2] and Megasphaera elsdenii [3, 4], Caproiciproducens [5, 6], and Ruminococcaceae bacterium [7]. Caproic acid is synthesized by the reverse β-oxidation of short-chain fatty acids (e.g., acetic acid C₂, propionic acid C₃, and butyric acid). Caproic acid has broad potential applications. It can be used directly as feed additives [8], antimicrobials [9], and plant growth promoters [10]. It can also be a precursor to various commodities, including fragrances, lubricants, paint additives, and pharmaceuticals [11–13]. Caproic acid has a higher energy density and stronger hydrophobicity than its short-chain fatty acids precursors [14]. Bio-based caproic acid production is an attractive alternative to the conventional petrochemical route, as it can reduce the dependency on fossil resources, lower greenhouse gas emissions, and utilize renewable biomass feedstocks.

Bio-based caproic acid production involves using biological processes and biotechnology to convert biomass into caproic acid or its precursors. Traditional fermentation processes are conducted at moderate temperatures, typically between 25°C and 35°C. New fermentation technology with thermotolerant microorganisms has been widely explored. Thermotolerant yeast strains can improve ethanol fermentation efficiency, allowing fermentation to occur at temperatures higher than 40°C [15]. Another example is thermotolerant bacteria, which possess high metabolic rates, higher end-product yield, and stable enzymes. They develop resistance to high temperatures by producing specialized proteins known as Chaperonins, which are thermostable [16]. Researchers also have actively sought thermotolerant microorganisms capable of thriving and producing valuable compounds like caproic acid at elevated temperatures [7, 17]. The advantage of using thermotolerant conditions is the possibility of conducting biotechnological processes at elevated temperatures, thus reducing the risk of contamination by mesophilic microorganisms, decreasing the viscosity of the reaction medium, increasing the bioavailability and solubility of organic compounds, and increasing the diffusion coefficient of substrates and products resulting in higher reaction rate [18].

Sewage sludge from wastewater treatment plants contains abundant organic matter, nutrients, and microorganisms. However, our understanding of the microbial diversity and functional capacity of sewage sludge is limited, especially regarding caproic acid-producing and thermotolerant bacteria. To address this knowledge gap, we screened, isolated, identified, quantified, and characterized the caproic acid-producing and thermotolerant bacteria from sewage sludge at Ube Eastern Wastewater Treatment Plant, Japan.

2.1 Inoculum source

The sewage sludge was collected from the Ube Eastern wastewater treatment plant in Japan, which manages and treats municipal sewage. Sewage sludge holds immense potential for housing diverse microbial populations with unique capabilities. The collected sludge samples were stored under controlled conditions, specifically at a temperature of 4°C, to preserve the microbial composition until further processing and experimentation.

2.2 Enriched and isolated caproic acid-producing bacteria

Sewage sludge samples were enriched in the basic anaerobic medium, supplemented with ethanol and lactic acid as electron donors and acetic acid, glucose, and xylose as carbon sources (Table 1). The composition of the basic anaerobic medium is modified based on Angelidaki et al. [19], incorporating specific components as follows (per 1,000 ml): 1 g/L NH₄Cl, 0.1 g/L NaCl, 0.1 g/L MgCl₂·6H₂O, 0.05 g/L CaCl₂·2H₂O, 0.3 K₂HPO₄·3H₂O, 0.001 g/L C₁₂H₆NO₄Na, 2.6 g/L NaHCO₃, 1 mL trace-metal solution, 10 µL vitamin mixture solution, and 1 g yeast extract. Substrates are prepared with a defined carbon source-to-electron donor ratio of 3:1 based on molarity. Sewage sludge of 10%w/v was introduced into the broth medium. The samples undergo anaerobic cultivation at an initial pH of 6.5 and a temperature of 35°C for 7 days. The experiment was performed in triplicate. The products and substrates from the fermentation broth are quantified using HPLC to measure caproic acid and other metabolites. Cultures displaying the highest caproic acid concentrations from the enrichment process are established for isolation. Make a dilution series for 10^− 6 to 10^− 9 from the best enriched culture. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. The plates were incubated under anaerobic conditions at 35°C for 7 days. Single colonies on the agar plate were selected and subcultured three times on the agar medium. The single colony was inoculated into a 100-ml broth medium with an ethanol and acetate ratio of 3:1 (molarity) as substrates at an initial pH of 6.5 and incubated under anaerobic conditions at 35°C for 7 days for screening its capability for caproic production.

