Carbon dioxide and trace oxygen concentrations impact growth and product formation of the gut bacterium Phocaeicola vulgatus

Abstract

Background

The promising yet barely investigated anaerobic species Phocaeicola vulgatus (formerly Bacteroides vulgatus) plays a vital role for human gut health and effectively produces organic acids. Among them is succinate, a building block for high-value-added chemicals. Cultivating anaerobic bacteria is challenging, and a detailed understanding of P. vulgatus growth and metabolism is required to improve succinate production. One significant aspect is the influence of different gas concentrations. CO₂ is required for the growth of P. vulgatus. However, it is a greenhouse gas that should not be wasted. Another highly interesting aspect is the sensitivity of P. vulgatus towards O₂. In this work, the effects of varying concentrations of both gases were studied in the in-house developed Respiratory Activity MOnitoring System (RAMOS), which provides online monitoring of CO₂, O_2, and pressure under gassed conditions. The RAMOS was combined with a gas mixing system to test CO₂ and O₂ concentrations in a range of 0.25-15.0 vol% and 0.0-2.5 vol%, respectively.

Results

Changing the CO₂ concentration in the gas supply revealed a CO₂ optimum of 3.0 vol% for total organic acid production and 15.0 vol% for succinate production. It was demonstrated that the organic acid composition changed depending on the CO₂ concentration. Furthermore, unrestricted growth of P. vulgatus up to an O₂ concentration of 0.7 vol% in the gas supply was proven. The viability decreased rapidly at concentrations larger than or equal to 1.3 vol% O₂.

Conclusions

The study showed that P. vulgatus requires little CO₂, has a distinct O₂ tolerance and is therefore well suited for industrial applications.

Background

The largest population of bacteria in the human body inhabits the intestine, with about 10^11–12 organisms per mL of colonic contents [1, 2]. The bacterial microbiota of the intestine facilitates the maturation of the immune system, the development of the gut and protects against colonization by pathogens. It also supports the human metabolism by breaking down indigestible polysaccharides into nutrients, vitamins, co-factors, amino acids, and short-chain fatty acids (SCFAs) [3,4,5]. The most common phylum in the human gut is Bacteroidota [2, 6]. Species of Bacteroidota achieve high yields of organic acids [7,8,9] and can be genetically modified [10, 11]. Phocaeicola vulgatus, initially classified as Bacteroides vulgatus [12], is one of the most abundant bacteria within the phylum of Bacteroidota [6]. Even though P. vulgatus has great potential as an industrial platform organism, it has not yet been used for biotechnological processes [10], because the species.

has not been sufficiently characterized in axenic culture. The cultivation of gut microbes is complex, as they are highly adapted to the gastrointestinal ecosystem [2, 13].

The gastrointestinal ecosystem is an environment that offers CO₂ in abundance, as the gas is a by-product of anaerobic fermentation. The CO₂ is then taken up by enterocytes or utilized by other microorganisms [14]. Another aspect of the intestine is the changing O₂ level. The gut epithelium is supplied with O₂ by the vasculature, and the bulk of the lumen is essentially anoxic [15]. Due to the O₂ gradient, even strict anaerobic gut bacteria need response mechanisms, if they encounter higher O₂ concentrations in the intestine or escape the gut environment. Species of Bacteroidota are classified as opportunistic pathogens with a broad range of oxygen tolerance, capable of infecting oxygenated tissues [2]. O₂ can diffuse into the bacterial cells and inactivate enzymes with a radical in the active center [15]. Another common mechanism of O₂-induced damage includes the formation of reactive oxygen species (ROS) in the form of superoxide and hydrogen peroxide [16]. ROS are formed, when molecular O₂ oxidizes reduced metals and thiols. Anaerobes protect themselves from ROS with the same defensive tactics initially identified in aerobes [15]. The arsenal of the genus Bacteroides against O₂-induced damage contains e.g., peroxidases, rubrerythrins, and catalases [15]. In an oxygenated environment, Bacteroidota switch to a stationary-like state to protect themselves from damage by ROS. In this condition, the translation of biosynthesis genes is downregulated, and growth is impaired [17, 18]. Furthermore, on a transcriptional level, downregulation of potential ROS-producing enzymes, such as fumarate reductase, occurs [19]. Fumarate reductase is responsible for the reduction of fumarate to succinate in P. vulgatus [3]. The genetically related Bacteroides fragilis species can also express cytochrome bd oxidase [20]. In this way, O₂ serves as a terminal electron acceptor in the respiratory chain. Ultimately, cytochrome bd oxidase can stimulate O₂-dependent growth in micro-aerobic conditions.

The carbon metabolism is a significant aspect of a better understanding of P. vulgatus. One of the three glycolytic pathways is used in related Bacteroides to obtain phosphoenolpyruvate (PEP), a key metabolite in glycolysis. PEP is then converted to products such as organic acids and gases [3]. This conversion is done using anaerobic respiration and fermentation via oxaloacetate, malate, and fumarate [3]. Bacteroides use anaerobic respiration, since it is generally more efficient than fermentation [3]. The main products of the anaerobic respiration of Bacteroides are acetate, propionate, succinate, lactate, formate, CO₂, and H₂ [3]. The high CO₂ levels in the gut are advantageous for anaerobic respiration [3]. Thereby, Bacteroides can establish a primitive electron transport chain based on reducing fumarate to succinate [3]. As a result, CO₂ is fixed to fumarate, and the bacterium can regenerate CO₂ from succinate under CO₂-limiting conditions [3]. Through this process, propionate can be produced [3]. Additionally, lactate is formed by reducing pyruvate via lactate dehydrogenase [10]. Prevotella copri, another P. vulgatus-related species, can convert pyruvate to formate, CO₂, Fd_red (possible site for hydrogen formation), and acetyl-CoA, which is converted in the next step to acetate [21].

