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Cited by 17Microbial Natural Products and BiosynthesisProtein purification and stabilityEnzyme Production and Characterization

Degradation Kinetics of Clavulanic Acid in Fermentation Broths at Low Temperatures

Open access
Gómez-Ríos, David, Ramírez-Malule, Howard, Neubauer, Peter, Junne, Stefan, Ríos-Estepa, Rigoberto·2019·Antibiotics
DOI 10.3390/antibiotics8010006PMC PMC6466556
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Abstract

Clavulanic acid (CA) is a β-lactam antibiotic inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus; yield and downstream process are compromised by a degradation phenomenon, which is not yet completely elucidated. In this contribution, a study of degradation kinetics of CA at low temperatures (−80, −20, 4, and 25 °C) and pH 6.8 in chemically-defined fermentation broths is presented. Samples of CA in the fermentation broths showed a fast decline of concentration during the first 5 h followed by a slower, but stable, reaction rate in the subsequent hours. A reversible-irreversible kinetic model was applied to explain the degradation rate of CA, its dependence on temperature and concentration. Kinetic parameters for the equilibrium and irreversible reactions were calculated and the proposed kinetic model was validated with experimental data of CA degradation ranging 16.3 mg/L to 127.0 mg/L. Degradation of the chromophore CA-imidazole, which is commonly used for quantifications by High Performance Liquid Chromatography, was also studied at 4 °C and 25 °C, showing a rapid rate of degradation according to irreversible first-order kinetics. A hydrolysis reaction mechanism is proposed as the cause of CA-imidazole loss in aqueous solutions.

AI snapshot

Written from the paper’s full text.

TL;DR

Clavulanic acid (CA) in chemically defined fermentation broths at pH 6.8 degraded rapidly in the first 5–6 h and then more slowly, with degradation strongly temperature-dependent and also affected by initial CA concentration. A consecutive equilibrium–irreversible first-order kinetic model fit the data with <5% deviation, and the derivatized CA–imidazole chromophore degraded faster than CA by first-order kinetics.

Key findings

  • CA loss was 8–12% during the first 5–6 h; at 25 °C the accumulated loss reached 35% at 42 h at the highest initial CAo, while at −80 °C less than 4% was lost in 42 h. Degradation at −20 °C was unexpectedly high.
  • Statistical analysis at 95% confidence (r² = 0.996) indicated that both temperature and CAo affect CA degradation, with temperature much more significant than CAo and the combined interaction.
  • The proposed equilibrium–irreversible first-order model gave k₂ values lower than Keq and Keq values lower than 1; for the irreversible step, Ea = 16.512 kJ/mol, A = 26.97 h⁻¹ (r² = 0.961), and ΔG° = 3.155 kJ/mol at 25 °C. Model predictions deviated from measured concentrations by less than 5% in all cases.
  • The CA–imidazole chromophore (CAI) degraded by first-order kinetics with rate constants 0.020 h⁻¹ at 4 °C (r² = 0.989) and 0.039 h⁻¹ at 25 °C (r² = 0.994); Ea = 21.85 kJ/mol, A = 262.43 h⁻¹ (r² = 0.997). Half-lives were 34.5 h at 4 °C and 17.8 h at 25 °C; at 25 °C, 56% was lost in the first 24 h and up to 82% at 45 h, while at 4 °C 40% decayed in 24 h and up to 60% in 46 h.

Methods

  • Batch fermentations of *Streptomyces clavuligerus* DSM 41826 were carried out in a 15 L stirred tank bioreactor (5 L filling volume) with chemically defined medium at pH 6.8, 28 °C, and 0.6 VVM. Supernatant samples taken at 36 h were adjusted to pH 6.8 and stored at −80, −20, 4, and 25 °C for 43 h. A factorial design varied concentration (supernatant, 1:2, 1:5) and temperature (four levels), with 12 runs by duplicate plus an additional high-CA sample (127 mg/L); samples were withdrawn at 3.1, 5.4, 18.3, 31.0, and 42.1 h, derivatized with 20% imidazole for 30 min at 30 °C and 800 rpm, and analyzed by HPLC-DAD.
  • CAI stability used a 2-squared factorial design with duplicates; CA samples at 127.0 mg/L (0.636 mM) and 61.7 mg/L (0.310 mM) were derivatized and stored at 4 and 25 °C, with aliquots withdrawn at five times over 46 h and analyzed by HPLC.
  • Statistical analysis was performed in Statgraphics Centurion XVII at 95% confidence. Kinetic parameters were calculated by linear regression with least squares, checked by r² and residual analysis; Keq was estimated using the Levenberg-Marquardt method for least squares minimization, and Arrhenius parameters were obtained by linear regression.

