A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus
© Ljunggren et al; licensee BioMed Central Ltd. 2011
Received: 21 January 2011
Accepted: 13 September 2011
Published: 13 September 2011
Caldicellulosiruptor saccharolyticus has attracted increased interest as an industrial hydrogen (H2) producer. The aim of the present study was to develop a kinetic growth model for this extreme thermophile. The model is based on Monod kinetics supplemented with the inhibitory effects of H2 and osmotic pressure, as well as the liquid-to-gas mass transfer of H2.
Mathematical expressions were developed to enable the simulation of microbial growth, substrate consumption and product formation. The model parameters were determined by fitting them to experimental data. The derived model corresponded well with experimental data from batch fermentations in which the stripping rates and substrate concentrations were varied. The model was used to simulate the inhibition of growth by H2 and solute concentrations, giving a critical dissolved H2 concentration of 2.2 mmol/L and an osmolarity of 0.27 to 29 mol/L. The inhibition by H2, being a function of the dissolved H2 concentration, was demonstrated to be mainly dependent on H2 productivity and mass transfer rate. The latter can be improved by increasing the stripping rate, thereby allowing higher H2 productivity. The experimentally determined degree of oversaturation of dissolved H2 was 12 to 34 times the equilibrium concentration and was comparable to the values given by the model.
The derived model is the first mechanistically based model for fermentative H2 production and provides useful information to improve the understanding of the growth behavior of C. saccharolyticus. The model can be used to determine optimal operating conditions for H2 production regarding the substrate concentration and the stripping rate.
The question of climate change, together with the increasing scarcity and cost of fossil fuels, has triggered research on the sustainable production of energy carriers, such as biofuels. Although a wide spectrum of alternative fuels and processes are available, it is still not known which of them will succeed in replacing fossil fuels in the long term. Processes for producing some biofuels, such as ethanol and methane, are already highly developed for commercial use, while others, including biohydrogen, require more basic research before they can be produced in an economically feasible way .
The current major drawbacks of fermentative hydrogen (H2) production are its low yield  and the requirement of gas stripping to remove H2 from the liquid . Dark fermentation can be performed at either moderate or elevated temperatures: the productivity is generally higher in the former case , whereas the latter provides higher yields . However, for an economically sustainable biohydrogen process, high productivity should be accompanied by high yields . One solution is to focus on the process design, such as the optimization of H2 removal and substrate concentration . A higher substrate concentration is required to avoid excess amounts of water, since a surplus of process water has a negative effect on both the environmental and economic aspects of the process . It is important to find alternatives to nitrogen (N2) sparging, as this dilutes the H2, leading to a lower-grade product or the need for expensive gas upgrading to remove the N2.
The extreme thermophile Caldicellulosiruptor saccharolyticus may be of industrial interest because of its ability to produce high yields of H2 from a wide variety of sugars [8, 9], ranging from pentose and hexose monomers  to complex (hemi)cellulosic materials [8, 11, 12]. However, the growth of this microorganism, as well as the production of H2 and acetic acid, is subject to substrate and product inhibition . In addition, it is sensitive to increased osmotic pressure , making it necessary to dilute the substrate. Moreover, C. saccharolyticus is inhibited by H2 , which is a general trait of H2-producing organisms. A high dissolved H2 concentration (H2aq) inhibits H2-generating hydrogenases [1, 2], leading to increased nicotinamide adenine dinucleotide, reduced/nicotinamide adenine dinucleotide (NADH/NAD) ratios, resulting in a metabolic shift toward reduced products such as lactate and ethanol [1, 3, 4] and thus decreased H2 productivity.
The critical partial H2 pressure () is usually the parameter coupled to growth inhibition and lactate formation [5, 13, 15–17], although it has been shown that there are mass transfer limitations that cause the fermentation medium to be supersaturated with H2 [18, 19]. Therefore, H2aq should be considered rather than when discussing inhibition by H2. Stripping with N2, which decreases H2aq , can significantly improve the H2 yield . Indeed, optimizing the process should consist of a compromise between minimizing the stripping gas flow rate, henceforth called the "stripping rate," and maintaining a suitable H2 yield and productivity . However, optimization must take H2 productivity into account, since it influences H2aq greatly; otherwise, the optimization will be relevant only to the specific system studied and cannot be applied to other biohydrogen systems. Furthermore, better insight into H2 productivity and liquid-to-gas mass transfer will help considerably in finding technical solutions allowing an economical and feasible fermentation process to be designed.