The SCFAs acetate, propionate, succinate, formate, and lactate, a short-chain hydroxy fatty acid, denoted as an SCFA in this study, are the main products of P. vulgatus. SCFAs are important for gut microbes, to regulate the production of redox equivalents in the anaerobic environment of the intestine [22]. Moreover, SCFAs benefit the human host and serve as signal molecules or energy substrates [23, 24]. Currently, most SCFAs for the chemical industry are produced based on fossil fuels. However, as Bacteroidota produce numerous SCFAs, there is the potential for a sustainable production.

Although only limited efforts for characterization of P. vulgatus in terms of CO₂ requirement and O₂ tolerance have been conducted so far, Franke and Deppenmeier [21] and Reilly [25] have shown that P. vulgatus requires CO₂ or bicarbonate supplementation for growth. However, as the study of Franke and Deppenmeier [21] focused on P. copri, they found a more pronounced CO₂ dependency of P. copri, compared to P. vulgatus. Furthermore, Baughn and Malamy [20] stated that P. vulgatus could cope with oxygen concentrations of 0.03 vol% without suffering damage. However, the optimum CO₂ level and maximal oxygen tolerance still need to be unraveled.

This study aims to advance the characterization of P. vulgatus under anaerobic cultivation conditions, determining the CO₂ requirement and O₂ tolerance for growth and organic acid production. The characterization was conducted utilizing the Respiration Activity MOnitoring System (RAMOS). The RAMOS is a small-scale shaken cultivation system that, in contrast to traditional serum flasks, allows for non-invasive online measurement of CO₂, O_2, and pressure [26,27,28]. Furthermore, the influence of different gas concentrations was determined by combining the RAMOS with a gas mixing system. Thereby, up to four different gas streams were supplied to the RAMOS. This study determines the feasibility of P. vulgatus as an efficient organic acid producer under different influences of CO₂ and O₂.

Results

Influence of CO₂ on growth and acid production

In the first set of experiments, the influence of different concentrations of CO₂ in the gas supply on the cultivation of P. vulgatus in shake flasks was tested. During the experiments, the CO₂ concentration in the gas supply varied between 0.25 and 15.0 vol% with the aid of the gas mixing system. For these cultivations, online gas transfer rates are depicted in Fig. 1.

Fig. 1

Effect of different CO₂ concentrations on gas transfer rates of P. vulgatus shake flask cultivations. Online data of (a) carbon dioxide transfer rate (CTR) and (b) total gas transfer rate (TGTR). Shadows indicate standard deviations of four biological replicates. Measurement of CO₂ was not possible above 5.0 vol% CO₂ in the ingas, due to limited sensor range. Different successively conducted experimental runs are indicated by different symbols in the legend (*,-,+,~,#). Experimental setup is illustrated in Fig. 5a. Hydrogen transfer rate (HTR) plotted over CTR corresponding to this experiment can be found in Fig. S1. Medium: DMM-G, c_Glucose = 6 g L^− 1, c_buffer = 50 mM MOPS, T = 37 °C, n = 100 rpm, V_L = 50 mL, initial OD_600nm = 0.2, initial pH after inoculation = 6.9–7.15, vvm = 0.2 min^− 1, different gas mixtures of CO₂ in N₂, as indicated in legend

No carbon dioxide transfer rate (CTR) is displayed for the CO₂ concentrations of 10.0 and 15.0 vol%, as the limit of the CO₂ sensors used in this study was reached at 5.0 vol%. The CTR curves in Fig. 1a indicate different values at the first measurement point after 1 h, depending on the CO₂ concentration in the gas supply. For 0.25 vol% CO₂ (black squares) in the gas supply, no substantial increase or decrease in the CTR is visible throughout the cultivation. The following concentration of 0.5 vol% CO₂ (red circles) slightly increases, before decreasing to 0 mmol L^− 1 h^− 1. At 0.75 (green triangles) and 1.0 vol% (blue inverted triangles) CO₂ in the gas supply, the CTR maximum rises with increasing CO₂ concentration and is attained after 8.8 h. For 3.0 vol% CO₂ (light blue diamonds), the CTR maximum is higher than the maximum of 1.0 vol%, but CO₂ production also increases earlier than for the lower CO₂ concentrations, and the maximum is reached earlier. With a CO₂ concentration in the gas supply of 4.0 vol%, the CTR maximum attains a value of 1.6 mmol L^− 1 h^− 1, the highest CTR maximum of all conditions. In conclusion, the higher the CO₂ concentration in the gas supply up to the highest measured concentration of 4.0 vol%, the higher the CTR maximum. Figure 1b presents the different total gas transfer rate (TGTR) progressions. Generally, the same trends represented for the CTR maxima can be seen for the TGTR maxima. The higher the CO₂ concentration in the gas supply, the higher the TGTR maxima. This trend continues until a CO₂ concentration of 10.0 vol% (orange triangles) is obtained. However, for 15.0 vol% CO₂ (green stars), the TGTR maximum is slightly lower, but with a higher standard deviation. In general, the CTR peaks are lower than the TGTR peaks. The highest measurable CTR maximum reaches 1.6 mmol L^− 1 h^− 1, while the corresponding TGTR maximum attains 3.5 mmol L^− 1 h^− 1. CO₂ only contributes between 27.2 and 45.7% to the total gas production, depending on the CO₂ concentration in the gas supply.