Limitations

  • Stated: experimental limitations regarding analysis of CA decomposition products meant only the time course of CA concentration could be properly followed.
  • Stated: the proposed model simplified decomposition to only one intermediate product and one decomposition product following equilibrium and irreversible first-order kinetics, so deviations between experimental and simulated data were expected.
  • Evident from the text: experiments used a single *S. clavuligerus* strain (DSM 41826), one chemically defined medium, and pH 6.8 only; CAI stability was tested only at 4 and 25 °C and two initial concentrations, so broader generalizability is not established.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/antibiotics8010006

Cites

38 of 38 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

12 of 50 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    CA chemical instability largely depends on pH owing to a carbonyl group linked to the β-lactam ring that is susceptible to acid- or base-catalyzed water attacks.

    “CA chemical instability largely depends on the pH-value owing to the presence of a carbonyl group linked to the β-lactam ring, which is susceptible to acid or basic catalyzed water attacks [17].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Carvalho, 2009, Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach, Biochem. Eng. J. source
  • Premisecitation

    Addition of inorganic salts (NaCl, Na2SO4, CaCl2, and MgSO4) increased CA instability.

    “The optimal conditions for achieving a low degradation rate were pH 6.0–7.2 and 20 °C; in contrast, addition of inorganic salts (NaCl, Na2SO4, CaCl2, and MgSO4) increased instability of CA, possibly due to the higher rate of collisions between molecules within the solution [17].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Carvalho, 2009, Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach, Biochem. Eng. J. source
  • Interpretationcitation

    The kinetic approach is consequent with a feasible chemical mechanism of reaction for CA in aqueous solutions characterized by several equilibrium steps.

    “This kinetic approach is consequent with a feasible chemical mechanism of reaction for CA in aqueous solutions, characterized by several equilibrium steps (Figure 4) [28,29].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [28] Haginaka, 1985, Degradation of clavulanic acid in aqueous alkaline solution: isolation and structural investigation of degradation products, Chem. Pharm. Bull. source

      The Degradation Mechanism section (p. 6) proposes a multistep mechanism for clavulanate degradation, supporting the claim that a feasible chemical mechanism exists. It does not characterize those steps as equilibria or establish that the proposed mechanism follows from the citing paper’s kinetic approach; the study concerns weakly alkaline aqueous solution, not aqueous solutions generally. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [29] Finn, 1984, Studies on the hydrolysis of clavulanic, J. Chem. Soc. Perkin Trans. source
    • The authors state this as an assumption: CA degradation follows a two-step mechanism: a reversible equilibrium step in which CA produces an active intermediate I*, followed by an irreversible reaction of I* with an additional CA molecule to form degradation product D.
  • Interpretationcitation

    The observed time courses of CA degradation are typical of consecutive equilibrium-irreversible reactions in which the irreversible reaction rate is considerably low compared to the equilibrium reaction (k2 < k1 < k−1).

    “The observed time courses of CA degradation are typical of consecutive equilibrium-irreversible reactions in which the reaction rate of the irreversible reaction is considerably low in comparison to the equilibrium reaction (i.e., k2 < k1 < k−1) [17].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Carvalho, 2009, Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach, Biochem. Eng. J. source

      Section 3.3 (page unavailable) models the observed CA degradation as a preliminary equilibrium followed by irreversible degradation, supporting the reaction sequence. It reports an equilibrium constant K and an irreversible-degradation constant k_d, but does not establish the claimed ordering k2 < k1 < k−1 or quantify the irreversible rate relative to the forward and reverse equilibrium rates. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • The authors state this as an assumption: For the consecutive equilibrium-irreversible reactions, k2 < k1 < k−1.
  • Findingcitation

    The calculated Ea for the irreversible process of CA degradation is close to previous reports for CA in fermentation broths.