This paper describes the development of a kinetic Monod-based model for cell growth and product formation by C. saccharolyticus, which takes the liquid-to-gas mass transfer into consideration. The derived model successfully simulates batch fermentations of C. saccharolyticus and predicts the influence of H2aq and osmotic pressure on growth as well as the metabolic shift to lactate production. To the best of our knowledge, this is the first time the inhibitory effects and liquid-to-gas mass transfer of H2 and carbon dioxide (CO2) have been included in a model for fermentative H2 production. The results obtained with the model are relevant for biohydrogen systems other than that described herein using C. saccharolyticus.
Cultivation and fermentation
C. saccharolyticus was cultivated in a N2 atmosphere in modified DSM640 medium as described previously . Cultures for inocula were grown overnight in 250-mL serum flasks containing 50 mL of modified DSM640 medium, together with a 0.4% carbon source and 0.02% cysteine.
Fermentations were performed in a jacketed 3-L reactor (Applikon, Schiedam, the Netherlands) at a working volume of 1 L. The pH was monitored using an ADI 1025 Bio Console Bio Controller (Applikon) and maintained at pH 6.6 (corresponding to neutral pH at 70°C) by the addition of sodium hydroxide (NaOH). The temperature was thermostatically maintained at 70°C ± 1°C, and the stirring rate was set to 350 rpm. Prior to inoculation, the medium was reduced by the addition of 0.1% cysteine. The fermentor was continuously stripped with N2.
Main characteristics of the eight fermentation experimentsa
Glucose concentration (g/L)
Stripping rate (L/hour)
Measurement of dissolved hydrogen concentration
The dissolved H2 concentration, H2aq, was determined in samples of cell suspensions from the bioreactor in serum flasks. The pressure in the flasks was reduced using a vacuum pump to withdraw the fermentation medium (30 to 40 mL) from the fermentor. The sealed flasks were placed in a water bath (20°C) with magnetic stirring to ensure good mixing. After one hour in the water bath, the pressure in the flasks was rapidly raised to atmospheric pressure by inserting a small needle through the cap, allowing air to enter the flasks. After 12 hours, the H2 concentration in the headspace of the serum flasks was measured as described below (Analytical methods). The volume of the collected liquid and the total volume of the individual serum flasks were measured to estimate the original H2aq in the sample.
Headspace samples were analyzed for CO2 and H2 using a Varian CP-4900 Micro GC gas chromatograph (Varian, Inc., Middelburg, the Netherlands) equipped with a thermal conductivity detector (100 mA). The results were analyzed using Galaxie Chromatography Workstation version 220.127.116.11 software (Varian, Inc.). The optical density of aerobic samples was measured at 620 nm using a spectrophotometer (Hitachi U-1100; Hitachi High-Technologies Corp., Tokyo, Japan). Concentrations of acetic acid, lactic acid and ethanol were analyzed using high-pressure liquid chromatography (Waters Corp., Milford, MA, USA). An Aminex HPX-87H ion exchange column (Bio-Rad Laboratories, Hercules, CA, USA) was used at 45°C with a mobile phase of 5 mM H2SO4 at a flow rate of 0.6 mL/minute. The chromatograph was equipped with a refractive index detector (RID-6A; Shimadzu, Kyoto, Japan).
The constants used in the model
10.3 L atm/K/mol
7.40 × 10-9 mol/L/Pa
2.70 × 10-7 mol/L/Pa
0.082 L atm/K/mol
Growth and product kinetics in C. saccharolyticus
where G, Ac and Lac are the concentrations of glucose, acetic acid and lactic acid, respectively, and CO2sol is the concentration of bicarbonate and carbonate, resulting from the CO2 produced by the bacterium. The stoichiometric factor 2 arises from the assumption that for each mole of acid produced, one mole of NaOH is added to maintain the pH. The background osmolarity, 0.10 mol/L, resulting from yeast extract and other nutrients was calculated based on osmolarity measurements presented previously .
where μ, X and rcd are the specific growth rate, the cell mass concentration and the cell death rate, respectively.
