Skip to main content

Growth, respiratory activity and chlorpyrifos biodegradation in cultures of Azotobacter vinelandii ATCC 12837


This study aimed to evaluate the growth, respiratory activity, and biodegradation of chlorpyrifos in cultures of Azotobacter vinelandii ATCC 12837. A strategy based on the modification of culture media and aeration conditions was carried out to increase the cell concentration of A. vinelandii, in order to favor and determine its tolerance to chlorpyrifos and its degradation ability. The culture in shaken flasks, using sucrose as a carbon source, significantly improved the growth compared to media with mannitol. When the strain was cultivated under oxygen-limited (5.5, 11.25 mmol L−1 h−1) and no-oxygen-limited conditions (22 mmol L−1 h−1), the growth parameters were not affected. In cultures in a liquid medium with chlorpyrifos, the bacteria tolerated a high pesticide concentration (500 ppm) and the growth parameters were improved even under conditions with a reduced carbon source (sucrose 2 g L−1). The strain degraded 99.6% of chlorpyrifos at 60 h of cultivation, in co-metabolism with sucrose; notably, A. vinelandii ATCC 12837 reduced by 50% the initial pesticide concentration in only 6 h (DT50).

Graphical Abstract

Key Points

  • A. vinelandii ATCC 12837 tolerates, grows, and degrades high concentrations of chlorpyrifos in vitro.

  • Respirometric parameters of A. vinelandii ATCC 12837 were not adversely affected by chlorpyrifos.

  • The use of a sucrose-enriched medium favored the biodegradation of chlorpyrifos by A. vinelandii.


One of the ecotoxicological problems caused by the intensive use of organophosphate pesticides (OP) is damage to non-target organisms. Pesticides can inhibit the growth of beneficial microorganisms, such as plant growth-promoting rhizobacteria (PGPR) (Walvekar et al. 2017), or reduce metabolic capacities related to their efficacy as inoculants (Sethi and Gupta 2013; Abo-amer et al. 2014; Muttawar and Wadhai 2014).

The evaluation of the effects of the most widely used OP worldwide on PGPR has gained interest because tolerant organisms could maintain their promoting activities, establish in contaminated sites, even used as potential decontaminating agents (Sumbul et al. 2020; Chitara et al. 2021). Tolerance and degradation to various pesticides by PGPR have been evaluated in the genera Azospirillum (Santos et al. 2020), Bacillus (Praveen Kumar et al. 2014), Klebsiella (Rani et al. 2019), Pseudomonas (Giri and Rai 2012), Serriata (Cycón et al. 2013), Ochrobactrum (Abraham and Silambarasan 2016) and Azotobacter (Chennappa et al. 2018a, b), the latter being one of the most important for agricultural proposes.

Azotobacter spp. are efficient in asymbiotic N2 fixation, P solubilization (Sethi and Gupta 2013), production of phytohormones (Chobotarov et al. 2017), siderophores (Shahid et al. 2019), vitamins (Revillas et al. 2000), synthesis of antimicrobial compounds (Nagaraja et al. 2016), production of metabolites of industrial interest such as the alginate and polyhydroxybutyrate (PHB) (Gurikar et al. 2016), as well as in the synthesis of enzymes involved in degradation processes of toxic substances (Chennappa et al. 2019).

Some Azotobacter species degrade aromatic compounds such as insecticides, fungicides, and herbicides (Castillo et al. 2011; Chennappa et al. 2016). These bacteria have particularly shown tolerance to endosulfan, phorate, carbendazim, chlorpyrifos (CP), pendimethalin, among others (Castillo et al. 2011; Chennappa et al. 2014a; Gurikar et al. 2016; Rani and Kumar et al. 2017), without showing growth inhibition (Chennappa et al. 2016). Also, there are reports describing the degradation of lindane (Anupama and Paul 2009), phorate (Moneke et al. 2010), endosulfan (Castillo et al. 2011), pendimethalin (Chennappa et al. 2018a), glyphosate (Mousa et al. 2021), and CP by Azotobacter isolates (Chennappa et al. 2019).