    “The activation energy (Ea) calculated for the irreversible process of degradation was 16.512 kJ/mol which is close to previous reports for CA in fermentation broths [10,17].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [10] Bersanetti, 2005, Kinetic studies on clavulanic acid degradation, Biochem. Eng. J. source
    • cites [17] Carvalho, 2009, Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach, Biochem. Eng. J. source
  • Classified as probably not a substantial claim. 17
  • May combine two claims. 35
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A pH of 6.8 is favorable for obtaining high titers of CA in submerged cultures.

    “Previous studies have shown that CA stability increases at slightly acidic pH values [10,11]; several authors found a pH of 6.8 as favorable for obtaining high titers of CA in submerged cultures [5,7,9,12,13,14,15,16].”
    Quote in the paperNot a substantial claim3 of 8 sources checked
    • cites [5] Ser, 2016, Fermentation conditions that affect clavulanic acid production in Streptomyces clavuligerus: A systematic review, Front. Microbiol. source
    • cites [7] Antonio, 2013, Production of clavulanic acid and cephamycin c by Streptomyces clavuligerus under different fed-batch conditions, Braz. J. Chem. Eng. source
    • cites [9] Neto, 2005, A study on clavulanic acid production by Streptomyces clavuligerus in batch, FED-batch and continuous processes, Braz. J. Chem. Eng. source

      The Fermentation Equipment and Cultivation Procedure section (page not specified) says the cultures were maintained at pH 6.8 ± 0.1, and the Abstract reports CA concentrations up to 404 mg/L. The paper does not compare pH values or establish that 6.8 is particularly favorable for high titers. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [12] Rosa, 2005, Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus, Bioprocess Biosyst. Eng. source
    • cites [13] Teodoro, 2006, Influence of feeding conditions on clavulanic acid production in fed-batch cultivation with medium containing glycerol, Appl. Microbiol. Biotechnol. source
    • cites [14] Bushell, 2006, Manipulation of the physiology of clavulanic acid biosynthesis with the aid of metabolic flux analysis, Enzyme Microb. Technol. source
    • cites [15] Cerri, 2012, Shear conditions in clavulanic acid production by Streptomyces clavuligerus in stirred tank and airlift bioreactors, Bioprocess Biosyst. Eng. source
    • cites [16] Costa, 2012, Production of clavulanic acid by Streptomyces clavuligerus in batch cultures without and with glycerol pulses under different temperature conditions, Biochem. Eng. J. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Carvalho et al. found that the optimal conditions for achieving a low CA degradation rate were pH 6.0-7.2 and 20 °C.

    “The optimal conditions for achieving a low degradation rate were pH 6.0–7.2 and 20 °C; in contrast, addition of inorganic salts (NaCl, Na2SO4, CaCl2, and MgSO4) increased instability of CA, possibly due to the higher rate of collisions between molecules within the solution [17].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Carvalho, 2009, Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach, Biochem. Eng. J. source

      Section 3.1 identifies pH 6.0–7.2 as the best range for CA stability. Section 3.2 describes 20–30 °C as an optimum temperature range, rather than singling out 20 °C, and reports that all four salts increased degradability. It does not attribute that salt effect to more frequent molecular collisions. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Zhong et al. proposed a mechanism of formation of the degradation product known as substance E involving several steps, including the irreversible reaction of an active intermediate with an additional molecule of CA.

    “In this regard, Zhong et al. [30] proposed a mechanism of formation of the degradation product known as substance E; this mechanism involves several steps, including the irreversible reaction of an active intermediate with an additional molecule of CA leading to the reported decomposition product.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] Zhong, 2014, Studies on the formation and forming mechanism of the related substance E in potassium clavulanate production by HPLC-MS/MS, Braz. J. Pharm. Sci. source

      The “Inference for the forming mechanism” section proposes a multistep route in which an active intermediate reacts with another molecule of clavulanic acid to form substance E. The Conclusions likewise describe formation of a new amide bond. Neither passage says the reaction is irreversible; page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    The significant concentration of intermediate I* after 6 h shifts the equilibrium towards CA, and hence the equilibrium reaction is the rate-limiting step.

    “The significant concentration of intermediate I* after 6 shifts the equilibrium towards CA and hence, the equilibrium reaction (Equation (1)) is the rate-limiting step [17].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Carvalho, 2009, Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach, Biochem. Eng. J. source
    • The authors state this as an assumption: CA degradation follows a two-step mechanism: a reversible equilibrium step in which CA produces an active intermediate I*, followed by an irreversible reaction of I* with an additional CA molecule to form degradation product D.