Table 1

Carbon source and electron donors in the medium for the enrichment of caproic acid-producing bacteria from sewage sludge
Treatment	compositions
1 2 3 4 5 6 7 8 9 10	Ethanol (5.5 g/L) and acetate (2.4 g/L) Ethanol (5.5 g/L) and glucose (7.2 g/L) Ethanol (5.5 g/L) and xylose (6.0 g/L) Lactic acid (10.8 g/L) and acetate (2.4 g/L) Lactic acid (10.8 g/L) and glucose 7.2 g/L) Lactic acid (10.8 g/L) and xylose (6.0 g/L) Ethanol (6.9 g/L) Acetate (8.9 g/L) Glucose (6.9 g/L) Lactic acid (13.5 g/L)

2.3 Identification and characteristics of strain M1NH

The strain M1NH with high caproic acid production was selected. First, the bacterial morphological shape and staining pattern were observed under a microscope. The biochemical characteristics of the designated representative strains were studied using Bergey’s Manual of Determinative Bacteriology [20]. To investigate the growth and metabolic activity of the M1NH strain, a physiological test was conducted under anaerobic conditions with 2.75 g/L ethanol and 1.2 g/L acetate. 10% of the fresh culture of M1NH was added to the medium. The medium was flushed with nitrogen gas and incubated at 35°C for 48 hours. The genomic DNA was extracted from strain M1NH using the NucleoSpin® kit (Macherey-Nagel, Germany) according to the NucleoSpin® Soil Manual. The extracted DNA was used as a template for 16S rRNA amplification with universal primers 27F (5’‑AGAGTTT-GATCCTGGCTCAG-3’) and 1492R (5’-CTACGGC-TACCTTGTTACGA-3’). The amplified products were sent to the Center for Gene Research, Yamaguchi University, Japan, for sequencing. The sequences were spliced by the basic local alignment search tool (BLAST) program available at the National Centre for Biotechnology Information (NCBI) database and compared with the data available in the NCBI 16S rDNA database to obtain the homologous sequence showing the most remarkable similarity with the sequence of the species to be tested. The phylogenetic tree was constructed by MEGA 5.2 software using the neighbor-joining (NJ) method.

2.4 Substrate and product inhibition of Clostridium strain M1NH

A loopful of Clostridium strain M1NH inoculum was introduced into individual serum bottles, each containing a fresh basic anaerobic medium. The initial absorbance (optical density) was assessed at a wavelength of 600 nm using a spectrophotometer. Subsequently, the bottles were subjected to incubation at a range of temperatures (25 to 55°C) and initial pH levels spanning from 3 to 10 for 48 hours. The absorbance was determined every 6 hours. A loopful of Clostridium strain M1NH inoculum was introduced into individual serum bottles, each containing a fresh basic anaerobic medium. After 48 hours, a loopful of inoculum from these incubated bottles was employed to inoculate culture media with varying concentrations of ethanol, acetate, and caproic acid. These cultures were adjusted at an initial pH of 6.5 and a temperature of 35°C for another 48-hour incubation period. Then, the absorbance was determined every 6 hours.