Fig. 2

Effect of different CO₂ concentrations on offline data of P. vulgatus shake flask cultivations. These data refer to the experiment shown in Fig. 1. Offline data of (a) HPLC analysis of produced organic acids, including formate, succinate, acetate and lactate and remaining glucose from four biological replicates with standard deviation. (b) Carbon balance in % as function of the CO₂ concentration in the gas supply. The start of the fermentation was set to 100%. No carbon balance is calculated for 10.0 and 15.0 vol% CO₂, as CO₂ could not be measured in this range. Initial samples were drawn after inoculation. (c) Final OD_600nm and final pH from four biological replicates with standard deviation. Experimental setup is illustrated in Fig. 5a. Medium: DMM-G, c_Glucose = 6 g L^− 1, c_buffer = 50 mM MOPS, T = 37 °C, n = 100 rpm, V_L = 50 mL, initial OD_600nm = 0.2, initial pH after inoculation = 6.9–7.15, vvm = 0.2 min^− 1, different gas mixtures of CO₂ in N₂, as indicated in legend

In Fig. 2, the offline data of the cultivation is shown. High-performance liquid chromatography (HPLC) measurements were performed for the key metabolites formate, succinate, acetate, lactate, and glucose. Propionate could not be detected in these experiments. Formate (light blue hexagons), succinate (brown triangles), and acetate (purple circles) production rise with increasing CO₂ concentration in the gas supply until 4.0 vol% (Fig. 2a). Lactate production (orange squares) is approximately two-fold higher with lower CO₂ concentrations of up to 3.0 vol% in the gas supply and decreases with higher CO₂ concentrations. The main acids produced are succinate, acetate, and lactate, with only small amounts of formate generated. The highest total amount of SCFAs is produced at 3.0 vol% CO_2, whereas the lowest is formed at 0.25 vol% CO₂. Glucose (pink stars) is completely consumed for conditions with 3.0 and 10.0 vol% CO₂ in the gas supply. Figure 2b depicts the molar carbon balance. The molar carbon balance (calculated according to Eqs. 1–2) is closed with a maximum deviation of 8.5%. The biomass accounts for 25 to 46% of the total carbon, depending on the CO₂ concentration in the gas supply. Moreover, low amounts of CO₂ are formed, reaching a maximum of 5.9% of the total carbon. In Fig. 2c, the final pH values, as well as the final optical density (OD_600nm), are displayed. The final pH values decline for increasing CO₂ concentrations between 0.25 and 3.0 vol%. From there, the final pH values rise until 10.0 vol% and decrease again for 15 vol% CO₂. Overall, the final pH values are low, between 4.8 and 6.7. The final OD_600nm reaches the lowest value for 0.25 vol% CO₂ with 1.8 and increases from there. It changes with no clear trend between 2.0 and 3.2 for the higher CO₂ concentrations in the gas supply.

Influence of O₂ on growth and organic acid production

To determine the O₂ tolerance of P. vulgatus in shake flasks, O₂ concentration in the gas supply was varied between 0 and 2.5 vol%. The CO₂ concentration in the gas supply was kept constant at 4.0 vol%.

Fig. 3

Effect of different O₂ concentrations on gas transfer rates of P. vulgatus shake flask cultivations. Online data of (a) carbon dioxide transfer rate (CTR) and (b) total gas transfer rate (TGTR) and (c) oxygen transfer rate (OTR). Shadows indicate standard deviations of four biological replicates. Different successively conducted experimental runs are indicated by different symbols in the legend (*,+,-,~). Dashed horizontal line in (c) indicates an OTR of 0 mmol L^− 1 h^− 1. Experimental setup is illustrated in Fig. 5b. Hydrogen transfer rate (HTR) plotted over CTR corresponding to this experiment can be found in Fig. S2. Medium: DMM-G, c_Glucose = 6 g L^− 1, c_buffer = 50 mM MOPS, T = 37 °C, n = 100 rpm, V_L = 50 mL, initial OD_600nm = 0.29, initial pH after inoculation = 6.9–7.1, vvm = 0.2 min^− 1, different gas mixtures of O₂ & 4% CO₂ in N₂, as indicated in the legend

In Fig. 3, the online gas transfer rates are shown. The CTR curves in Fig. 3a start at approximately the same value after 1 h for all tested O₂ concentrations. The CTR curves of the lower O₂ concentrations between 0 vol% (black squares) and 0.7 vol% (purple diamonds) rise until reaching their maximum after 6 h, with no distinct trend between the four concentrations. The CTR curves of higher O₂ concentrations between 1.3 (orange hexagons) and 2.5 vol% (brown pentagons) directly decline, until all curves reach 0 mmol L^− 1 h^− 1. The TGTR curves, depicted in Fig. 3b, display the same trends. As already observed in the previous experiments, the maxima of the TGTR curves are substantially higher than those of the CTR curves, with the TGTR attaining a maximal value of 4.3 mmol L^− 1 h^− 1 (0.7 vol%, purple diamonds). The corresponding CTR maxima of 0.7 vol% O₂ reaches 1.9 mmol L^− 1 h^− 1. In Fig. 3c, the progression of the oxygen transfer rate (OTR) is presented for the O₂ concentrations between 0.2 and 2.5 vol%. For 0 vol%, no calculation of the OTR was possible. The O₂ concentrations of 0.2 (red circles) and 0.4 vol% (green triangles) remain at an OTR of approximately − 0.1 mmol L^− 1 h^− 1 throughout the cultivation. The higher O₂ concentrations of 0.7 to 2.5 vol% indicate a rising OTR curve in the positive range over the first hours. 2.0 vol% (blue stars) and 2.5 vol% (brown pentagons) O₂ reach an OTR maximum after 2.8 h, while 0.7 vol% (purple diamonds) and 1.3 vol% (orange hexagons) O₂ conditions do not indicate a clear OTR maximum. In the following cultivation time, the OTR curves of 0.7 to 2.5 vol% O₂ decrease until reaching about 0 mmol L^− 1 h^− 1 at the end of the cultivation. However, it must be noted that the detection limit of the OTR is reached here.