  • May combine two claims.

    Premisecitation

    N-alkyl-imidazoles are hydrolytically unstable and react under water-catalyzed, base-catalyzed, or acid-catalyzed reaction mechanisms.

    “N-alkyl-imidazoles are hydrolytically unstable and they react under water-catalyzed, base-catalyzed (Figure 9), or acid-catalyzed (Figure 10) reaction mechanisms [33,34].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [33] 1983, Hydrolysis rates of some acetylimidazole derivatives, Can. J. Chem. source

      The Abstract describes water-catalyzed hydrolysis of an acetylimidazolium ion, and the Results and discussion report pH-dependent hydrolysis of N-acetyl derivatives, including N-acetyl-N′-methylbenzimidazolium acetate (pages unavailable in our copy). The rate laws show acid-, pH-independent-, and hydroxide-dependent contributions for particular acetyl derivatives, but the paper does not establish that N-alkyl-imidazoles generally are hydrolytically unstable or undergo all three mechanisms. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [34] Joule, J.A., and Mills, K. (2010). 1,3-Azoles: Imidazoles, thiazoles and oxazoles: Reactions and synthesis. Heterocyclic Chemistry, John Wiley & Sons, Inc.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    Initial CA concentrations in supernatant samples were 65.48 ± 0.04 mg/L (high level), 25.29 ± 0.03 mg/L (medium level), 16.33 ± 0.04 mg/L (low level), and an additional sample had CAo = 126.67 ± 0.04 mg/L.

    “The initial CA concentrations (CAo) in supernatant samples included in the experimental design were 65.48 ± 0.04 mg/L (high level), 25.29 ± 0.03 mg/L (medium level), and 16.33 ± 0.04 mg/L (low level). An additional sample of higher concentration (CAo =126.67 ± 0.04 mg/L) was included in the study.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The rate of CA decomposition tended to slow down markedly as CAo decreased, which was confirmed in the statistical analysis.

    “Interestingly, the rate of decomposition tended to slow down markedly as CAo decreased, which was also confirmed in the statistical analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    At −80 °C, the exhibited degradation rate was minimum, with less than 4% lost in 42 h.

    “In contrast, at the lowest temperature that was evaluated (−80 °C), the exhibited degradation rate was minimum, since less than 4% was lost in 42 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Samples of CA in fermentation broths showed a fast decline in concentration during the first 5 h followed by a slower but stable reaction rate in subsequent hours.

    “Samples of CA in the fermentation broths showed a fast decline of concentration during the first 5 h followed by a slower, but stable, reaction rate in the subsequent hours.”
    • Also stated in: § 2.1. Clavulanic Acid Degradation · plain
      “The experimental data presented in Figure 1 showed that the degradation proceeded at the highest rate during the first 5–6 h.”
    • Also stated in: § 4. Conclusions · plain
      “The samples of CA in fermentation broth exhibited a fast decline of concentration during the first 5 h followed by a slower but stable reaction rate in the subsequent hours, which had been also observed in previous works.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For CAI, the Arrhenius parameters (r2 = 0.997) gave a calculated Ea of 21.85 kJ/mol and a frequency factor (A) of 262.43 h−1.

    “Regarding the Arrhenius parameters (r2 = 0.997), the calculated Ea was 21.85 kJ/mol, which is also close to the calculated value for the irreversible step for CA, the corresponding frequency factor (A) was 262.43 h−1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The calculated kinetic rate constants for CAI were 0.020 h−1 (r2 = 0.989) at 4 °C and 0.039 h−1 (r2 = 0.994) at 25 °C.

    “The calculated kinetic rate constants were 0.020 h−1 (r2 = 0.989) and 0.039 h−1 (r2 = 0.994) at 4 °C and 25 °C.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The chromophore CAI is unstable over time, following a different kinetic mechanism to untreated CA.

    “Considering the structural difference between the CAI and CA, it was observed that the chromophore is also unstable over time, following a different kinetic mechanism to the untreated CA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The dynamics of CA concentration at different temperatures do not fit irreversible first-order or pseudo-first-order models; rather they are similar to those observed in two consecutive equilibrium-irreversible first-order reactions.

