Comprehensive Study and Modeling of Acetic Acid Effect on Trichoderma reesei Growth

Abstract

Acetic acid is one of the major potential inhibitors of cellular growth resulting from acid pretreatment of lignocellulosic biomass. To use lignocellulose hydrolysate in biorefineries it is necessary to assess their effect on the microorganisms likely to be used. An attractive option is to use a part of these hydrolysates for on-site cellulase production. Based on an industrial process that included a fast initial growth phase, the effect on Trichoderma reesei grown on glucose was assessed and modeled at different concentrations and pH. Acetic acid had a strong effect on T. reesei growth rate and yield, which correlated only with the concentration of the undissociated form in solution. The specific growth rate was accurately modeled by two different classic inhibition models as a function of pH and total acetic acid concentration. Co-consumption of glucose and acetic acid was observed, so that the culture medium was gradually detoxified by the cells. With or without glucose, acetic acid was mineralized into carbon dioxide at a similar specific rate, but no growth was observed without glucose. A 2.5-fold increase in the maintenance coefficient was observed, due to the need for glucose consumption to preserve cell integrity, which corresponded to a one-third decrease in the overall biomass yield. The resulting models can be used to simulate T. reesei growth on acetic acid-containing media and to choose the optimum pH for efficient growth on lignocellulosic biomass hydrolysates.

Introduction

Cellulose, hemicellulose, and lignin—closely gathered in lignocellulosic materials—are the most abundant polymers on Earth. They are naturally degraded by plant pathogens and saprophytes, which produce a large array of enzymes with activity against plant cell walls.¹ Cellulose and hemicellulose are hydrolyzed by cellulases and hemicellulases into fermentable monomer sugars such as glucose and xylose. This natural process can be reproduced at industrial scale in biorefineries to transform renewable lignocellulosic materials biochemically into many different chemicals.² However, the high loading of cellulolytic enzyme required for complete lignocellulose hydrolysis is an issue for the development of cost-effective processes. The filamentous fungus Trichoderma reesei is recognized as the best microorganism for industrial-scale cellulase production because of its high secretion capacity.³

To decrease cellulase supply costs, cellulase production can be performed on-site using lignocellulosic materials rather than purified sugars as carbon substrates. Pretreated lignocellulosic materials contain degradation compounds, however, such as weak organic acids and furanic and phenolic derivatives that may have inhibitory activity against microorganisms.^4,5 Acetic acid, formed by deacetylation of the hemicellulose polymers during physicochemical or enzymatic hydrolysis, is one of the major degradation compounds in lignocellulosic hydrolysates with concentrations reaching 50–100 mM (3–6 g/L).^6,7 It is therefore necessary to assess the effect of acetic acid on microorganisms likely to be used in biorefineries. A previous study of cellulase production by T. reesei showed that acetic acid alone had no effect on global cellulase activity.⁸ However, the process chosen for that study—batch mode on pretreated cellulosic material—is hard to achieve at industrial scale because of aeration and stirring issues in the highly viscous cellulosic media. Moreover, the reported cellulase activities were much lower than previously reported for other, better industrial processes.⁹

A highly efficient industrial process for cellulase production by T. reesei has been developed by IFP Energies nouvelles and validated at pilot scale (30m³).^10,11 It consists of two successive phases; first, cellular growth in batch mode with excess carbon source for about 24 h; followed by cellulase production in fed-batch mode under carbon flux limitation using an inducing carbon source (eg, lactose, for about 170 h). The initial growth phase is critical because the formation of T. reesei mycelia increases broth viscosity, which impacts mass transfer.¹² It is generally performed at a low pH (∼4.8) to limit bacterial contamination. Lignocellulosic hydrolysates containing soluble carbon sources can be used for the fed-batch phase, with comparable results.¹³ However, using these hydrolysates for cellular growth in batch mode is more difficult because of the higher concentration of cell growth-inhibiting degradation compounds. This study focused on the initial growth phase and strategies to reach a high cell concentration for the following production phase.

In solution, weak acids exist in equilibrium between the undissociated protonated form and the dissociated anionic form, in proportions that depend on pH and the equilibrium constant (pK_A) of the acid. When undissociated, weak acids are soluble in lipids and can diffuse across cell membranes and accumulate in cytoplasm, where they dissociate because of the almost neutral intracellular pH. Two mechanisms have been proposed to explain their inhibitory effect. According to uncoupling theory, proton release due to dissociation is neutralized by cell membrane ATPase to preserve the cellular proton gradient, which may consume much of the ATP normally devoted to biosynthesis.¹⁴ According to anion accumulation theory, diffusion of undissociated acid occurs until equilibrium is reached, which depends on the difference between extracellular and intracellular pH.¹⁵ Acid concentrations are then very high in cytoplasm, which may directly inhibit glycolytic enzymes. In both theories, the extracellular concentration of the undissociated form is of the highest importance, since only this form can diffuse across the cell membrane.