2.4 Optimization condition for caproic acid production by Clostridium strain M1NH

The selected caproic acid-producing culture (strain M1NH) was subjected to adaptive one-factor-at-a-time optimization in the optimization phase. This method systematically explored the impact of pH, temperature, and carbon source-to-electron donor ratio on caproic acid production (Table 2). For pH, levels of 5.5, 6.0, 6.5, 7.0, and 7.5 were investigated, while temperature variations of 30°C, 35°C, 37°C, and 40°C were tested at optimum pH. Carbon source to electron donor ratio was tested at 4:1 (160 mM:40 mM), 3:1 (120 mM:40 mM), 2:1 (80 mM:40 mM), and 1:1 (40 mM:40 mM) under optimal pH and temperature. High-performance liquid chromatography (HPLC) was employed to quantify caproic acid concentrations. The optimized conditions were determined based on the highest caproic acid production, and the selected combination of factors was validated through repetition and utilized for further experiments.

Table 2

Optimization parameters for caproic acid production of *Clostridium* strain N1HM
Factor	Variables
pH	5.5	6.0	6.5^*	7.0	7.5
Temperature	30°C	35°C^*	37°C	40°C
Carbon to electron donor ratio	4:1 (160 mM:40 mM)^*	3:1 (120 mM:40mM)	2:1 (80 mM:40 mM)	1:1 (40 mM:40 mM)
^*Optimized factor to be taken for forthcoming experiment

3.1 Enrichment and isolation of caproic acid-producing bacteria

The experiment aimed to enrich caproic acid-producing bacteria under different substrate conditions, including lactic acid, glucose, xylose, ethanol, and acetate, either alone or in combination. Five substrates (ethanol-glucose, ethanol-acetate, ethanol-xylose, lactic acid-glucose, and lactic acid-xylose) could produce caproic acid from enriched cultures (Fig. 1). The highest caproic acid yields 47% of the substrate, producing 2.7 g/L observed in ethanol-acetate fermentations. Researchers have reported producing caproic acid from a mixed culture utilizing ethanol and acetate in a batch reactor. In the stable-phase batch fermentation conducted by Liu et al. [21], they achieved the highest caproate concentration, reaching 3 g/L with a 41% yield. In a separate study by Bao et al. [22], caproate production reached 2.3 g/L, accompanied by a yield of 6.7%. Steinbusch et al. (2011) produced 8.17 g/L of caproate using a mixed culture, achieving an impressive 60% yield based on the substrate. Ethanol-acetate as a substrate also resulted in moderate valeric and butyric acid yields of 2% and 7% of the substrate, respectively. Interestingly, the ethanol-acetate combination also showed a considerable acetic acid yield of 12% of the substrate. This was followed by an enrichment medium containing 10.8 g/L lactic acid and 7.2 g/L glucose, which produced 0.6 g/L caproic acid. The results suggest that the combination of ethanol and acetate is more effective in promoting caproic acid-producing bacteria. The higher yield of caproic acid indicates that the microorganisms in the culture are more efficient at utilizing ethanol and acetate as substrates.

In contrast, the ethanol-xylose combination exhibited a relatively lower caproic acid yield of 2%. However, it displayed a substantial butyric acid yield (40%), indicating a shift in product profile based on the carbon source. Additionally, the combination yielded a noteworthy amount of propionic acid (3%) and a moderate amount of valeric acid (2%). The influence of different carbon sources was also evident in the case of lactic acid. When combined with glucose, lactic acid yielded caproic acid (6%), along with notable amounts of butyric acid (28%) and acetic acid (7.37%). On the other hand, the lactic acid-xylose combination yielded caproic acid of 3%, along with other VFAs such as butyric acid (17%) and propionic acid (6.67%). The standalone substrates, ethanol and acetate, did not yield caproic acid, highlighting the importance of combining suitable carbon sources for productive fermentation and enriched caproic acid-producing bacteria. Among these, ethanol demonstrated a propensity for acetic acid production (7.14%), while acetate showed a moderate butyric acid yield (2%). The glucose substrate favored acetic acid production (12.5%), while xylose favored butyric acid (52%) and displayed a higher proportion of caproic acid (7.59%). Ethanol and acetate are carbon sources effective for enriched caproic acid-producing bacteria. Furthermore, enriched culture from ethanol and acetate as carbon sources was isolated for pure strain.