In contrast, other authors have reported adverse effects for Azotobacter spp. (Askar and Khudhur 2013; Chennappa et al. 2013; Walvekar et al. 2017; Kumar et al. 2019); e.g. reduced growth rate in the presence of CP (Menon et al. 2004), glyphosate (Moneke et al. 2010) and endosulfan (Castillo et al. 2011), inhibition of diazotrophic activity (Menon et al. 2004; Chennappa et al. 2019), reduced respiration rate with glyphosate, pendimethalin and fomesafen (Chennappa et al. 2013, 2014b; Wu et al. 2014), cell damage and loss of viability after exposure to different concentrations of glyphosate and atrazine (Shahid et al. 2019).

The genus Azotobacter can exhibit varied behaviors depending on the species and strains, growth conditions, type of pesticide, and contaminant concentrations; therefore, it is useful to evaluate the effect of these factors on model organisms such as Azotobacter vinelandii (Noar and Bruno-Bárcena 2018); A. vinelandii is a strictly aerobic free-living bacterium with growth and metabolite production, both in vitro and in soil, closely related to physicochemical parameters (Lenart 2012; Plunkett et al. 2020), nutrient concentration and availability (essentially carbon and nitrogen sources) (Tejera et al. 2005; Then et al. 2016), microbial interactions (Bhosale et al. 2013), exposure to toxic substances (Chennappa et al. 2019) and oxygenation levels (Peña et al. 2007; Castillo et al. 2013), the latter being one of the critical parameters because of the high oxygen rate consumption of Azotobacter spp. On this regard, some aspects of the respiration in A. vinelandii have been evaluated widely concerning its growth and polymers synthesis (Lozano et al. 2011; Castillo et al. 2020). Culture factors such as the oxygen transfer rate (OTR) and respiratory quotient (RQ) are crucial in describing the physiological state under different growth conditions. They are related to parameters such as the specific growth rate and metabolite production (Gómez-Pazarín et al. 2015). Additionally, it can be useful for monitoring degradation processes (Kahraman and Altin 2020). However, information on the effects of pesticides on the growth and respirometric profile of A. vinelandii is scarce.

Although there are some studies focused on the role of Azotobacter species in tolerance and degradation of pesticides, the information about the effect of OP like CP in the A. vinelandii growth and respiratory activity is limited. Therefore, this study aimed to evaluate the growth, respiratory activity, and biodegradation of chlorpyrifos in cultures of Azotobacter vinelandii ATCC 12837. A strategy based on the modification of culture media and aeration conditions was carried out to increase the cell concentration of A. vinelandii, in order to favor and determine its tolerance to chlorpyrifos and its degradation ability.

Materials and methods


Experiments were carried out using A. vinelandii ATCC 12837. Cells were cryopreserved at − 70 °C in 40% (w/w) glycerol solution and maintained by monthly subculture on Burk´s-sucrose (BS) agar slopes and stored at 4 °C (Peña et al. 2011).

Preparation of inoculum

The inoculum was prepared as follows: A. vinelandii cells were grown at 29 °C in 250 mL Erlenmeyer flasks, containing 50 mL of BS medium for 24 h at 200 rpm. Flasks were incubated until they reached a biomass concentration of 1 g L−1 (measured by dry weight). The liquid culture was diluted at 10% with a fresh BS liquid medium. This suspension was used as inoculum. Each flask was inoculated with 0.1 g L−1 of biomass.

Culture media

Four different media were used for A. vinelandii culture with the following composition (g L−1): (1) BS: sucrose 20, yeast extract (Difco™ BS, USA) 3, K2HPO4 0.66, KH2PO4 0.16, NaCl 0.2, MgSO4·7H2O 0.2, CaSO4 0.05, Na2MoO4·2H2O 0.0029, FeSO4·7H2O 0.027, MOPS (Sigma Aldrich, USA) [50 mmol]. (2) BS2: the same BS composition except by sucrose (2 g L−1). (3) NBRC: Mannitol 5, yeast extract (Difco™ BS, USA) 3, K2HPO4 0.7, KH2PO4 0.1, MgSO4·7H2O 1, MOPS (Sigma Aldrich, USA) [50 mmol]. (4) NBRCm: the same composition except by mannitol (21.3 g L−1). The initial pH was adjusted to 7.2 using NaOH 2N solution. To avoid precipitation during autoclaving, the FeSO4·7H2O and Na2MoO4·2H2O solutions were separated from the other medium components during sterilization (121 °C, 35 min). The C:N ratio (g mol/g mol) of the BS, BS2; NBRCm, and NBRC media were 29, 5.9, 29, and 21, respectively.