    “In this case, the dynamics of CA concentration at different temperatures do not fit with irreversible first-order or pseudo-first-order models; rather they are similar to those observed in two consecutive equilibrium-irreversible first-order reactions (Equations (1) and (3)).”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: CA degradation follows a two-step mechanism: a reversible equilibrium step in which CA produces an active intermediate I*, followed by an irreversible reaction of I* with an additional CA molecule to form degradation product D.

  • May combine two claims.

    Findingown data

    The corresponding frequency factor (A) was 26.97 h−1 with a correlation coefficient (r2) of 0.961.

    “The corresponding frequency factor (A) was 26.97 h−1 with a correlation coefficient (r2) of 0.961.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The results indicate a loss of 56% of CAI complex during the first 24 h, reaching a maximum of 82% at 45 h and 25 °C.

    “The results indicate a loss of 56% of CAI complex during the first 24 h, reaching a maximum of 82% at 45h and 25 °C.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The estimated values of equilibrium constant confirmed the initial assumption of k2 < k1 < k−1, since all calculated values for k2 were lower than Keq, and all estimated values of Keq were lower than 1.

    “The estimated values of equilibrium constant confirmed the initial assumption of k2 < k1 < k−1, since all the calculated values for k2 were lower than Keq, and all the estimated values of Keq were lower than 1.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: For the consecutive equilibrium-irreversible reactions, k2 < k1 < k−1.

  • May combine two claims.

    Findingreasoning

    The CAI chromophore exhibited lower stability in time than CA itself, possibly due to the susceptibility of N-alkyl-imidazoles to hydrolysis in aqueous solutions.

    “Finally, the CAI chromophore, commonly used for spectrophotometric and HPLC analysis of CA samples, exhibited lower stability in time than the CA itself, possibly due to the susceptibility of N-alkyl-imidazoles to hydrolysis in aqueous solutions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    Values of the irreversible rate constant (k2) were previously determined as kobs,1.

    “Values of the irreversible rate constant (k2) were previously determined as kobs,1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Statistical analysis with 95% confidence (r2 = 0.998) indicated that solely temperature had a significant effect on the final concentration of the analyzed CAI, showing a typical Arrhenius-type behavior.

    “Statistical analysis with 95% of confidence (r2 = 0.998) indicated that solely temperature had a significant effect on the final concentration of the analyzed CAI, showing a typical Arrhenius-type behavior.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The half-lives of the CAI complex were 34.5 h at 4 °C and 17.8 h at 25 °C.

    “The half-lives of the complex were 34.5 h and 17.8 h at 4 °C and 25 °C, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 4 °C, the rate of CAI degradation is significant, showing a 40% decay in 24 h and a maximum of 60% in 46 h.

    “However, at this temperature, the rate of degradation is also significant showing a 40% decay in 24 h and a maximum of 60% in 46 h.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Brethauer et al. proposed a first-order kinetic model in which the kinetic constant increased with increasing initial CA concentration, indicating that CA accelerated its own decomposition.

    “A first-order kinetic model was proposed where the kinetic constant increased while increasing the initial CA concentration, indicating that CA accelerated its own decomposition [19].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Brethauer, 2008, Clavulanic acid decomposition is catalyzed by the compound itself and by its decomposition products, J. Pharm. Sci. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Bersanetti et al. found that CA degradation at 10, 20, 25, 30, and 40 °C and pH 6.2 and 7.0 fitted an irreversible first-order kinetics relating the degradation rate constant to temperature.

    “Bersanetti et al. [10] investigated CA degradation at 10, 20, 25, 30, and 40 °C and pH values of 6.2 and 7.0. The results fitted an irreversible first-order kinetics accounting for the relationship between the degradation rate constant and the temperature.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Bersanetti, 2005, Kinetic studies on clavulanic acid degradation, Biochem. Eng. J. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous studies have shown that CA stability increases at slightly acidic pH values.

    “Previous studies have shown that CA stability increases at slightly acidic pH values [10,11]; several authors found a pH of 6.8 as favorable for obtaining high titers of CA in submerged cultures [5,7,9,12,13,14,15,16].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [10] Bersanetti, 2005, Kinetic studies on clavulanic acid degradation, Biochem. Eng. J. source
    • cites [11] Marques, 2009, Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060, Enzyme Microb. Technol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Amino acids and other metabolites or compounds present in culture media affect CA degradation, attributed to polar groups attacking the four-ring lactam carboxyl group of CA to open the β-lactam ring.