Here μmax is the maximum specific growth rate, G is the glucose concentration and KG is the glucose affinity parameter. OSMcrit, H2aqcrit, and nμ are the critical osmolarity, critical dissolved H2 concentration and the exponential parameters describing the level of inhibition, respectively. The critical parameters represent the values at which inhibition is 100%. The kinetic inhibition expressions, as presented by Han and Levenspiel , are valid only for concentrations of the inhibiting agents (OSM or H2aq) lower than the respective critical concentrations. Elevated osmolarity inhibits the growth of C. saccharolyticus , which may lead to a metabolic shift toward the production of lactic acid .
Here RAcF, and RLacFOSM are the factors relating the production rates of acetic acid and lactic acid to the growth rate; INHIB and ACT are the inhibition and activation factors, respectively (see below, Product formation) YGAc and are the stoichiometric yield coefficients of the reactions described above (equation 1); and is the estimated H2 yield coefficient (mol H2/mol glucose). The following inhibition mechanisms were studied.
Here is the overall volumetric mass transfer coefficient for H2 and is the H2 saturation concentration.
where is the overall volumetric mass transfer coefficient for CO2; CO2aq and are the concentration of dissolved CO2 and the saturation concentration of CO2, respectively; K1 and K2 are the dissociation constants; and CO2sol is the total concentration of carbonates. The last two expressions in the CO2 mass balance (equation 18) are related to the reaction of CO2 with water to form carbonates (see Additional file: Liquid-to-gas mass transfer and chemistry of CO2__Theory_experiments_and_results). The expressions and in equations 17 and 18 describe the liquid-to-gas mass transfer of H2 and CO2, respectively (see Additional file: Liquid-to-gas mass transfer and chemistry of CO2__Theory_experiments_and_results).
where YGX is the cell mass yield coefficient parameter (which is the model notation of the constant k in equation 3) and YGAc and YGLac are the stoichiometric yield coefficients (mol/mol substrate) describing the theoretical yields of cell mass, acetic acid and lactic acid, respectively.
Gas phase balances
Here V l and Vg are the liquid and gas volume; k l a is the volumetric mass transfer coefficient for the various compounds; H2aq and CO2aq are the dissolved concentrations of H2 and CO2; and are the dissolved concentrations of the compounds at equilibrium; is the flow rate of N2 into the fermentor; and , and are the effluent flow rates of N2, CO2 and H2, respectively.
Other equations used
Since the pH was controlled during fermentation, it was kept constant at 6.6 in the model. For more details, see Additional file 1: Liquid-to-gas mass transfer and chemistry of CO2__Theory_experiments_and_results.
where k l a i0 is the volumetric mass transfer coefficient at a stripping rate of , k l a i is the volumetric mass transfer coefficient at a stripping rate of and γ is an experimentally determined exponential coefficient.
Experimental design and estimation of kinetic parameters
Here the diffusion coefficients for CO2 () and H2 () are 1.98 × 10-5 and 4.65 × 10-5 cm2/second, respectively, according to Pauss et al. . Only the most important results in the estimation of the mass transfer parameters are given here; the detailed results and the methodology can be found in Additional file: Liquid-to-gas mass transfer and chemistry of CO2__Theory_experiments_and_results.