Three strains were isolated from an ethanol and acetate enrichment culture. Strain M1NH exhibited the highest caproic acid production, with a production of 2.5 g/L and a yield of 44% (Fig. 2). On the other hand, strain M2NH displayed a lower caproic acid production of 0.6 g/L, and strain M3NH exhibited the lowest caproic acid production of 0.2 g/L. Regarding butyric acid production, strain M1NH showed a minimal production of 0.1 g/L, while strain M2NH exhibited a higher production of 0.9 g/L. Interestingly, strain M3NH displayed a comparable butyric acid production of 0.8 g/L, similar to strain M2NH. The results highlight the strain-dependent variations in caproic acid and butyric acid production. Strain M1NH emerges as the most promising candidate for caproic acid production, demonstrating the highest production. Therefore, strain M1NH was selected for further studies. Further analysis and characterization of these strains can provide insights into the underlying mechanisms governing caproic acid and butyric acid production. Such information can be instrumental in optimizing fermentation processes for enhanced bio-based chemical production.

3.2 Characteristics and phylogenetic classification of strain M1NH

Strain M1NH is a Gram-positive, rod-shaped bacterium exhibiting anaerobic growth and spore-forming capability. The morphology of the strain reveals a large, smooth, and cream-colored appearance. Several biochemical tests were conducted to assess its physiological and biochemical attributes. The strain tested positive for urease, catalase, and oxidase activities while showing a negative response in the hydrogen sulfide test. These findings indicate the strain's adaptability to environments containing these compounds, which is crucial for its potential application in caproic acid production. A physiological tree was constructed to elucidate further the strain's phylogenetic placement by aligning its 16S rDNA sequence with sequences from the GenBank database. The analysis revealed that the closest relative of Strain M1NH was identified as Clostridium sp. (GenBank: KP754675.1), with a sequence similarity of 92% (Fig. 3). This insight into its phylogenetic relationship provides valuable information about the strain's evolutionary context and taxonomic classification.

3.4 The growth and inhibition of Clostridium M1NH

Understanding the growth kinetics of Clostridium M1NH is crucial for optimizing production processes. Growth of Clostridium M1NH was significantly increased in OD₆₀₀ between 12 and 36 hours, indicating exponential growth. This phase is characterized by rapid cell division as the culture adapts to the medium. After 36 hours, the growth rate slowed and stabilized. Cell culture enters the stationary phase, where the cell death rate balances the cell division rate. In the later stages, particularly between 96 and 120 hours, there was a noticeable decrease in OD₆₀₀, suggesting a decline in cell viability or metabolic activity. This could be due to nutrient depletion or accumulation of waste products. Maintaining optimal pH and providing adequate temperature can enhance cell growth. The data suggests that the Clostridium M1NH has an optimal pH range for growth around pH 6-6.5 (Fig. 4a). The highest OD₆₀₀ value was observed at pH 6.5 at 36 hours of incubation, and the growth rate is 0.14 h^− 1, suggesting that this pH level is the most favorable for growth. At pH 6.5, the culture has the shortest doubling time (approximately 5.3 hours). Growth is severely inhibited at acidic pH levels, with minimal or no growth observed at pH 3, 4, and 5. Growth is also inhibited at alkaline pH levels, with reduced growth at pH 8 and 9 and no growth at pH 10. As the pH becomes more acidic or alkaline, the growth rate decreases, and the doubling time increases. The microorganisms are less efficient at utilizing nutrients and reproducing outside the pH range of 5.5-7. Several studies reported the effect of initial pH Clostridium species growth and inhibition. Low pH conditions reduced Clostridium difficile growth, sporulation, and motility [23]. A study reported that the highest growth and organic acid production was found at pH 7.0 by Clostridium propionicum [24]. Moderate changes in pH can modify the ionization of amino-acid functional groups and disrupt hydrogen bonding. This can lead to changes in the folding of the molecule, promoting denaturation and destroying activity [25].