We studied the effect of acetic acid on T. reesei growth and yield at laboratory scale, with the aim of developing a model based on the results. In particular, pH was closely monitored, as it is known to have a substantial impact on the effects of weak acids.

Materials and Methods

Strain

T. reesei CL847 (Cayla Company, Toulouse, France) is a cellulase hyperproducer strain obtained from QM 9414 strain by several steps of mutagenesis and selection.¹⁶ It is comparable to other hyperproducer strains, including Rut-C30.⁹ Spores were conserved in cryotubes at −80°C with 50% glycerol.

Culture Medium Composition

The medium composition was the same for flask and bioreactor cultivations: corn-steep solid, 1.5 g/L; KOH, 1.66 g/L; H₃PO₄ 85%, 2.5 mL/L; (NH₄)₂SO₄, 2.8 g/L⁻¹; MgSO₄·7H₂O, 0.6 g/L; CaCl₂·2H₂O, 0.6 g/L; FeSO₄·7H₂O, 60 mg/L; MnSO₄·H₂O, 12 mg/L; ZnSO₄·7H₂O, 16 mg/L; CoNO₃·6H₂O, 18 mg/L; and H₃BO₃, 2 mg/L.

For flask cultivations, this medium was supplemented with 15 g/L glucose and acetic acid depending on the experimental protocol, followed by pH adjustment with NaOH 30%. For bioreactor cultivations, the pH was adjusted with NH₄OH 20%.

Salt solutions and glucose solutions were sterilized for 20 min at 121°C (separately to avoid Maillard reaction). Acetic acid solutions were sterilized by filtration at 0.2 μm to avoid stripping when autoclaving.

Cultivations

For closed flask cultivations, 160-mL penicillin flasks with 20-mL working volume were closed with an airtight stopper and incubated in a rotary shaker at 150 rpm and 30°C. For CO₂ accumulation analysis, 250-μL gas samples were taken from the headspace with a syringe through the stopper. This protocol was validated by comparison with bioreactor cultivations and gave similar biomass growth rate and yield.

Bioreactor cultivation was performed in a 7-L ez-Control bioreactor (Applikon Biotechnology BV, Schiedam, The Netherlands) with 2-L working volume. Pre-culture was performed in a Fernbach flask with 250-mL culture medium, inoculated with 10⁶ spores, then incubated 72 h in an Infors (Bottmingen, Switzerland) rotary shaker (150 rpm, 30°C). pH was automatically adjusted to 5.75 with 5.5 N NH₄OH solution. Aeration rate was fixed at 60 sL/h and agitation was regulated to maintain the dissolved oxygen concentration at a minimum 40% of its saturation value. For continuous mode, culture medium was supplemented with acetic acid or a mixture of glucose and acetic acid, then fed at 60 mL/h. Constant volume was provided by constant weight regulation.

Analyses

Culture medium was filtrated using Whatman (Kent, UK) GF/C filters. For biomass concentration determination, biomass cake was washed with distilled water, then dried at 105°C until constant weight.

Glucose concentration was measured in supernatants by high-performance liquid chromatography (HPLC). Separation was carried out using a Varian (Palo Alto, CA) Metacarb 87P column with mobile phase milliQ water (Millipore, Billerica, MA) at a flow rate of 0.4 mL/min, 80°C, and pressure ∼32 bar; detection was carried out with Waters (Milford, MA) 2414 refractive index detector.

Acetic acid concentration was measured by HPLC. Separation was carried out using Aminex HPX-87H column (Bio-Rad, Hercules, CA) at 60°C with mobile phase 10 mM sulfuric acid (0.6 mL/min, 15 bar); detection was carried out with SpectraSYSTEM^™ RI-150 refractive index detector (Thermo Fisher Scientific, Waltham, MA).

For flask cultivations, CO₂ content in the flask headspace was measured off-line by gas chromatography using a Varian 3800 chromatograph with thermal conductivity detector, after calibration using a 5% CO₂/95% N₂ mix. The mass of the CO₂ accumulated in the headspace was then calculated assuming constant headspace volume and constant pressure equal to 1 atm in flasks (respiratory quotient about 1 for this aerobic microorganism). For bioreactor cultivations, CO₂ content in the gas outflow was measured on-line by infrared absorbance with a Duet analyzer (Advanced Biosystems Ltd, Roebuck, SC).