The estimated model parameters for 5 g of glucose/L experimentsa
6 and 7
95% CI (±)
95% CI (±)
95% CI (±)
95% CI (±)
9 × 10-3
1.6 × 10-2
5.6 × 10-2
2 × 10-4
4.0 × 10-3
2 × 10-2
8 × 10-2
7 × 10-2
9 × 10-2
9 × 10-2
9 × 10-2
5 × 10-3
1.9 × 10-3
4 × 10-4
1.8 × 10-3
3 × 10-4
2.3 × 10-3
7 × 10-4
1.3 × 10-3
2 × 10-4
2.7 × 10-3
5 × 10-4
Finally, the cell death rate (rcd; equation 5), was calculated directly from the experimental data by determining, for each experiment, the slope of the biomass curve as it declined and calculating an average value of the slope based on all experiments. The overall affinity constant (KG; equation 6) was taken from the literature on bacteria with the same type of glucose transporter system, that is, the ATP-binding cassette transport system (values are given in Table 2) [10, 27, 28].
where LSQ is the least squares quadratic, that is, the sum of the squared residuals, Yexp,i,tand Ymodel,i,t, are the experimental and model values, respectively, at time t for each compound i, that is, glucose, acetic acid, lactic acid, cell mass and H2. The parameter estimations were carried out and the 95% confidence intervals were calculated using the MATLAB functions nlinfit and nlparci, respectively (MathWorks, Natick, MA, USA).
Results and Discussion
The order in which the parameters are estimated is important. Since the mass transfer influences fermentation, and thus the corresponding kinetic parameters, the mass transfer parameters were estimated prior to determining the fermentation-related kinetic parameters.
Estimation of the volumetric mass transfer coefficient
where is the stripping rate. For more detailed results on mass transfer, see Additional file: Liquid-to-gas mass transfer and chemistry of CO2__Theory_experiments_and_results.
The six 5 g/L experiments (that is, experiments 1 and 3 through 7) differed with respect to growth rate and H2 yield (Figures 1, 2, 3, 4, 5 and 6). The reason for the variations is not clear. However, the two sets of experiments, carried out in parallel using inocula from the same preculture (experiments 3 and 4 as well as experiments 5 through 8), showed a strong resemblance. Batch experiments are generally characterized by poor reproducibility, as has also been observed with Escherichia coli . Owing to the variations between the experiments, each of the 5 g/L experiments was used separately to estimate all parameters except OSMcrit, nμ and RLacFOSM, which were estimated using the 10 g/L experiments (experiments 2 and 8 and Figures 7 and 8). In addition, acetic acid and H2 were not produced at a stoichiometric ratio (1:2) in any of the experiments, which is in agreement with prior observations in continuous culture [15, 21] where the H2/acetic acid ratio depended on the dilution rate. Instead, less acetate was produced in relation to H2, and the H2 yield was thus considered a parameter in the model. A possible explanation of the nonstoichiometric yield is that acetic acid is used as a building block by the cells.
The estimated maximum growth rate of C. saccharolyticus varied between the different experiments; however, the values were similar for experiments carried out in parallel (that is, experiments 3 and 4 and 5 through 8) (Table 3). The μmax determined for glucose ranged from 0.21 to 0.36/hour and corresponded well with earlier findings, which varied from 0.2 to 0.5/hour [14, 21]. Vrije et al.  found different values of μmax obtained in washout experiments with C. saccharolyticus, depending on the growth history of the culture. The reason for the variation in growth rates has not yet been elucidated, but the quality of the inocula used seems to depend on its growth history. The variation in growth rate was considered in the present model by not using an average value of μmax, while average values were used for all the other parameters (described in more detail below, Kinteic parameters: inhibition by dissolved hydrogen).
Together with changes in the product yields, the variations in growth rates strongly suggest that an as yet unknown factor influences the metabolism. Also, it is likely that the highest value of μmax determined in this study (0.36/hour) is not the true maximum, that is to say, the real μmax that can be achieved for glucose by this organism. This could mean that the growth of C. saccharolyticus is normally suppressed, as has been observed for E. coli . The cause of the suppression of the growth of C. saccharolyticus is not understood and must be further studied to gain a better understanding of the physiology of the microorganism and to improve the model .