Culture conditions

Cultures were carried out in 250 mL Erlenmeyer flasks at 200 rpm and maintained at 29 °C for 72 h in an orbital incubator with a shaking diameter of 2.5 cm. In addition to the flasks used for online measurements of respiration activity, cultures were developed in some parallel flasks, three of which were regularly withdrawn (every 6, 12, or 24 h) and submitted to off-line analytical measurements. Cells of A. vinelandii were grown in 250 mL Erlenmeyer flasks containing 50 mL of BS, NBRC, and NBRCm media and the culture conditions previously described. The effect of different aeration conditions was evaluated by growing the cells of A. vinelandii in 250 mL Erlenmeyer flasks at different filling volumes, containing 10, 20, and 50 mL of BS medium and cultivated as previously described. In order to evaluate the CP effect, cultures were carried out in 250 mL Erlenmeyer flasks containing 50 mL of BS and BS2 culture media with 0, and 500 ppm of technical grade CP (Clorver® 480 EC Versa Agrochemicals, Mexico) and cultivated under the conditions previously described. Uninoculated media with the same concentration of CP were used as a control.

Measurements of respiration activity

Oxygen transfer rate (OTR) and respiratory quotient (RQ) were determined by a respiration activity monitoring system (RAMOS) (Anderlei and Büchs 2001). During the measuring phase, this device measures the decrease of oxygen partial pressure in the gas phase of closed 250 mL flasks with a sensor mounted in the neck of each flask. From the slope of the oxygen partial pressure curve, the system calculates the OTR (Gomez-Pazarín et al. 2015). RQ was estimated from the quotient between the molar ratio of cumulative CO2 production to cumulative O2 utilization (Anderlei et al. 2004). The specific oxygen uptake rate (qO2) was obtained from the quotient between the OTRmax value and the total protein content as previously described by Díaz-Barrera et al. (2011, 2021).

Analytical determinations

Biomass and alginate concentrations were determined gravimetrically (Peña et al. 1997). The number of colonies forming units mL−1 (CFUs) was estimated by plate count (Strobel et al. 2018). Sucrose was assayed for reducing power with 3,5 dinitrosalicylic acid (DNS reagent) (Sigma Aldrich, USA) (Miller 1959). Samples were previously hydrolyzed using β-fructofuranosidase as described by Peña et al. (2011). The protein concentration was determined by the Lowry method using bovine serum albumin as standard (Lowry et al. 1951).

All experiments were carried out by triplicate, and the results presented are the averages of independent samples. When needed, figures and tables show the mean values and standard deviations among replicates. Statistical analysis was carried out using an ANOVA with a multiple comparison Tuckey test (alpha < 0.05).

Determination of chlorpyrifos (CP) and 3,5,6-trichloro pyridine-2-phenol (TCP)

The extracts resulting from the CP experiments described above (subsection 2.4 Culture conditions) were evaluated to identify and quantify CP and its main metabolites (3,5,6-trichloro pyridine-2-phenol (TCP), O, O-diethyl thiophosphate (DETP), and chlorpyrifos oxon). The samples were filtered through a 25 mm and a 0.22-μm polyvinylidene fluoride (PVDF) membrane and then were diluted 100 and 1000 times with mobile phase prior to CP and its metabolites detection.