Fig. 4

Effect of different O₂ concentrations on offline data of P. vulgatus shake flask cultivations. These data refer to the experiment shown in Fig. 3. Offline data of (a) HPLC analysis of produced organic acids including propionate, formate, succinate, acetate and lactate and remaining glucose from four biological replicates with standard deviation. (b) Carbon balance in % as function of the O₂ concentration in the gas supply. The start of the fermentation was set to 100%. Initial samples were drawn after inoculation. (c) Final OD_600nm and final pH from four biological replicates with standard deviation. Experimental setup is illustrated in Fig. 5b. Final OD_600nm and final pH each differed statistically significant for the different oxygen concentrations, for final OD_600nm: p < 0.001 and for final pH: p < 0.001. Medium: DMM-G, c_Glucose = 6 g L^− 1, c_buffer = 50 mM MOPS, T = 37 °C, n = 100 rpm, V_L = 50 mL, initial OD_600nm = 0.29, initial pH after inoculation = 6.9–7.1, vvm = 0.2 min^− 1, different gas mixtures of O₂ & 4% CO₂ in N₂, as indicated in the legend, N = 4

In Fig. 4, the offline data of this set of experiments are depicted. HPLC analysis of SCFAs and glucose is outlined in Fig. 4a. Almost no propionate (dark blue reverse triangles) and formate (light blue hexagons) are formed between 0 and 0.7 vol% O₂, and production stops completely with higher O₂ concentrations. Succinate concentrations (brown triangles) decrease slightly between 0.2 and 0.7 vol% O₂. At more elevated O₂ concentrations, the concentrations decline rapidly. Acetate concentrations (purple circles) remain constant between 0 and 0.7 vol% O₂ and show a substantial decrease at higher O₂ concentrations. Lactate production (orange squares) is highest at 0.4 vol% O₂ and strongly lowers between 1.3 and 2.5 vol% O₂. Almost no acids are formed at O₂ concentrations higher than 0.7 vol%. The remaining glucose (pink stars) decreases between 0 and 0.7 vol% O₂. Only low amounts of glucose have been consumed for O₂ concentrations between 1.3 and 2.5 vol%. The molar carbon balance presented in Fig. 4b is closed with a maximal deviation of 6.5%. Cell dry weight (CDW) accounts on average for 32.8% of the total carbon for O₂ concentrations between 0 and 0.7 vol% and 6.6% at O₂ concentrations between 1.3 and 2.5 vol%. As in the set of experiments before, CO₂ contribution to the total carbon is low, with a maximum of 6.4%. Figure 4c illustrates the final pH values and the final OD_600nm. A Mann-Whitney-U-Test was conducted to assess the influence of different oxygen concentrations on the final OD_600nm and final pH values. Final OD_600nm and final pH values were split in two groups for the statistical analysis, low (0-0.7 vol%) and high oxygen concentration levels (1.3–2.5 vol%). The distributions between both groups differed, according to Kolmogorov-Smirnov p < 0.001. The final OD_600nm and final pH values differed statistically significant for the two oxygen concentration levels. The final pH values reveal a significant increase between 0.7 and 1.3 vol% O₂, while the data of the final OD_600nm displays a substantial decrease between those O₂ concentrations. To exclude the pH value as a responsible parameter for the observed results, the influence of different O₂ concentrations was validated in another set of experiments with higher initial pH values (Fig. S3).

Discussion

Influence of CO₂ on growth and organic acid production

With increasing CO₂ concentration in the gas supply up to a concentration of 10.0 vol% (Fig. 1), CO₂ production rises. However, interpretation of CO₂ production via CTR must be conducted cautiously, as pH changes can also contribute to CTRs. While pH values decrease, CO₂ is released due to the chemical CO₂/HCO₃⁻ balance [29]. During cultivation, the pH values decrease caused by the strain’s organic acid production. With increasing CO₂ concentrations, especially observable from 3.0 vol%, the maximum of the CTR and TGTR curves is reached faster. This behavior can be observed until a CO₂ concentration of 10.0 vol% is obtained. Lower CO₂ concentrations, especially 0.25-1.0 vol%, prolong the lag phase of gas formation. Caspari and Macy [30] observed a prolonged lag phase for genetically related Bacteroides fragilis for CO₂/HCO₃⁻ concentrations below 10 mM (corresponding to about 12 vol% CO₂ in the gas supply). They further observed that the maximum growth rate and cell yield decreased. Interestingly, the limiting CO₂ concentrations observed for B. fragilis are substantially higher than those observed for P. vulgatus in this study. Furthermore, Franke and Deppenmeier [21] observed that P. vulgatus is less dependent on CO₂ and HCO₃⁻ as the genetically related Prevotella copri. While P. copri only reached maximal biomass formation above 20 mM HCO₃⁻, P. vulgatus reached maximal growth yields at lower HCO₃⁻ concentrations of ~ 10 mM. Reilly [25] studied the CO₂ optimum for P. vulgatus cultivating on agar plates and found the optimum between 0.25 and 40 vol% CO₂ in the gas supply. Above 40 vol% CO₂, growth was inhibited. This study already observed reduced gas production at 15 vol% CO₂.