Kinetic parameters: inhibition by dissolved hydrogen
The average, maximum and minimum values of the parameters based on the confidence intervalsa
2.2 × 10-3 mol/L
9.6 × 10-4 mol/L
4.1 × 10-3 mol/L
Dissolved hydrogen: degree of supersaturation and its effect
The measured dissolved hydrogen concentration under different fermentation conditions compared with the modela
0.28 ± 0.01 mmol/L
0.8 ± 0.5 mmol/L
0.34 ± 0.02 mmol/L
0.9 ± 0.5 mmol/L
H2aq at equilibrium
12.5 ± 0.3
12 ± 7
12.5 ± 0.4
34 ± 19
The experiment with the lowest stripping rate (experiment 4) showed the highest production of lactic acid (Figure 3). The other two experiments with low stripping rates (experiments 6 and 7) (Figures 5 and 6) showed only a slight increase in lactic acid production compared to the experiments with high stripping rates (experiments 1, 3 and 5) (Figures 1, 2 and 4) and very little lactic acid production compared to experiment 4. This can be explained by the lower growth rates resulting in lower H2 productivity, and hence lower values of H2aq, in experiments 5 through 7 (Figures 4, 5 and 6). Experiments 3 and 4 both showed high initial growth rates characterized by a high value of μmax and hence high H2 productivity, leading to higher values of H2aq. In experiment 4, with a low stripping rate (0.78 L/hour), H2aq reached a plateau faster than it did in the other experiments (Figure 9). At that stage, the culture reached maximum H2 productivity (data not shown), which resulted in linear rates of glucose consumption and H2 and acetic acid production (Figure 4). This behavior was reflected by the model (Figure 4) and can be explained by growth inhibition by H2. When H2 productivity reaches a critical value in the fermentation system, the cells respond with a reduction in the specific growth rate, and hence a plateau is observed. This plateau was reached much faster in experiment 4 than in the other two low-stripping-rate experiments (experiments 6 and 7), which was due to a combination of the higher initial growth rate and H2 productivity and the lower stripping rate (Table 1).
The organism thus seems to respond to an increase in H2aq by adjusting its growth rate and directing its metabolism toward the production of lactate, thus fine-tuning H2 productivity to prevent H2aq values detrimental to the cell. On the basis of both the experimental data and the model, it is clear that dissolved H2 has a negative effect on C. saccharolyticus and that H2 productivity and the stripping rate have a considerable impact on H2aq. This can explain why fermentation with high H2 productivity commonly results in low H2 yields [15, 21, 32, 33] and why an increase in the stripping rate tends to increase H2 yield and productivity .
Predicting the effect of dissolved hydrogen concentration
Furthermore, the current model cannot simulate a system without sparging, since k l a is based on the stripping rate (equation 24). However, Figure 10 provides an excellent basis for further discussions, since the relation between , and H2 productivity still holds, even without stripping. For instance, with C. saccharolyticus at a total pressure of 1 atm, no stripping, a productivity of 10 mmol H2/L/hour, and a gas composition of 60% H2 and 40% CO2, a value of of at least 7.5/hour is required to avoid serious H2 inhibition (Figure 10).
Kinetic parameters: inhibition by osmolarity
Experiments 2 and 8 were carried out with almost identical initial conditions, but they progressed quite differently, probably due mainly to different inocula and a slight difference in the initial glucose concentration. It was evident that H2aq as the sole inhibitor could not adequately describe the system at elevated substrate concentrations (Figures 7 and 8). Indeed, the model fitted the data from experiments 2 and 8 very well after introducing growth inhibition by osmotic pressure.
The effect of the assumption of μmax for experiment 2 on the estimates of the parameters associated with osmolaritya
Experiment 2 μmax
Although this rather simple model describes the effect of osmolarity well, some aspects of it cannot be reproduced by this type of unstructured, mechanistic model. It is evident from Figure 8 that the growth rate, and hence the glucose consumption and acetic acid production rates, are not affected at the initiation of growth, since the model excluding inhibition by osmolarity fits the first three points perfectly. However, at an osmolarity of about 0.2 mol/L, there is a shift toward a reduction in the growth rate, glucose consumption, and acetic acid production rate, as well as an increase in lactic acid production, rapidly at first, followed by a slow decline of all of them. It is not possible to model this effect, that is, a rapid shift followed by a slow gradual decrease, using a simple inhibition expression. One could assume a very high exponential factor, but that would predict only the beginning of the experiment; however, as inhibition came into effect, the decrease in the growth rate to zero would not be slow and gradual, but very considerable and rapid.