The optimal temperature for the growth of the Clostridium M1NH is 35–40°C, with a growth rate of 0.11 h^− 1 and a doubling time of approximately 6.5 hours. The strain can also tolerate and grow reasonably well within a temperature range of 30°C to 41°C, where growth rates are relatively stable (Fig. 4b). At temperatures below 30°C and above 41°C, the OD₆₀₀ decreases significantly. At 55°C, the strain altogether ceased to reproduce. This indicates that the strain has difficulty growing outside of 30°C to 41°C. There is a noticeable decline in OD₆₀₀ as the temperature increases beyond 41°C. This suggests that the strain is more thermotolerant and has high growth at temperatures of 41°C. At fermentation of Clostridium carboxidivorans, higher temperatures (33°C-37°C) promoted rapid growth and caused cellular agglomeration, and lower temperatures (25°C-29°C) avoided agglomeration but resulted in slow growth [26]. Low temperatures can slow down or stop enzyme function, cause hardening of lipids, and increase the viscosity of fluids [27]. High temperatures can cause the denaturation of proteins and enzymes [28].

A high concentration of ethanol concentration (> 1%) inhibits the growth rate of the Clostridium M1NH and increases its doubling time (Fig. 4c). At 0% ethanol (no ethanol added), the OD₆₀₀ is the highest, indicating healthy and robust growth of the strain under these conditions. As the ethanol concentration increases from 1–5%, there is a significant decrease in OD₆₀₀, indicating reduced cell growth. This suggests that ethanol is detrimental to the strain, and they struggle to grow as ethanol concentration increases. At a 6% ethanol concentration, the OD₆₀₀ drops to 0, indicating that the strain has ceased to grow or reproduce. This concentration is inhibitory enough to halt their growth entirely. A study on Escherichia coli showed that ethanol at 30 g/L decreased the growth yield and experimentally enhanced the specific death rate [29]. Ethanol inhibition can be caused by end-product inhibition or chaotropic-induced stress, resulting in increased membrane fluidization and disruption of macromolecules [29, 30].

High acetate concentration (> 1%) also inhibits the growth rate of the Clostridium M1NH. It increases its doubling time (Fig. 4d). At 0% acetate (no acetate added), the culture has the highest exponential growth rate of approximately 0.10 h^− 1. As the acetate concentration increases from 1–5%, there is a progressive decrease in the exponential growth rate. This indicates that acetate negatively impacts the strain's ability to reproduce rapidly. The growth rate decreases from 0.08 h^− 1 at 1% acetate to 0.03 h^− 1 at 5% acetate. This suggests that at 5% acetate, growth is nearly or entirely inhibited. Higher acetate concentrations can lead to an increase in the doubling time of the microorganisms. At 0% acetate, the doubling time is approximately 7.38 hours; at 5% acetate, it increases to around 27.13 hours. A similar observation discussed the fermentation kinetics of Clostridium tyrobutyricum cultures after being classically adapted for growth at 26.3 g/L acetate. It showed a lag time of 25 hours and log phase growth of 0.07 h^− 1 [31]. The mechanism of action of high concentrations of acetate as an inhibitor of bacterial growth is due to the acidification of the cytoplasm, which can lead to the disruption of cellular processes. Acetate can also inhibit the activity of enzymes involved in energy metabolism, such as ATP synthase and succinate dehydrogenase [32].