    “It has been reported that amino acids and other metabolites or compounds present in the culture media affect the CA degradation, their effect is attributed to the polar groups probably attacking the four-ring lactam carboxyl group of CA to open the β-lactam ring [18,20].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [18] Ishida, 2006, Degradation of clavulanic Acid During the cultivation of Streptomyces clavuligerus; Instability of clavulanic Acid by Metabolites and Proteins from the Strain, J. Microbiol. Biotechnol.
    • cites [20] Roubos, 2002, Clavulanic acid degradation in Streptomyces clavuligerus fed-batch cultivations, Biotechnol. Prog. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Marques et al. observed the highest rates of CA formation and degradation at 32 and 40 °C; the course of CA concentration fitted irreversible first-order kinetics and temperature dependence followed Arrhenius-type behavior.

    “The highest rates of CA formation and degradation were observed at relatively high temperatures (32 and 40 °C). The course of CA concentration fitted irreversible first-order kinetics and the temperature dependence followed an Arrhenius-type behavior [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Marques, 2009, Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060, Enzyme Microb. Technol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Costa and Badino observed that temperature reduction had a stronger impact on CA production than on the reduction of CA degradation.

    “The authors observed that temperature reduction had a stronger impact on CA production rather than on the reduction of CA degradation [16].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Costa, 2012, Production of clavulanic acid by Streptomyces clavuligerus in batch cultures without and with glycerol pulses under different temperature conditions, Biochem. Eng. J. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    β-lactam compounds are susceptible to acid-base catalysis and contain groups such as amino or hydroxyl that can act as catalysts leading to self-catalyzed decomposition.

    “The β-lactam compounds are susceptible to acid–base catalysis and contain groups such as amino or hydroxyl, which can act as catalysts leading to a self-catalyzed decomposition [19].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Brethauer, 2008, Clavulanic acid decomposition is catalyzed by the compound itself and by its decomposition products, J. Pharm. Sci. source

  • May combine two claims.

    Interpretationreasoning

    The equilibrium-irreversible reaction model fits not only hypothetical chemical reaction mechanisms involving equilibrium steps but also potential enzymatic degradation of CA in fermentation broths at temperatures higher than 4 °C, since Michaelis-Menten kinetics represents a special case of this kind of reactions.

    “The equilibrium-irreversible reaction model fits not only the hypothetical chemical reaction mechanisms involving equilibrium steps, but also the potential enzymatic degradation of CA in fermentation broths at temperatures higher than 4 °C, since Michaelis-Menten kinetics represents a special case of this kind of reactions.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: CA degradation follows a two-step mechanism: a reversible equilibrium step in which CA produces an active intermediate I*, followed by an irreversible reaction of I* with an additional CA molecule to form degradation product D.

  • May combine two claims.

    Interpretationreasoning

    The trend of the time course of degradation for CAI does not suggest an equilibrium process as for CA, possibly due to structural chemical modification; other degradation products are formed following a different mechanism.

    “The trend of the time course of degradation for the CAI does not suggest an equilibrium process as for the case of CA, possibly due to the structural chemical modification; other degradation products are formed following a different mechanism.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationnone given

    The reaction rate of degradation is dependent on several factors like pH, medium composition, and temperature, which explains the high variability in the values of constant rates available in literature.

    “The reaction rate of degradation is dependent on several factors like pH, medium composition and temperature, which explains the high variability in the values of constant rates available in literature.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The degradation rate at −80 °C is almost null, thus this condition is appropriate for long-term storage of supernatants and stock solutions.

    “The degradation rate at −80 °C is almost null, thus, this condition is appropriate for long-term storage of supernatants and stock solutions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisereasoning

    Derivatization of CA is required to generate the chromophore clavulanate-imidazole (CAI), which is detectable at a wavelength of 311 nm.

    “Therefore, derivatization of CA is required to generate the chromophore clavulanate-imidazole (CAI), which is detectable at a wavelength of 311 nm.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Twelve experimental runs were performed by duplicate.