Batch fermentations of C. saccharolyticus on glucose were successfully simulated using Monod kinetics extended to include liquid-to-gas mass transfer and inhibition by H2aq and osmolarity. In agreement with previous measurements [18, 19], the model predicted high oversaturation of H2 in the liquid, which was also confirmed experimentally.
To the best of our knowledge, we have demonstrated for the first time the possibility of predicting H2aq and its effect on the fermentation in a laboratory-scale bioreactor as a function of stripping rate and productivity using the derived model. With this tool, it was possible to conclude that the inhibition of growth and H2 production depend on both H2aq and osmolarity. The model described the inhibition by both H2aq and osmolarity satisfactorily and can hence be used to predict the optimal stripping rate and substrate concentration. It also shows how C. saccharolyticus responds to increased osmolarity and H2aq. In addition, the modeled relationship between mass transfer, H2 productivity and H2aq is not specific to C. saccharolyticus, but can also be applied to other systems. To widen the applicability of the model even further, future studies should aim at incorporating the effect of the stirrer speed and the absence of stripping on the liquid-to-gas mass transfer.
Ac: acetic acid concentration (mol/L); CO2aq: CO2 concentration in the liquid phase (mol/L); : saturation concentration (mol/L); CO2sol: solubilized CO2 (mol/L); : flow rate of CO2 out of the fermentor (L/hour); : flow rate of H2 out of the fermentor (L/hour); : flow rate of N2 into the fermentor (L/hour); : flow rate of N2 out of the fermentor (L/hour); G: substrate concentration (mol/L); H2aq: H2 concentration in the liquid phase (mol/L); H2aqcrit: critical H2 concentration in the liquid phase (mol/L); : saturation concentration (mol/L); : Henry's constant for H2 (L atm/mol); : Henry's constant for CO2 (L atm/mol); K1: dissociation constant for CO2 into bicarbonate; K2: dissociation constant for bicarbonate into carbonate; : mass transfer coefficient for H2 (per hour); : mass transfer coefficient for CO2 (per hour); KG: saturation constant (mol/L); Lac: lactic acid concentration (mol/L); nμ: inhibition coefficient for inhibition by osmolarity; : inhibition coefficient for inhibition by H2; OSM: osmolarity parameter (mol/L); OSMcrit: critical osmolarity parameter (mol/L); : partial pressure of H2 (Pa); : critical partial pressure of H2 (Pa); Ptot: total pressure (Pa); PPi: pyrophosphate; LSQ: least squares quadratic; R: gas constant (L atm/K/mol); RAcF: maximum Ac production rate (mol/g cell mass/hour); : maximum Lac production rate caused by H2 (mol/g cell mass/hour); RLacFOSM: maximum Lac production rate caused by osmotic pressure (mol/g cell mass/hour); rcd: death rate of cells (per hour); T: temperature (Kelvin); Vg: gas volume in liquid (L); Vl: liquid volume in reactor (L); X: cell mass concentration (mol/L); YGAc: stoichiometric acetic acid yield (mol acetic acid/mol substrate); : stoichiometric CO2 yield (mol CO2/mol substrate); : stoichiometric H2 yield (mol H2/mol substrate); YGLac: stoichiometric lactic acid yield (mol lactic acid/mol substrate); YGX: biomass yield (mol cell mass/mol substrate); α: cell-growth-associated constant; αAc: cell-growth-associated constant for Ac production rate (mol Ac/mol cells); : cell growth-associated constant for CO2 production rate (mol CO2/mol cells); : cell growth-associated constant for H2 production rate (mol H2/mol cells); αLac: cell-growth-associated constant for Lac production rate (mol Lac/mol cells); β: non-cell-growth-associated constant; γ: exponential coefficient for mass transfer correlation; μ: specific growth rate (per hour); μmax: maximum specific growth rate (per hour).
The authors gratefully acknowledge the Commission of the European Communities, Sixth Framework Programme, Priority 6, Sustainable Energy Systems (019825 HYVOLUTION), as well as the Swedish Energy Agency for their financial support.
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