Each standard (chlorpyrifos 99.5% N-11459 (Chem Service, USA); 3,5,6-trichloro-2-piridinol (TCP) 99.5% (33972-BCBZ8746) (Sigma Aldrich, USA); DETP (Sigma Aldrich, USA); chlorpyrifos oxon (Sigma Aldrich, USA)) and samples were automatically injected through a Sample-Manager system–FTN Acquity® to equipment of Ultra Performance Liquid Chromatography (UPLC) Acquity® Serie H (Waters Corporation, USA) equipped with a column Acquity® UPLC BEH C18 1.7 µm, 2.1 × 50 mm, in a volume of 5.0 µL (Waters Corporation, USA). The column temperature was kept at 40 °C. The chromatographic conditions were as follows: The mobile phase A was ammonium formate 5 mM, pH 3.0, and mobile phase B was methanol + ammonium formate 5 mM + 0.1% of formic acid at a constant flow rate of 0.35 mL min−1, with the following gradient: starting with 83% of solvent A and 17% of solvent B, reaching the 90% of solvent B at 5.5 min and remaining there for 2 min and returning to its first constitution at 7.51 min and remaining there for 2.5 min. With a total running time of 10 min. The autosampler injection needle was rinsed with a mobile phase after each injection. Nitrogen was used as the desolvation gas at a flow rate of 1000 L h−1. The desolvation temperature was 600 °C and the source temperature was 150 °C. Argon was used as the collision gas at a flow rate of 0.14 mL min−1.

The identification and quantification were performed by means of ESI+ (CP) and ESI (TCP) mode in a Mass Spectrometer Xevo TQ-S and workstation with MassLynx™ 4.1 software (Waters Corporation, USA). Ions were monitored using Multiple Reaction Monitoring (MRM) (Additional file 1: Table S1).

Mathematical analysis

The specific growth rate (μ) was calculated considering the growth from 0 to 12 h of cultivation, the period at which the culture was growing exponentially. The equation used was: dX/dt = μX where μ is the specific growth rate (h−1) and X is the cell concentration (g L−1) (Klimek and Ollis 1980). The percentage of degradation and the degradation time in which the pesticide concentration was reduced by 50% was calculated (Abraham and Silambarasan 2016) and reported as DT50 values. The CP concentration profiles in each of the experiments were fitted to a pseudo-first-order degradation equation Ct = C0*e-kt where Ct is the concentration of the component at time t, C0 is the initial concentration, k is the degradation constant, and t is the time.


Growth and respiratory activity of A. vinelandii under different culture media

The respiratory activity parameters of A. vinelandii developed in NBRC, NBRCm, and BS media are shown in Fig. 1. Both the OTR and RQ profiles were different depending on the amount and type of carbon source present in the different media evaluated. In the three evaluated media, there were notable differences during cultivation time and presented characteristic profiles of oxygen limiting conditions distinguished by a higher sustained OTR value during the cell growth period (OTRmax). For the NBRC medium with mannitol as carbon source and a C:N ratio 21 (Fig. 1a), variations in OTR were observed at the beginning of cultivation until reaching an average OTRmax of 2.65 mmol L−1 h−1 between 24 and 55 h of culture. With the NBRCm medium (Fig. 1b), increasing the mannitol content (ratio C:N 29), the OTRmax increased at 5.87 mmol L−1 h−1 as expected because of the increase of the carbon source, indicating a high respiration activity and extending the culture time up to 80 h. Finally, in the BS medium (with sucrose as carbon source and a C:N ratio of 29) (Fig. 1c), an exponential increase in OTR was observed from 0 to 12 h, reaching an average OTRmax of 5.52 mmol L−1 h−1 until 36 h, when it decreased and then increased again until 48 h. Concerning the RQ values, in the NBRCm and BS media, similar values were obtained, both above 1 (1.2 and 1.1, respectively) indicating a less oxidative metabolic activity; whereas, the medium with a lower carbon concentration (NBRC) reached an average RQ of 0.7.