Acid Equilibrium Calculations

Concentrations of undissociated acetic acid (AcH) and dissociated acetic acid (Ac^–) were calculated assuming ideal solutions; the activities were considered equal to concentrations (no impact on ionic strength). AcH and Ac^– concentrations were calculated using Equation 1 and Equation 2, respectively, as functions of pH, pK_A (4.76), and the total concentration of acetic acid in both forms ([Ac]_total): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}[AcH] = [Ac] _ {total} \frac {1} {1 + 10^{pH - pK_A}} \qquad{{Equation} \ 1}\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}[Ac^ {-}] = [Ac] _ {total} \frac {10^ {pH - pK_A}} {1 + 10^ {pH - pK_A}} \quad\quad{{Equation} \ 2}\end{align*} \end{document}

Results and Discussion

Effect of pH on T. Reesei Growth Rate

Since the effects of acetic acid were assessed at different pH, it was first necessary to test the influence of initial pH on T. reesei grown on glucose without acetic acid using the closed flask protocol. The CO₂ production rate was calculated by measuring the mass of CO₂ that accumulated in the headspace of the flasks after 24 h (before oxygen exhaustion). The results showed a large optimal pH range between 4.5 and 6.0, and a slight decrease for lower pH (Fig. 1). At pH 3.5, the CO₂ production rate was 27% lower than at pH 5.0. The biomass production yield was constant at 0.38±0.04 g_X/g_S at all pH values tested. Since biomass and CO₂ are the only two products formed during growth on glucose—no co-products such as organic acids—CO₂ production rate was a good measure of the biomass growth rate.

Fig. 1.

Effect of initial pH on T. reesei growth. CO₂ production rate on glucose was measured using the closed flask protocol for different initial pH values (CO₂ production rate=mass of CO₂ accumulated in the headspace after 24 h).

Protocol for Acetic Acid Assessment

The effect of acetic acid on T. reesei growth (on 15 g/L glucose) was assessed for different initial pH conditions and different total acetic acid concentrations, to observe a range of distributions between the dissociated form Ac^– and the undissociated form AcH. Seventeen different initial conditions were compared, with an initial pH between 4.56 and 5.67 and total initial acetic acid concentration between 20-130 mM, in addition, one control culture without acetic acid was evaluated in duplicate (Fig. 2).

Fig. 2.

Initial conditions for acetic acid assessment in closed flasks. The 18 initial conditions for acetic acid assessment are represented depending on the initial pH (y axis) and the total initial acetic acid concentration (x axis).

According to Fig. 1, the influence of initial pH on T. reesei growth in this protocol would be very limited, with ∼7% decrease in growth at the lower pH 4.56, and less than 3% for the other pH levels in the experimental range. The observed effects would only be caused by the presence of acetic acid.

The 19 flasks were inoculated, and CO₂ production was followed during 28 h, at which time the oxygen was almost totally consumed in the control flasks. Owing to the additional buffer effect of acetic acid, pH was almost constant during culture (max. 0.38 pH-unit increase). Glucose and acetic acid consumption and CO₂ and biomass production were measured. Carbon balance remained close to 100%, ranging from 90% to 130%, which is satisfactory for this small-scale protocol.

Effect on CO₂ Production

CO₂ production was not correlated with total initial acetic acid concentration or with initial pH (Fig. 3A–B). Using Equations 1 and 2, the equilibrium concentrations of AcH and Ac^– could be calculated. CO₂ production was not correlated with Ac^– concentration (Fig. 3C), but a clear correlation was observed with the concentration of the undissociated form AcH (Fig. 3D). This result was consistent with the common inhibition mechanism, for which the uncharged, undissociated form can diffuse through the membrane to the cytoplasm.

Fig. 3.

Effect of acetic acid on CO₂ production in flasks. CO₂ production (in mgC) during T. reesei growth on glucose is represented depending on four experimental conditions: total initial acetic acid concentration (A); initial pH (B); Ac¯ dissociated form concentration (C); and AcH undissociated form concentration (D).