    “Twelve experimental runs were performed by duplicate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For the study of CAI stability, a 2-squared factorial design with duplicates was used.

    “For the study of CAI stability, a 2-squared factorial design with duplicates was used.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    A factorial experimental design was proposed with concentration and temperature as factors varying at three and four levels, respectively.

    “A factorial experimental design was proposed; concentration and temperature were defined as factors varying at three and four levels, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Once the open intermediate is formed, several irreversible possibilities can occur, such as nucleophilic attacks by nitrogen on other CA molecules or amino acids, imine formation via decarboxylation, or attack of other substances present in the medium.

    “Once the open intermediate is formed, several irreversible possibilities can occur like nucleophilic attacks by nitrogen on other CA molecules or amino acids, imine formation via decarboxylation or attack of other substances present in the medium [31].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Soroka, 2015, Hydrolysis of clavulanate by Mycobacterium tuberculosis β-lactamase BlaC harboring a canonical SDN motif, Antimicrob. Agents Chemother. source

  • May combine two claims.

    Interpretationown data

    Although the proposed model simplified the decomposition of CA to only one intermediate product and one decomposition product, it allowed prediction of the CA concentration pattern in a rather reliable manner.

    “Although the proposed model simplified the decomposition of CA to only one intermediate product and one decomposition product, it allowed to predict the CA concentration pattern in a rather reliable manner.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The proposed degradation model simplifies the decomposition of CA to only one intermediate product and one decomposition product following equilibrium and irreversible first-order kinetics.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Marques et al. pointed out that ΔG° values for CA formation are slightly higher than those for CA degradation and that at moderate temperatures the degradation of CA would be practically unavoidable.

    “Marques et al. [11] pointed out that ΔG° values for CA formation are slightly higher than those for CA degradation and at moderate temperatures the degradation of CA would be practically unavoidable.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Marques, 2009, Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060, Enzyme Microb. Technol. source

  • May combine two claims.

    Premisenone given

    During derivatization, the β-lactam ring of CA is opened, yielding decarboxylation and formation of an amido group.

    “During derivatization the β-lactam ring of CA is opened, yielding decarboxylation and formation of an amido group as shown in Figure 7.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationcitation

    The fact that ΔG° values for CA formation are slightly higher than those for degradation explains the accumulation of CA in the fermentation broth while the microorganism is active despite continuous degradation.

    “This fact explains the accumulation of CA in the fermentation broth while the microorganism is active despite the continuous degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    During the first 5-6 h of exposition, the equilibrium reaction favors the formation of intermediate I*, which is converted irreversibly into product D at a lower reaction rate, so the irreversible reaction is the rate-limiting step.

    “During the first 5–6 h of exposition, and according to Equation (1), the equilibrium reaction is favoring the formation of intermediate I*, which is converted irreversibly into product D at lower reaction rate, thus the irreversible reaction (Equation (2)) is the rate-limiting step.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: CA degradation follows a two-step mechanism: a reversible equilibrium step in which CA produces an active intermediate I*, followed by an irreversible reaction of I* with an additional CA molecule to form degradation product D.

  • May combine two claims.

    Interpretationreasoning

    The irreversible hydrolysis of the CAI complex might lead to underestimations of CA concentration and/or overestimation of CA degradation rates due to the waiting time of derivatized samples until analyses are performed.

    “The irreversible hydrolysis of the complex might lead to underestimations of CA concentration of samples and/or overestimation of CA degradation rates due to the waiting time of the derivatized samples until the analyses are performed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    CA is poorly retained in C-18 reverse-phase columns for HPLC and does not produce distinctive peaks.

    “CA is poorly retained in C-18 reverse-phase columns for High Performance Liquid Chromatography (HPLC) and it does not produce distinctive peaks.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The model supports the hypothesis of degradation as a consequence of susceptibility to nucleophilic attacks at specific points of the molecule, which might also coincide with hypothetical enzyme-catalyzed reactions at specific conditions.

    “The model is also able to account for the change in the reaction rate depending on the initial CA concentrations and supports the hypothesis of degradation as consequence of a susceptibility to nucleophilic attacks at specific points of the molecule, which also might coincide with hypothetical enzyme catalyzed reactions at specific conditions.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    Kinetic parameters were calculated by linear regression of experimental data with the least squares method, and consistency was checked by determination of correlation coefficient (r2) and residual analysis.