Fig. 1
figure 1

Evolution of the oxygen transfer rate (OTR) and respiration quotient (RQ) in cultures of A. vinelandii grown in NBRC (a), NBRCm (b), and BS (c) media

The growth of A. vinelandii determined by CFUs and total protein in the different culture media is shown in Fig. 2. The maximum values of CFUs mL−1 were 1.4 × 1010 at 48 h in the BS medium, followed by NBRCm medium (9.16 × 109) and NBRC (1.25 × 109) at 72 h (Fig. 2a). Similarly, total protein content (Fig. 2 b) increased exponentially up to 48 h and it was higher in BS medium compared to NBRCm medium containing the same C:N ratio (29); whereas cultures with NBRC medium (C:N ratio 21) showed notably lower growth and protein content.

Fig. 2
figure 2

Growth of A. vinelandii in different culture media. CFUs (a) and protein content (b) of A. vinelandii cultures in shake flasks in BS (black circle), NBRCm (black diamond suit), and NBRC (Black square) medium. Data are presented as the mean and standard deviation from three experiments

Finally, the kinetic and respirometric parameters of A. vinelandii culture on the different media are summarized in Table 1. It is clear from the values of the table that a higher μ, number of CFUs, and protein content were reached in the cultures with the BS medium when OTRmax was 5.52 mmol L−1 h−1. It is important to point out that the highest alginate content was obtained in the NBRCm medium, indicating that with mannitol as a carbon source, compared to sucrose medium (BS), the alginate synthesis was improved (5.17 g L−1); whereas in BS medium, the alginate production was lower (0.97 g L−1alginate).

Table 1 Kinetic and respirometric parameters of A. vinelandii cultured in shake flasks in different culture media and conditions

Growth and respiratory activity of A. vinelandii under oxygen and non-oxygen-limited conditions

Figure 3 shows the respiratory activity parameters of A. vinelandii developed in BS medium with different filling volumes 10 (a), 20 (b), and 50 mL (c). As it was expected, the OTRmax values increased considerably, decreasing the filling volume, obtaining values of 5.5, 11.45, and 22 mmol L−1 h−1 with 50, 20, and 10 mL, respectively. With 50 and 20 mL, a typical oxygen limitation OTR profile was obtained for A. vinelandii cells.

Fig. 3
figure 3

Evolution of the oxygen transfer rate (OTR) and respiration quotient (RQ) in cultures of A. vinelandii grown in BS liquid medium with 10 (a), 20 (b), and 50 mL (c) of filling volumes

On the other hand, in the cultures with 10 mL of filling volume, a typical non-oxygen-limited profile was observed. In that case, the OTRmax was reached at 20 h of culture, followed by a drop in the respiration rate, indicating the decrease in oxidative activity due to the rapid depletion of the sucrose. In the case of the cultures using 20 mL of filling volume, the same drop was presented at 20 h but increased again from 20 to 27 h. Finally, with 50 mL of filling volume, a previously described oxygen limitation profile was exhibited, showing an exponential increase in OTR during the first 6 h of culture and a stationary stage that remained until the carbon source in the medium was exhausted, prolonging the culture until 55 h. In contrast, the RQ was not modified by the filling volume, being in all cases RQ of 1.02–1.07.

As it is presented in Fig. 4, the maximum growth values determined by the parameters of maximum biomass (9.7 g L−1), CFUs mL−1 (1 × 1010), and total protein (2.78 mg mL−1) were obtained in the lowest OTR condition, i.e., in the BS medium with 50 mL filling volume between 48 and 72 h of culture. Similarly, the kinetic parameters (Table 1) with that condition showed a higher growth rate of 0.12 h−1 and thus a shorter doubling time (5.7 h) even under O2-limiting conditions (5.5 mmol L−1 h−1). It is clear that, the growth of strain ATCC 12837 in BS medium under limited oxygenation conditions did not significantly affect growth, and also presented better cell viability, protein content, and lower qO2.