Effect on Substrate Consumption and Biomass Production

Similar to the finding for CO₂ production, glucose consumption, acetic acid consumption, and biomass production were only correlated with the concentration of the undissociated form AcH, regardless of the initial pH or the total acetic acid concentration (Fig. 4). As previously observed by Szengyel and Zacchi, acetic acid and glucose are co-consumed; in our study, about 0.5 g/L acetic acid (∼8 mM) was consumed for every 0.5–3 g/L glucose consumed (Fig. 4A–B).⁸ Acetic acid consumption was not due to limited glucose, since glucose (initial concentration of 15 g/L) was never exhausted. The effect of acetic acid on growth was considerable; biomass production and specific growth rate were halved after AcH concentrations of 4 mM and 7 mM, respectively (about 1.6 g/L and 3.0 g/L total acetic acid concentration at pH 5.5, respectively) (Fig. 4D).

Fig. 4.

Effect of acetic acid on substrate consumption [glucose (A) and acetic acid (B) concentrations] and biomass production [biomass concentration produced (C) and specific growth rate (D)] during T. reesei growth in closed flasks on glucose with acetic acid. Consumption and production are presented versus the concentration of the undissociated form AcH of acetic acid, single parameter, showing good correlation. For specific growth rate (D), two models are represented by solid lines: power law model (black) and exponential model (gray).

The effect of acetic acid on the specific growth rate was accurately modeled by two different mathematical expressions: a power law model (Equation 3) with I_max=38 mM and n=3.4; or an exponential model (Equation 4) with K_I=9.5 mM (Fig. 4D , black and gray solid lines, respectively), both with μ_max=0.087/h. (The biomass specific growth rate observed without acetic acid was consistent with measurements in bioreactor cultivation for this strain.) In these models, I was the concentration of the inhibiting undissociated form AcH, which can be calculated using Equation 1: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\mu = \mu_ {\max} \left(1 - \frac {I} {I_ {\max}} \right) ^n \qquad\quad\quad\quad{{Equation} \ 3}\end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\mu = \mu_ {\max} \exp \left(- \frac {I} {K_I} \right) \qquad\quad\quad\quad{{Equation} \ 4}\end{align*} \end{document}

When carbon balance was adequate (12 flasks between 90-110%), the results were used to calculate carbon yields (Fig. 5). Biomass yield was 0.6 gC/gC on glucose without acetic acid, which was consistent with previous measurements.^17,18 With acetic acid in the medium, biomass yield decreased quickly to a value close to 0.4 gC/gC. This meant that the cells consumed some substrate to overcome acetic acid inhibition without associated growth, which lowered global biomass yield. So acetic acid had a double effect on T. reesei growth, decreasing both growth rate and growth yield. However, it was observed that acetic acid was slowly metabolized, so T. reesei appeared able to achieve slow detoxification of the medium.

Fig. 5.

Effect of acetic acid on carbon yields during T. reesei growth in flasks. Biomass production yield (diamonds); CO₂ production yield (squares); glucose consumption yield (triangles); acetic acid consumption yield (circles).

Continuous Culture on Acetic Acid

For precise measurement of acetic acid degradation kinetics, it was decided to perform a continuous culture in a bioreactor with feeding either by acetic acid alone or a mixture of glucose and acetic acid. To minimize growth inhibition by acetic acid, the setpoint pH was chosen at 5.75, one unit above the pKa (4.76), so less than 10% of the total acetic acid in the medium was in the undissociated form. Moreover, since inhibition lowered the specific growth rate, a low dilution rate was chosen, at 0.03/h, or about one-third the maximal specific growth rate (μ_max).

Four successive modes were tested during this culture. First, a classic growth phase was performed in batch mode on 10 g/L glucose until glucose exhaustion (0–23 h). Then the culture medium was fed with 10 g/L acetic acid in continuous mode with a dilution rate of 0.03/h (23–48 h). Since acetic acid accumulated in the medium, a culture in batch mode on acetic acid (2.8 g/L initial) was performed from 48 to 56 h. Finally, the culture medium was fed with a mixture of 10 g/L acetic acid and 5 g/L glucose in continuous mode with a dilution rate of 0.03/h (56–103 h). Concentration monitoring is shown Fig. 6 and rate calculations is presented in Table 1. Carbon balances were correct in each mode, ranging from 81% to 107%.

Fig. 6.

Concentration monitoring during continuous culture with acetic acid. Residual glucose (triangles); biomass (diamonds); total residual acetic acid (squares).

Table 1.