The TGTR is higher than the CTR, confirming the formation of another gas besides CO₂. Gas chromatography (data not shown) has proven that the only other gas besides CO₂ is H₂, which has also been revealed in other studies for P. vulgatus [31,32,33]. In Fig. S1, the hydrogen transfer rate (HTR) for 0.75 to 4.0 vol% CO₂, calculated from TGTR and CTR, is plotted over the CTR. The decreasing slope with increasing CO₂ concentration points out that while the CO₂ concentration in the gas supply decreases, more H₂ is formed, in relation to CO₂. Since the metabolic pathways of P. vulgatus are not yet fully understood, it is unclear, what causes this shift from CO₂ to H₂ production.

Since at a concentration of 0.25 vol% CO₂, the OD_600nm is lower than for the higher CO₂ concentrations, some amount of CO₂ seems to be utilized for biomass production. Caspari and Macy [30] could as well observe for B. fragilis a decreasing final OD_600nm for concentrations of ~ 12 vol% CO₂ or lower in the gas supply. The pH (Fig. 2) decreases strongly at all CO₂ concentrations higher than 0.25 vol%. Final values are in an inhibitory range for P. vulgatus. The literature demonstrates that pH values below 6.0 have a growth inhibitory effect on P. vulgatus, and growth stops entirely at a pH value below 5.3 [4, 34]. The second lowest final pH is reached at 3.0 vol% CO₂, affiliating with the highest organic acid production. The lowest final pH is reached at 15.0 vol% CO₂, probably caused by the high CO₂ concentration, as it does not correlate with the highest organic acid production. The low final pH is a factor for growth inhibition, and another factor may be product inhibition by the organic acids formed by P. vulgatus.

At increasing CO₂ concentrations, succinate, acetate, and formate production are rising (Fig. 2). Lactate formation increases with decreasing CO₂ concentration. The high lactate formation at low CO₂ concentrations may be an easy way for P. vulgatus to balance the production of redox equivalents [22]. Lactate production requires only the enzyme lactate dehydrogenase and is the most straightforward metabolic pathway [10]. Enhanced lactate production at lower CO₂ concentrations and an increased acetate concentration at elevated CO₂ concentrations were also observed in the study of Caspari and Macy [30] for B. fragilis. Succinate formation rises with increasing CO₂ concentration in the gas supply, since the organism must first accumulate CO₂ to start succinate formation [3]. Under CO₂ deficiency, succinate could be converted to propionate, to release bound CO₂ and utilize the CO₂. However, in this study, no propionate could be detected with HPLC measurements, even at low CO₂ concentrations, e.g., 0.25 vol%. At low CO₂ concentrations, little succinate was produced, so almost no succinate was available for conversion to propionate. Acetate and formate do not increase as much as succinate with elevated CO₂ concentration. Metabolic limitation is evident for all cultivations with CO₂ concentrations below 3.0 or above 10.0 vol%, because glucose is not completely metabolized.

Although succinate production increased with increasing CO₂ concentration, with 15.0 vol% CO₂ in the gas supply, P. vulgatus achieved only 0.36 mol succinate/mol glucose. Therefore, P. vulgatus is less efficient than other succinate producers, such as A. succinogenes with 1.42 mol succinate/mol glucose [35] or A. succiniciproducens with 1.33 mol succinate/mol glucose [36]. P. vulgatus forms high amounts of acetate and lactate besides succinate and low amounts of formate, resulting in a yield for total SCFA of 1.1 mol acid/mol glucose. The total SCFA yield is close to the succinate yield of the beforementioned producers. The carbon balance is closed for these experiments, demonstrating that all major products contributing carbon to the balance have been considered. In this study, the optimal CO₂ production for acid production by P. vulgatus was 3.0 vol%. Biomass growth was highest in the range of 0.5 to 4.0 vol%.

Influence of O₂ on growth and organic acid production

P. vulgatus reveals an unimpaired growth in the range of 0-0.7 vol% O₂ in the gas supply (Fig. 3). Within this range, there is little deviation between all measured values, online and offline. An abrupt decline in viability is visible in the data, while increasing the O₂ concentration from 0.7 to 1.3 vol%. This decline is evident for all measured values (Figs. 3 and 4). The exhibited O₂ tolerance is higher than the published data of Bacteroides species, which specifies the O₂ tolerance between 0.03 and 0.4 vol%. Only for Bacteroides melaninogenicus a higher O₂ tolerance of up to 2.5 vol% was evaluated [15, 20]. It should be noted that the addition of L-cysteine as a reducing agent to the medium can reduce the oxidative damage on P. vulgatus. The relatively high O₂ tolerance of P. vulgatus helps the species to maintain its high proportion in the gut, as O₂ can be encountered in low concentrations of < 1 vol% [15, 37, 38]. However, the decline in viability, acid, and gas production above 0.7 vol% O₂ is due to the damage that O₂ causes to anaerobic bacteria. Molecular O₂ diffuses into the cell and inactivates enzymes with a radical in its active center [15]. Another mechanism may be the formation of ROS, which can react with many molecules in the cell and lead to DNA, lipid, and disulfide bond damage [16].