Fig. 4
figure 4

Biomass (a), protein content (b), and CFUs growth kinetics (c) of A. vinelandii cultures in 250 mL shake flasks at different filling volumes 10 mL (Black circle), 20 mL (black diamond suit) and 50 mL (black square). Data are presented as the mean and standard deviation from three experiments

Growth and respiratory activity of A. vinelandii in media with chlorpyrifos

The respiratory activity parameters of A. vinelandii developed in BS and BS2 media with and without CP are shown in Fig. 5. In the first case, when the bacterium was cultured without a decrease in the carbon source (sucrose) in both BS medium without the contaminant (a) and BS with CP (b), clear differences in OTR at the first 12 h of culture were observed. Particularly in the BS medium with CP (Fig. 5b), the increase in the OTR until reaching the maximal was slower compared to the medium without pesticide (BS) (Fig. 5a) in which during the first 6 h the maximum OTR was reached. However, the OTRmax values were higher in the medium with CP (7.9 mmol L−1 h−1) in contrast to the BS medium (5.88 mmol L−1 h−1), and a prolongation of the respiratory activity up to more than 60 h of culture was observed, suggesting a higher metabolic activity. The average RQ values for the medium with CP were slightly lower (1.07) compared to the BS medium (1.12), in both cases greater than 1. In the second case, by decreasing the concentration of the carbon source (BS2) tenfold and with the addition of the pesticide (B2 + CP) (Fig. 5c and d) a similar behavior to the previous one was obtained in terms of the increase in OTRmax for the medium with pesticide (6.9 mmol L−1 h−1) compared to the BS2 medium (5.38 mmol L−1 h−1) and a delayed activity in the increase of OTR in the first hours of culture, possibly linked to the adaptation of the bacteria to the presence of the contaminant. Finally, the RQ values for BS2 and BS2 + CP media were 0.90 and 0.89, respectively.

Fig. 5
figure 5

Evolution of the oxygen transfer rate (OTR) and respiration quotient (RQ) in cultures of A. vinelandii grown in media: BS (a), BS + 500 ppm CP (b), BS2 (c), and BS2 + 500 ppm CP (d)

Regarding the growth of A. vinelandii in BS medium with and without CP, Fig. 6 shows the biomass (a), protein content (b), and CFUs (c), as well as sucrose consumption of all treatments. The BS + CP and BS2 + CP media obtained higher biomass production in relation to the controls without pesticide. The media with 20 gL−1 sucrose showed exponential growth until 60 h of culture, while with 2 gL−1 sucrose, the exponential phase ended at 12 h of culture. Similarly, the higher total protein content and CFUs mL−1 were recorded in the media with CP at 60 and 24 h for BS + CP and BS2 + CP media, respectively.

Fig. 6
figure 6

Biomass (a), protein content (b), CFUs (c), and Sucrose consumption (d) of A. vinelandii growth in BS (white square), BS + CP (black square), BS2 (white square) and BS2 + CP (black circle). Data are presented as the mean and standard deviation from three experiments

On the other hand, sucrose consumption (d) was slightly faster in the media without pesticide compared to the media with CP, indicating the use of alternative sources present in the medium with pesticide. Finally, the kinetic parameters showed a growth rate without statistical differences for the media with pesticide and their respective controls, as well as a lower qO2 in the BS + CP medium (Table 1).

Tolerance and biodegradation of chlorpyrifos

The tolerance and biodegradation of CP and its major metabolite TCP were assessed using A. vinelandii ATCC 12837 in liquid culture. Although the ATCC 12837 strain was exposed to a concentration of 500 ppm of CP, this demonstrated not only tolerance to the compound but also increased growth and respiratory activity as described above in both BS and BS2 media.

According to the analysis of detection and quantification of CP and its intermediates in the supernatants of A. vinelandii cultures, both in BS and BS2 medium, a decrease in the concentration of the pesticide was determined in the medium (Fig. 7). Strain ATCC 12837 completely degraded the pesticide (500 mg L−1 of CP) with a DT50 of 6 h with BS medium. Whereas in the BS2 medium, when sucrose was reduced, the strain degraded 330 mg L−1 of CP and the time to reach DT50 was 30 h. The above was influenced by reduced growth in the BS2 medium with low sucrose content. The degradation percentages of the strain after 60 h of culture were 99.5% and 66.8% in BS and BS2 media, respectively.