Rates and Carbon Balance Calculations During the Different Modes of Continuous Culture with Acetic Acid

		h^-1	VOLUME RATE (g/L/h)					SPECIFIC RATE (mg/g_x/h)
		D	r_Glu	r_Ac	r_X	r_CO2	CARBON BALANCE (%)	q_Glu	q_Ac	μ	q_CO2
Batch on glucose	0 to 23h	0						−125		71
Continuous mode on acetate	23 to 32h	0.03		−0.13	−0.02	0.23	103		−26	−3	44
	32 to 48h	0.03		−0.15	0.00	0.24	107		−41	0	67
Batch on acetate	49 to 56h	0		−0.18	0.00	0.20	81		−65	0	72
Continuous mode glucose+acetate	56 to 75h	0.03	−0.15	−0.11	0.05	0.24	86	−58	−41	19	94
	75 to 103h	0.03	−0.15	−0.11	0.06	0.21	81	−65	−47	27	92

From 23–56 h without glucose feeding, a specific growth rate close to zero was observed; biomass concentration quickly decreased during continuous mode because of washing out and was constant during batch mode. No protein production was observed. T. reesei growth was impossible on acetic acid alone, but acetic acid was degraded and mineralized in CO₂ with a specific rate of about 40 mg/g_X/h. This result had not been previously described, since monomer sugars or cellulose were always present in culture medium.⁸

From 56–103 h with glucose feeding, the specific growth rate was not null (∼0.025/ h), but lower than the set dilution rate (0.03/h) because of the growth inhibition by acetic acid; thus, biomass concentration decreased slightly. During this phase, acetic acid was still mineralized at the same specific rate of 40 mg/g_X/h, indicating that it was unaffected by glucose metabolism and biomass growth.

Assuming a classic Pirt law model (Equation 5) with maximal yield Y_X°=0.6 g_X/g_Glu (as previously measured in flasks), the maintenance coefficient was calculated at 20–25 mg_Glu/g_X/h. This value was much higher than usually measured (10 mg_Glu/g_X/h) because of supplementary glucose consumption to preserve cell integrity.¹⁹ \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}q_ {Glu} = \frac {\mu} {Y_X^ \circ} + m \qquad\qquad\qquad\qquad{{Equation} \ 5}\end{align*} \end{document}

Conclusions

The effect of acetic acid on T. reesei growth rate and yield was quantitatively studied using two different protocols. A broad screening in flasks was performed to assess the impact of the solution equilibrium between the dissociated and undissociated forms. Only the concentration of the undissociated form of acetic acid correlated with an effect on T. reesei growth, as expected according to classic inhibition mechanism theory. Therefore, increasing culture medium pH would be the best way to lower inhibition, but it may increase bacterial contamination risk at industrial scale. With knowledge of the acetic acid concentration in a lignocellulosic biomass hydrolysate, the inhibition model resulting from this study would be useful for determining the pH value required for a desired specific growth rate.

Medium detoxification was observed with acetic acid mineralization into CO₂ at a good specific rate. Surprisingly, this degradation rate was similar with or without glucose, suggesting that these two metabolisms were uncorrelated. Thus, acetic acid degradation can hardly be used to detoxify lignocellulosic biomass hydrolysates before fermentation, since the sugars will also be consumed by T. reesei, as previously observed.²⁰ The measured degradation rate can be used to predict medium detoxification during biomass growth. Medium detoxification will result in a progressive increase of the specific growth rate throughout the biomass growth phase.

High glucose consumption for maintenance was observed, with a 2.5-fold increase of the maintenance coefficient, which was consistent with the uncoupling theory (ATP consumption without associated growth to preserve the proton gradient across the cell membrane). Added to the decrease in the specific growth rate, this resulted in about a one-third decrease of the overall biomass yield. The classic Pirt law model can be used to predict glucose consumption during growth phase, and then the required initial glucose concentration for a desired biomass concentration at the end of the initial growth phase. This biomass concentration will govern productivity during the second phase of cellulase production.

Footnotes

Acknowledgments

This study was part of Projet Futurol, a project supported by OSEO Innovation.

Author Disclosure Statement

No competing financial interests exist.

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Comprehensive Study and Modeling of Acetic Acid Effect on Trichoderma reesei Growth

Abstract

Introduction

Materials and Methods

Strain

Culture Medium Composition

Cultivations

Analyses

Acid Equilibrium Calculations

Results and Discussion

Effect of pH on T. Reesei Growth Rate

Protocol for Acetic Acid Assessment

Effect on CO2 Production

Effect on Substrate Consumption and Biomass Production

Continuous Culture on Acetic Acid

Conclusions

Footnotes

Acknowledgments

Author Disclosure Statement

References

Effect on CO₂ Production