As observed in the experiments with changing CO₂ concentrations, the TGTR is substantially higher than the CTR. Comparing HTR with CTR (Fig. S2) discloses that the O₂ concentration has only a neglectable influence on the CO₂/H₂ ratio, compared to the influence of the CO₂ concentration in the gas supply.

Interestingly, despite the anaerobic character of P. vulgatus, a positive OTR (oxygen consumption) could be measured (Fig. 3). However, the OTR values are lower than those of aerobic microorganisms [27]. The OTR favor the assumption that O₂ was reacting with medium components (e.g. L-cysteine) or was utilized in small amounts by P. vulgatus during the first hours of the cultivation. The enzyme cytochrome bd oxidase is a possible consumer of O₂ [20], which enables the use of molecular O₂ as a final electron acceptor instead of fumarate. In addition, cytochrome bd oxidase could act as a buffer enzyme to ensure electron flow through the anaerobic respiratory chain, despite O₂ being present and prevent O₂ from damaging other components in the cell. The study of Baughn and Malamy [20] finds evidence for unrestricted growth of P. vulgatus up to 0.03 vol% O₂. The decrease in viability, when the O₂ concentration reaches values above 0.7 vol%, is caused by the O₂ damage exceeding the capacity of P. vulgatus protective mechanisms. In order to examine the oxidative stress response of P. vulgatus, L-cysteine should not be added to the medium. In this study, growth and organic acid production of P. vulgatus in a best-case scenario was investigated. Therefore, L-cysteine was added to the medium.

Considering the acid production (Fig. 4), succinate and propionate production decrease strongly with O₂ concentrations of 0.7 vol% or higher. One possible explanation for this decrease is the O₂-induced downregulation of the potentially ROS-forming enzyme fumarate reductase, which is necessary to produce succinate and propionate. For future experiments, expression studies for fumarate reductase are planned. The same behavior is observed for acetate production, as the enzymes crucial for acetate production are damaged by O₂ [15]. The damaged acetate pathway leads to a lack of redox equivalents in the form of Fd_red. The lack of redox equivalents could be the reason why lactate production is increased for 0.4 and 0.7 vol% O₂. Lactate formation is an easy way for P. vulgatus to balance the production of redox equivalents [22].

The final pH is low, with a value of avg. 4.8 for the O₂ range 0-0.7 vol% (Fig. 4c). The pH value influences P. vulgatus growth behavior. Therefore, the pH value as a responsible parameter for the observed results was excluded (Fig. S3). The slight increase in glucose consumption and final OD_600nm from 0 to 0.7 vol% O₂ (Fig. 4a and c) lead to the conclusion that more glucose is used to produce biomass. Due to the impact of O₂, the enzymes to produce SCFAs might be damaged or downregulated. Nevertheless, through the low amounts of produced SCFAs and the low growth, some metabolic activity is indicated even at O₂ concentrations above 0.7 vol%. The activity probably occurred during the first hours of cultivation, when the protective mechanisms of P. vulgatus had not yet reached their capacity.

Conclusions

Concluding this study, the optimum of tested CO₂ concentrations for total organic acid and for succinate production by P. vulgatus is 3.0 vol% and 15.0 vol%, respectively. The O₂ tolerance lies above 0.7, but below 1.3 vol%. P. vulgatus is inhibited by pH, the produced SCFAs, or a combination of both. The species could achieve higher titers of succinate in a pH-controlled fermentation, as demonstrated by Isar et al. [39] and Isar et al. [40] for B. fragilis. Other important next steps are genetic modifications, which have been proven to increase lactate production for P. vulgatus by Lück and Deppenmeier [10]. However, acid production must be shifted from acetate and lactate to succinate, the most valuable product. To evaluate the exact O₂ tolerance of P. vulgatus, a continuous fermentation with a gradually raised O₂ concentration over time would pose the best solution. Additionally, it would be important to have a medium without reducing agents, e.g. L-cysteine.

Determining the CO₂ requirement and O₂ tolerance for growth and organic acid production of P. vulgatus exhibits the potential for an industrial application. However, the species cannot yet compete with established industrial SCFA producers. The species requires little CO₂ and has a certain O₂ tolerance. These results may contribute to a faster optimization of P. vulgatus as an organic acid producer and display that strictly anaerobic bacteria can tolerate more O₂ than expected.