The effect of caproic acid concentration on the growth rate was tested as a byproduct inhibition. At 0% caproic acid, the OD₆₀₀ has the highest growth rate of approximately 0.10 h^− 1, indicating optimum strain growth under these conditions (Fig. 4e). This is the baseline condition with no inhibitory effects. As caproic acid concentration increases from 0.5–2%, there is a progressive decrease in the OD₆₀₀, indicating reduced cell growth. Interestingly, the growth rate between 1.5% and 2% caproic acid concentrations remains relatively stable. This suggests that the microorganisms may have adapted to some extent to tolerate caproic acid concentrations within this range. At higher than 2.5% caproic acid concentration, the OD₆₀₀ drops to 0, indicating that the strain has ceased to grow. This concentration is inhibitory enough to halt their growth entirely and is therefore considered toxic to the strain. A dose of 4 mM caproic acid is sufficient to decrease the growth rate of Saccharomyces cerevisiae by 85% [33], while a dose of 40 mM caproic acid becomes completely inhibitory growth of Escherichia coli [34]. 1% of caproic acid becomes completely inhibitory growth of Staphylococcus aureus and Escherichia coli [35]. Furthermore, the inhibitory concentration of caproate is much lower than the inhibitory concentration of butyrate in neutral pH, as demonstrated with C. kluyveri [36]. The exact mechanism of action is not fully understood. However, it is suggested that the fatty acid derivatives might interact with the microbial cell membrane, leading to increased permeability and leakage of cell contents [34, 37]. These effects can lead to cell death or reduced growth. A caproic acid-producing strain increased membrane leakage as the product titer increased, but no change in membrane fluidity [34]. However, more research is needed to understand the underlying molecular mechanisms fully.

3.5 Optimization for caproic acid production from Clostridium M1NH

The optimum initial pH for caproic acid production was investigated by conducting experiments at different initial pH values ranging from 5.5 to 7.5 at 40°C. The results of caproic acid production at different pH levels over a 12-day fermentation period are presented in Fig. 5a. Caproic acid production was initiated at all pH levels from day 2 of fermentation. As observed, pH 6.5 demonstrated the highest initial caproic acid production, with a value of 0.7 g/L. At pH 7 and 7.5 exhibited relatively higher initial caproic acid production, with values of 0.5 g/L and 0.2 g/L, respectively, compared to the low pH (5.5 and 6.0). As the fermentation progressed, caproic acid production increased across all pH levels. On day 4, pH 6.5 exhibited the highest caproic acid production of 2.8 g/L, followed closely by pH 7.0 with a production of 2.3 g/L. A pH of 6.0 and 7.5 displayed intermediate caproic acid production, while a pH of 5.5 exhibited the lowest production. The stationary phase was reached on day 4. The trend of increasing caproic acid production continued until day 6, with pH 6.5 maintaining its production of 2.95 g/L. pH levels 7.0 and 7.5 were closely followed with production of 2.7 and 2.3 g/L, respectively. The caproic acid production at pH 6.0 reached 2.5 g/L, while pH 5.5 exhibited the lowest production of 2.0 g/L. From day 8 to day 12, caproic acid production remained relatively stable at all pH levels. The pH 6.5 consistently maintained the highest caproic acid production, ranging from 2.21 to 2.98 g/L. The results suggest that pH 6.5 is conducive to increased caproic acid production, followed by pH 6.0 and 7.0. While caproic acid production was observed at all pH levels, the variations in production emphasize the importance of pH optimization for enhancing caproic acid fermentation. Yan et al. [38] also found that the optimum initial pH for caproic acid production by C. kluyveri was determined to be 6.41. San-Valero et al. [39] found that C. kluyveri produced maximum caproic acid when the pH was maintained at 6.8. In addition, pH affected the dissolution state of CO₂ in the fermentation system, and CO₂ was a very important inorganic carbon source in the synthesis and metabolism of C. kluyveri. The influence of pH on metabolic pathways, enzyme activities, and microbial growth dynamics likely contributes to the observed differences in caproic acid production [40].