Fig. 7
figure 7

Chlorpyrifos degradation by A. vinelandii growth in BS + CP (black square) and BS2 + CP (black circle)

According to our screening analysis of CP and its main metabolites by UPLC/MS–MS, no accumulation of TCP or formation of other intermediate compounds (DETP or chlorpyrifos oxon) was detected in A. vinelandii growth supernatants with 500 ppm CP. Under the conditions tested, the bacteria apparently can metabolize CP and use it for growth and energy. In addition, a higher percentage of degradation was observed when grown on a nutrient-rich medium (BS medium).


Growth and respiratory activity of A. vinelandii in different culture media

Regarding to the respiratory activity A. vinelandii grown under different culture media, the OTR and RQ were dependent on the amount and type of carbon source available (Fig. 1), a similar behavior that has been observed previously (Noguez et al. 2008). This is explained because both parameters are substrate-dependent (Kahraman and Altin et al. 2020). Factors such as oxygen availability and the amount or type of carbon source modify the metabolic response of aerobic organisms to oxidize compounds and produce CO2 which can be monitored by OTR and RQ values (Gomez-Pazarín et al. 2015).

In this context, characteristic profiles of oxygen limiting conditions were observed in the three evaluated media, similar to those previously reported for cultures of A. vinelandii (Peña et al. 2011). Those profiles are characterized by maintaining a sustained OTRmax value during the culture time and RQ values of 1 or higher, as those obtained in the NBRCm and BS media. RQ values higher than 1 are generally attributed to anaerobic or microaerophilic conditions, where the oxygen availability is not sufficient to oxidize the carbon source present in the media, whereas, values below 1 are related to aerobic processes (Dilly 2001, 2003; Lamy et al. 2013).

In our results, the OTRmax values and the growth of A. vinelandii determined by CFUs and total protein in the different culture media were higher when C:N ratio of 29 was used (Fig. 2). It is known that high concentrations of organic carbon in the form of sugars, alcohols, and organic acids (25%) are used to improve the growth of Azotobacter (Tejera et al. 2005). In contrast, Castillo et al. (2017) found that using ratios between 16 and 32 gC gN−1, there were no significant differences in the growth of A. vinelandii when using sucrose and yeast extract as carbon and nitrogen sources, respectively. In our results with A. vinelandii ATCC 12837, the type of carbon source and the increase of the C:N ratio, positively impacted the cell growth, viability, and respirometric parameters, even in oxygen limiting conditions.

In this line, recently Díaz-Barrera et al. (2021) reported that, under oxygen limitation conditions (5.0 ± 0.9 mmol L−1 h−1) and no-nitrogen fixation, similar to those carried out in our study, A. vinelandii ATCC 9046 channeled the carbon source mainly to the production of biomass and intracellular polymers like PHB.

The above is consistent with that reported by Peña et al. (2007, 2011), who obtained higher viability, biomass production, and specific growth rate with the increase in the OTR in cultures in shaken flasks (OTRmax 6 mmol L−1 h−1 compared to an OTRmax of 2.5 mmol L−1 h−1). Also, when A. vinelandii was grown at OTRmax of 5.5 mmol L−1 h−1, (similar to that obtained in our study for BS and NBRCm medium) the carbon source was mainly directed to growth with an increase in the biomass concentration, polymer, and CO2 production, which may be affected by the strain qO2 (Diaz-Barrera et al. 2011). That is coherent with our results because a lower qO2 was obtained in the BS medium, the same with the higher growth and respiratory activity.

Growth and respiratory activity of A. vinelandii under oxygen and non-oxygen-limited conditions

The OTRmax values in the cultures of A. vinelandii in BS medium increased considerably by decreasing the filling volume (Fig. 3), obtaining a typical oxygen limitation and non-oxygen limitation profiles as previously reported in cultures of A. vinelandii in stirrer tank and shaken flasks (Peña et al. 2007; Díaz-Barrera et al. 2007; Moral et al. 2016). In contrast, the RQ values were not modified by the filling volume. These data contrast with those previously reported for A. vinelandii ATCC 9046 (Peña et al. 2011), where at higher OTR, RQ values are less than 1 and conversely at low OTR, higher than 1. This could be related to a lower respiration rate observed in the strain ATCC 12837 used in the present study. For the case of A. vinelandii ATCC 9046 strain, it has been documented that it possesses mechanisms that regulate its respiration efficiency depending on the modifications of its respiratory chain by the activation or deactivation of terminal oxidases. These oxidases respond to environmental and nutritional changes such as oxygen availability to maximize energy conservation or produce intracellular or extracellular polymers and it may vary slightly among strains (Castillo et al. 2020).