Methods

Strain and media

The research group of Prof. Deppenmeier (Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany) kindly provided the strain Phocaeicola vulgatus DSM 1447, obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Brain heart infusion medium (BHI) for cryogenic stocks was acquired as BD Difco™ (Thermo Fisher, Waltham, USA). BHI powder contained: 7.7 g L^− 1 calf brain extract, 9.8 g L^− 1 beef heart extract, 10 g L^− 1 protease peptone, 2 g L^− 1 dextrose, 5 g L^− 1 sodium chloride, and 2.5 g L^− 1 disodium phosphate, dissolved in deionized water. An active growing BHI culture was used to prepare cryogenic stocks after 24 h of cultivation by mixing 50 vol% culture broth with 50 vol% anaerobic sucrose solution (500 g L^− 1) and freezing 1.8 mL aliquots at -80 °C. For all main and precultures, a defined minimal medium with glucose (DMM-G) was used. DMM-G composition was based on Varel and Bryant [41] and Lück and Deppenmeier [10] with 3-(N-morpholino)propanesulfonic acid (MOPS) buffer instead of bicarbonate buffer. If not stated otherwise, DMM-G medium components were obtained from Carl Roth (Karlsruhe, Germany). The medium consisted of 13 individual stock solutions: Base components (pH 7.4), glucose, calcium chloride, magnesium chloride, iron(II) sulfate, SL6-trace elements, Wolin’s vitamin solution, butyrate, vitamin K1, hemin, resazurin (Thermo Fisher, Waltham, USA), L-cysteine hydrochloride, and MOPS buffer (pH 7.4). Stock solutions were stored separately, as premature mixing would have caused precipitation. The base components stock comprised ammonium chloride, dipotassium phosphate, monopotassium phosphate, and sodium chloride. The SL6-trace elements included boric acid, cobalt(II)chloride hexahydrate, copper(II)chloride dihydrate, manganese(II)chloride tetrahydrate (Merck, Darmstadt, Germany), nickel(II)chloride, sodium molybdate dihydrate and zinc sulfate heptahydrate (Merck, Darmstadt, Germany) and were set to pH 7.4 with 5 M sodium hydroxide. The Wolin’s vitamin stock solution contained α-lipoic acid, biotin, folate (Sigma Aldrich, St. Louis, USA), nicotinamide, p-aminobenzoic acid (Sigma Aldrich, St. Louis, USA), pantothenic acid (AppliChem, Darmstadt, Germany), pyridoxine hydrochloride (Sigma Aldrich, St. Louis, USA), riboflavin (Sigma Aldrich, St. Louis, USA), thiamine hydrochloride and vitamin B12. Table S1 lists the final concentrations of all components in the DMM-G medium. Base components, glucose, calcium chloride, magnesium chloride, iron(II) sulfate, and SL6-trace elements stocks were sterilized at 121 °C for 20 min. The remaining heat-sensitive stock solutions were sterile-filtered with 0.22 μm polyethersulfone filters (Merck, Darmstadt, Germany). To prevent premature oxidation, reducing agent L-cysteine was sterile-filtered and stored anaerobically in a serum bottle with a nitrogen atmosphere. Wolin’s vitamin solution, vitamin K1, hemin, and resazurin stock solutions were stored light-protected at 4 °C after sterilization. All other stock solutions were stored at room temperature.

Cultivation conditions

Precultures were grown in serum bottles with a total volume of 250 mL. The serum bottles were filled with 50 mL DMM-G medium and sealed gas-tight with a rubber stopper and clamp. Afterward, the serum bottles were gassed with N₂ for 20 min to ensure an anaerobic atmosphere. In the next step, CO₂ was added to the serum bottles with a sterile syringe to obtain a CO₂ headspace concentration of 10 vol%. Afterward, 0.1 mL L-cysteine solution was added as a reducing agent, and in the final step, the medium was inoculated with 500 μL cryogenic culture, both with a sterile syringe. The serum bottles were inoculated in a temperature-controlled shaker for 24 h at 37 °C with a shaking diameter of 50 mm and a shaking frequency of 100 rpm. The main experiments were performed in a RAMOS device designed by Anderlei and Büchs [27]. The RAMOS is a non-invasive online monitoring device for measuring CO₂, O_2, and pressure for up to eight shake flasks. Anderlei and Büchs [27], Anderlei et al. [28], and Munch et al. [26] provide a schematic overview of the RAMOS setup and gas measurement phases as well as the calculation of the carbon dioxide transfer rate (CTR), oxygen transfer rate (OTR) and total gas transfer rate (TGTR). Measurement of the increase of produced gases is conducted with pressure sensors (26PCA, Honeywell, Charlotte, USA) and infrared carbon dioxide sensors (MSH-P − CO2, 126 Dynament, Mansfield, UK). The RAMOS device is a proven system and has already been operated with syngas [42] or ethylene [43] in the ingas. As the gas measurement phases needed to be adapted to the specific microorganism, time and gas flows were set for both CO₂ and O₂ experiments as follows: 20 min measurement phase without gas flow, 2.38 min high gas flow rate at 22.5 mL min^− 1, and 40 min low gas flow rate at 10 mL min^− 1. Before inserting the shake flasks in the RAMOS device, they were filled with 45 mL sterile DMM-G medium and gassed overnight with the respective cultivation gas at 37 °C in a shaker (ISF1-X, Adolf Kühner AG, Birsfelden, Switzerland) at 100 rpm, with a shaking diameter of 50 mm. The system was tested for gas tightness to ensure anaerobic conditions and to prevent false gas measurements. As a reducing agent, 0.1 mL L-cysteine was inserted with a sterile syringe into each flask before inoculation with 5 mL preculture. Initial samples were drawn after inoculation, and final samples at the end of the cultivation.

Gas mixing system

The gas mixing system consists of up to four mass flow controllers (MFCs) and one control unit, which can be connected to the RAMOS. Therefore, the signal from the RAMOS controls the gas mixing system, to switch between the aforementioned different gas measurement phases.