    “Kinetic parameters were calculated by linear regression of experimental data with the least squares method; consistency was checked by determination of correlation coefficient (r2) and residual analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    At −20 °C the degradation rate in the time range explored is rather significant, but this condition is not expected to remain during a long time, since crystallization of the solution will avoid the progress of the reaction.

    “At −20 °C the degradation rate in the time range explored is rather significant, but this condition is not expected to remain during a long time, since crystallization of the solution will avoid the progress of the reaction.”
    Quote in the paperSubstantial claim
47 of 47

  • The authors state this as an assumption.

    Kinetics of CA degradation in fermentation broths at low temperatures have not been explored so far.

    “Kinetics of CA degradation in fermentation broths at low temperatures have not been explored so far.”
    “As far as we know, kinetics of CA degradation in fermentation broths at low temperatures have not been explored.”

  • The authors state this as an assumption.

    CA degradation follows a two-step mechanism: a reversible equilibrium step in which CA produces an active intermediate I*, followed by an irreversible reaction of I* with an additional CA molecule to form degradation product D.

    “Based on this analysis, the following kinetic mechanism of CA degradation was considered: a first step occurs in equilibrium, in which CA produces an active intermediate (I*), hereinafter this intermediate reacts irreversibly with an additional CA molecule to form the degradation product (D).”

  • The authors state this as an assumption.

    For the consecutive equilibrium-irreversible reactions, k2 < k1 < k−1.

    “The observed time courses of CA degradation are typical of consecutive equilibrium-irreversible reactions in which the reaction rate of the irreversible reaction is considerably low in comparison to the equilibrium reaction (i.e., k2 < k1 < k−1) [17].”
    “The estimated values of equilibrium constant confirmed the initial assumption of k2 < k1 < k−1, since all the calculated values for k2 were lower than Keq, and all the estimated values of Keq were lower than 1.”

  • The authors state this as an assumption.

    The standard condition for ΔG° is the previously determined equilibrium at 25 °C.

    “The standard Gibbs free energy of activation for the reversible reaction (ΔG°) was determined assuming the previously determined equilibrium at 25 °C as the standard condition yielding a value of 3.155 kJ/mol.”

  • The authors state this as an assumption.

    The proposed degradation model simplifies the decomposition of CA to only one intermediate product and one decomposition product following equilibrium and irreversible first-order kinetics.

    “The proposed model of degradation simplified the decomposition of CA to only one intermediate product and one decomposition product following equilibrium and irreversible first-order kinetics; hence, deviations were expected between experimental and simulated data.”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Irreversible reaction is cited as Equation (2) instead of (3)

    Minor

    Equation (2) defines the equilibrium constant; the irreversible reaction is labeled Equation (3).

1 of 1

Cites

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4 of the 42 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Gómez-Ríos, D., Ramírez-Malule, H., Neubauer, P., Junne, S., & Ríos-Estepa, R.
Affiliations
  • Universidad de Antioquia (CO)
  • Universidad del Valle (CO)
  • Technische Universität Berlin (DE)
  • Grupo de Bioprocesos, Departamento de Ingeniería Química, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín 050010, Colombia
Corresponding author
  • Gómez-Ríos, David
  • Ramírez-Malule, Howard
ORCID
Venue
Antibiotics
Publisher
MDPI AG
Published
2019-01-17
Volume
8
Issue
1
Pages
6
ISSN
2079-6382 (ISSN-L)
DOI
10.3390/antibiotics8010006
PMC
PMC6466556
OpenAlex
W2909235948
Cited by
17
References
42
Citations by year
2026: 2 · 2024: 1 · 2023: 2 · 2022: 1 · 2021: 5 · 2020: 3 · 2019: 3
FWCI
1.57
Type
journal article
Language
en
Open access
Licence
Locations
  • Antibiotics (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • Institutional Repository University of Antioquia (University of Antioquia) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Departamento Administrativo de Ciencia, Tecnología e Innovación (111577657246 CT 432-2017)
Topics
  • Health Sciences › Medicine › Pharmacology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
Keywords
clavulanic acid, degradation kinetics, low temperature, Streptomyces clavuligerus, fermentation broth, hydrolysis mechanism, first-order kinetics, antibiotic degradation, downstream processing

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-13.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-13.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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