Different experiments were performed at various incubation temperatures ranging from 30°C to 40°C, as shown in Fig. 5b. Caproic acid production was initiated at all temperatures from day 2 of fermentation. Among the temperatures studied, 35°C exhibited the highest initial caproic acid production of 0.7 g/L. Temperatures of 37°C and 40°C also displayed relatively higher initial caproic acid production of 0.5 and 0.3 g/L, respectively, compared to the lower temperature of 30°C. As the fermentation progressed, caproic acid production increased across all temperatures. On day 4, the temperature of 40°C showed the highest caproic acid yield of 2.7 g/L, followed closely by 35°C and 37°C with 2.6 and 2.3 g/L, respectively. The caproic acid production at 30°C reached production of 0.7 g/L. From day 6 to day 12, caproic acid production continued to rise at all temperatures. The temperature of 30°C displayed the lowest caproic acid production, reaching a final production of 3.2 g/L. Temperature levels of 37°C and 40°C exhibited comparable caproic acid production, ranging from 2.78 to 2.81 g/L. Notably, caproic acid production at 30–35°C reached the highest production of 3.1–3.2 g/L by the end of the fermentation period. A strain of Ruminococcaceae bacterium CPB6, affiliated with Clostridium cluster IV, prefers a temperature ranging from 30 to 40°C for caproic acid production [7]. These results indicate that 30–35°C is optimal for caproic acid production by Clostridium M1NH. While caproic acid production was observed at all temperatures, the variations in yield emphasize the significance of temperature optimization for maximizing caproic acid fermentation.

The caproic acid production by Clostridium M1NH under different substrate molarity ratios over a 12-day fermentation period is summarized in Fig. 5c. The variations in substrate molarity ratios (4:1, 3:1, 2:1, and 1:1) were investigated to assess their impact on caproic acid production. At the initiation of the fermentation process (day 0), caproic acid production was not detected across all substrate molarity ratios. This suggests that caproic acid production requires a certain period for microbial adaptation and metabolic activation before becoming detectable. As the fermentation progressed, caproic acid production became evident at day 2 for all substrate molarity ratios. Among the ratios studied, the 4:1 and 3:1 molarity ratios displayed the highest initial caproic acid production of 0.87 g/L each, followed by the 2:1 ratio of 0.6 g/L. The 1:1 ratio exhibited the lowest initial caproic acid production of 0.62 g/L. Subsequently, caproic acid production continued to increase across all substrate molarity ratios. By day 4, the 4:1 ratio has the highest caproic acid production with 1.31 g/L, closely followed by the 3:1 and 2:1 ratio of 1.3 and 1.0 g/L, respectively. The 1:1 ratio produced the lowest caproic acid production of 0.85 g/L at day 4. Increased caproic acid production was sustained from day 6 to day 12. The 4:1 substrate molarity ratio exhibited the highest caproic acid production at day 12, reaching 3.49 g/L. The 3:1 and 2:1 ratios maintained caproic acid production of 3.21 and 1.2 g/L, respectively. In contrast, the 1:1 ratio had the lowest caproic acid production, culminating in 0.81 g/L at day 12. The molarity ratio 4:1 (120 mM: 30 mM) has the highest caproic acid production of 3.5 g/L, indicating that this ratio is optimal for caproic acid production. The production of caproic acid decreased as the ethanol-to-acetate molarity ratio decreased. A relatively high concentration of ethanol versus acetate was reported to influence caproate production positively. An ethanol-to-acetate ratio of 6:1 has been reported to enable maximum caproic acid [41]. Another study found that a 3:1 ratio had the highest n-caproate concentration; however, a lower substrate ratio of 2:1 gave a more inferior product yield [21]. These results emphasize the role of substrate molarity ratio in influencing caproic acid production by Clostridium M1NH. The findings suggest that higher substrate molarity ratios (4:1 and 3:1) favor enhancing caproic acid production. The availability of a more significant number of carbon-rich substrates may provide the necessary precursors and energy sources to support increased caproic acid biosynthesis.

3.6 Comparison with other strains

Clostridium M1NH is an anaerobic and thermotolerant strain of Clostridium that grows best at 35–40°C and can grow up to 48°C. Additionally, the strain prefers a temperature of 35°C for caproic acid production. It is also a high producer of caproic acid, with a production of 3.5 g/L from ethanol and acetate as substrates. Clostridium M1NH is also tolerant to 2% (w/v) caproic acid. Other bacteria that can produce caproic acid include Megasphaera spp., C. kluyveri, Ruminococcaceae CPB6, and Clostridium sp. (Table 3). These bacteria are also anaerobic and thermotolerant but have different substrate preferences and optimum temperatures. Ruminococcaceae CPB6 prefers a temperature range of 30–40°C for caproic acid production from lactate. The maximum caproic acid of Clostridium M1NH during 12 days of fermentation was 1.6-fold higher than that of C. kluyveri during 33 days of fermentation when ethanol and acetate were used as the electron donor [2].