Fig. 5

Schematic illustration of the experimental setup of the gas mixing system. Change of the (a) CO₂ or (b) O₂ concentration in the gas supply. In case of (b), the dilution of N₂ and CO₂ by O₂ remains very low. Four mass flow controllers (MFC) were used with following ranges, for (a): MFC 1 & 3: 50–500 mL/min (calibrated with N₂), MFC 2: 5–50 mL/min (calibrated with O₂), MFC 4: 0.5-5 mL/min (calibrated with N₂) and for (b): MFC 1: 5–50 mL/min (calibrated with O₂), MFC 2 & 3: 50–500 mL/min (calibrated with N₂) and MFC 4: 2–20 mL/min (calibrated with N₂). This setup was chosen, as experiments at two different gas compositions can be performed with four shake flasks each

The schematic setup of the gas mixing system with gas supply lines can be found in Fig. 5a for different CO₂ concentrations and Fig. 5b for different O₂ concentrations. The setup was designed so that four shake flasks within the RAMOS can be operated with one gas concentration and the other four with a second gas concentration. After adjusting the gas supply lines, the gas flows were set prior to the experiments. Desired gas concentrations were configured as a percentage of the total maximum flow of the MFC on the control unit. Afterward, the settings of the MFCs were tested by measuring the total flow from the gas mixing system with a gas flow calibrator, Defender 530 + L (Mesa Laboratories, Inc., Lakewood, USA). Before the experiment, a calibration curve was created for the CO₂ and O₂ sensors within the RAMOS device. With the help of the calibration curve, the concentrations set by the gas mixing system of CO₂ and O₂ were checked and, if necessary, adjusted.

Hydrogen transfer rate

Besides CO₂, also H₂ is produced. As no other gases are formed, the hydrogen transfer rate (HTR) was calculated by subtracting the CTR from the TGTR.

Offline analysis

Initial and final samples were collected and directly used for OD_600nm measurement at a wavelength of 600 nm with a Genesys 20 spectrophotometer (Thermo Scientific, Germany). Samples were diluted with 9 g L^− 1 NaCl. To correlate the optical density and CDW, the equation $CDW = 0.563 \cdot O{D_{600nm}}$, derived in [44, in revision] for P. vulgatus, was used. Samples not used for optical density measurement were centrifuged at 18,000 rpm for 5 min. The supernatant was used for HPLC and pH measurement. The pH was measured with a pH electrode (Mettler-Toledo, Columbus, USA). The remaining sample supernatant was stored at -80 °C for further HPLC analysis. Therefore, samples were thawed and filtered with 0.2 μm cellulose acetate filters (Merck, Darmstadt, Germany). The SCFAs, acetate, succinate, lactate, propionate, formate, and remaining glucose were measured by HPLC. The HPLC device (Dionex, Sunnyvale, USA) was equipped with an organic acid resin column of 300 × 8 mm dimensions (CS-Chromatography, Langerwehe, Germany) and set to 60 °C. As an eluent, 5 mM H₂SO₄ at a flow rate of 0.8 mL min⁻¹ was applied. UV/VIS and a refractive index detector were used during HPLC measurement.

Carbon balances

Carbon balances were calculated for all experiments with the following Eq. 1:

$$Carbo{n_{inX}}\left[ {\frac{{mmol}}{L}} \right] = \frac{{Carbon\,molecule{s_{inX}}\left[ - \right]}}{{{M_X}\left[ {\frac{g}{{mmol}}} \right]}} \cdot {c_X}\left[ {\frac{g}{L}} \right]$$

(1)

Where X is the specific compound, c is the concentration [g L^− 1], M_X is the molar mass of the specific compound [g mol^− 1], Carbon molecules_{in X} is the number of carbon atoms in the specific compound [-], and Carbon_{in X} is the molar carbon concentration for the compound [mmol L^− 1].

The compounds glucose, acetate, lactate, succinate, propionate, formate, CO_2, and biomass of every sample were considered. Initial and final concentrations of glucose, acetate, lactate, succinate, propionate, and formate were measured by HPLC. The microbial biomass of P. vulgatus cells was based on data from Franke and Deppenmeier [21] of P. copri microbial biomass. Molar carbon from CO₂ was calculated from the CTR integral based on equations in Munch et al. [26]. First, the volumetric molar carbon [mmol L^− 1] for each compound was calculated, and then the values were combined to obtain the total volumetric molar carbon content for every sample. Finally, to achieve relative values for the carbon content of the compounds, the molar carbon value was divided by the total carbon of the sample, as shown in Eq. 2:

$$Carbo{n_{Samplen}}\left[ \% \right] = \frac{{Carbo{n_{in\,X,Sample\,n}}\left[ {\frac{{mmol}}{L}} \right]}}{{Total\,Carbo{n_{Sample\,n}}\left[ {\frac{{mmol}}{L}} \right]}}$$

(2)

Where Sample n is designated to a specific sample number in a specific experiment, Carbon_{in X, Sample n} is the volumetric molar carbon of the specific compound in Sample n [mmol L^− 1], and Total Carbon_{Sample n} is the sum of all carbon in this Sample n [mmol L^− 1].

Software

All graphs were created with OriginPro® version 2021 from OriginLab Corporation (Massachusetts, USA).

Statistical analyses

Statistical Analyses were performed in order to assess the influence of different oxygen concentrations on different cultivation parameters by Mann-Whitney-U-Test using OriginPro® version 2021 from OriginLab Corporation (Massachusetts, USA). Final OD_600nm and final pH values were split in two groups for the statistical analysis, low (0-0.7 vol%) and high oxygen concentration levels (1.3–2.5 vol%). To determine if the distributions between both groups differed, the Kolmogorov-Smirnov-Test was conducted.