Table 3

Comparison of caproic acid production of *Clostridium* M1NH with previously reported strains
Strain	Substrate	Caproic acid production (g/L)	Fermentation time (d)	Temperature	Reference
Clostridium sp. M1NH	Ethanol and acetate	3.5	12	35–40°C	This study
C. kluyveri	Ethanol and acetate	2.5	33	37	[2]
Ruminococcaceae CPB6	Lactate	16.6	11	30–40°C	[7]
Megasphaera sp. MH	Fructose	9.7	1	37°C	[42]
Megasphaera elsdenii ATCC 25940	Glucose	11.4	5-8.3^a	37°C	[43]
C. kluyveri 3231B	Ethanol	12.8	3	39°C	[17]
Clostridium. sp. BS-1	Galactitol	0.98	3-16^a	37°C	[44]
^a Fed-batch or product removal during fermentation

The isolation of a thermotolerant Clostridium strain M1NH from sewage sludge with a remarkable ability to produce caproic acid opens new avenues for sustainable chemical production. The utilization of ethanol-acetate as an efficient substrate, yielding 3.5 g/L of caproic acid, adds a layer of innovation in substrate choice. It facilitates effective conversion and offers opportunities for integrating waste alcohol and acetate streams from other industrial processes. The robustness of Clostridium M1NH is further exemplified by its tolerance to high concentrations of ethanol, acetate, and caproic acid, as well as its adaptability to a wide pH range and elevated temperatures. These physiological traits make it a strong candidate for industrial-scale applications, particularly in locations with thermal fluctuations and suboptimal substrate purity. The optimal conditions delineated pH of 6.5, temperature between 35–40°C, and a substrate molarity ratio of 4:1 (ethanol:acetate) provide a practical fermentation design and optimization guideline. From a future perspective, it would be valuable to investigate the metabolic pathways and regulatory mechanisms governing caproic acid production in strain M1NH. This could enable genetic or metabolic engineering approaches to boost yield and tolerance further. Additionally, conducting life-cycle analyses would be crucial for assessing the environmental impact and overall sustainability of the process. Finally, integrating this bioconversion process within a biorefinery context, leveraging synergies with other biomass-based transformations could set the stage for a circular economy model that turns waste into wealth.

Acknowledgments

The authors would like to thank the Research Center for Thermotolerant Microbial Resources and Faculty of Engineering, Yamaguchi University, Japan and the National Research Council of Thailand (NRCT) through the Talented Mid-Career Research Grant (Grant No. N41A640088) for the financial support.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authorship contribution

Edy Kurniawan: Visualization, Conceptualization, Formal analysis, Investigation, Methodology, Software, Writing - original draft, Writing - review and editing. Sompong O-Thong: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - review and editing. Tsuyoshi Imai: Funding acquisition, review and editing.

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Caproic acid production through fermentation with a thermotolerant Clostridium strain M1NH

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Inoculum source

2.2 Enriched and isolated caproic acid-producing bacteria

2.3 Identification and characteristics of strain M1NH

2.4 Substrate and product inhibition of Clostridium strain M1NH

2.4 Optimization condition for caproic acid production by Clostridium strain M1NH

3. Results and discussions

3.1 Enrichment and isolation of caproic acid-producing bacteria

3.2 Characteristics and phylogenetic classification of strain M1NH

3.4 The growth and inhibition of Clostridium M1NH

3.5 Optimization for caproic acid production from Clostridium M1NH

3.6 Comparison with other strains

4. Conclusion

Declarations

References

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