On the other hand, previous studies with A. vinelandii in shaken flasks, under high and low aeration conditions, showed that changes in oxygen availability had a considerable impact on the growth profiles (Castillo et al. 2013), especially on growth, measured as biomass, and protein production (Peña et al. 2011). In the study of García et al. (2018), the highest protein yield of A. vinelandii (0.15 g protein g glucose−1) was obtained in the cultures developed under the lowest OTR (2.4 mmol L−1 h−1). In our case, the maximum growth values were also obtained at the lowest OTR condition (Fig. 4). Other authors have reported that when OTRmax was reduced, the μ value also decreased, although without changes in the final biomass concentration (Peña et al. 2007, 2011; Díaz-Barrera et al. 2021). In our study, the growth of strain ATCC 12837 in BS medium under limited oxygenation conditions did not significantly affect growth, and also presented better cell viability, protein content, and lower qO2. The last is a relevant characteristic to consider as a scale-up criterion, owed to the high requirements that characterize other strains like A. vinelandii ATCC 9046, and are usually a limiting condition for process scale-up.

Growth and respiratory activity of A. vinelandii in media with chlorpyrifos

Due to the typically high respiration rates of A. vinelandii, oxygen limitations and preferences for carbon sources usually occur in the early stages of fermentation (Peña et al. 2007). Besides, in the presence of toxic substances, a period of adaptation or reduced respiration rates could occur early in the culture (Chennappa et al. 2013). In our case, we observed differences in OTR values in the cultures of A. vinelandii developed in BS and BS2 media with CP at the first 12 h of cultivation, in OTRmax, and the prolongation of respiratory activity, relative to their respective control conditions (Fig. 5).

It is important to point out that, this is the first time when OTR and RQ online values have been estimated for A. vinelandii in response to the presence of CP in a liquid medium with sucrose. The OTRmax values recorded were significantly higher in media with the pesticide, and prolongation of respiratory activity was observed in both conditions (BS and BS2). The above suggested an increase in metabolic activity related to the addition of the pesticide as a carbon source.

Regarding the RQ values, the average values were slightly lower by decreasing tenfold the carbon concentration (BS2) with and without the addition of pesticide. The latter was related with the decrease of the carbon availability and presence of pesticide, which resulted in an oxygen non-limiting condition, where the lower metabolic activity and CO2 production was reflected in the RQ value (Lamy et al. 2013). All the above was also then supported by growth parameters.

In the cultures of A. vinelandii in the presence of CP, the growth was better than when the pesticide was not used (Fig. 6). This suggests the use of CP as a carbon source by A. vinelandii ATCC 12837, since it not only tolerated the high concentration of the pesticide (500 ppm) but also had a significantly higher growth compared to the reference treatments, as well as a lower qO2 in the BS + CP medium. This is consistent with other Azotobacter strains that did not show any in vitro growth impairment in the presence of CP (Chennappa et al. 2014a). On the other hand, slightly faster sucrose consumption in the media without pesticide, indicating the use of alternative sources present in the medium with CP.

Although other Azotobacter strains have shown tolerance to CP (Gurikar et al. 2016; Chennappa et al. 2019), this is the first time that respirometric parameters are measured and related to the growth of A. vinelandii in presence of a pesticide. In the present study, we highlight that strain ATCC 12837 growth in a high CP concentration in contrast with previously reported (100 ppm and 1–5%) (Mac-Rae and Celo 1974; Chennappa et al. 2019); without adversely affecting its growth or respiratory activity. In contrast, according to Mac-Rae and Celo (1974), despite showing tolerance, the oxygen consumption rate of A. vinelandii was considerably reduced when using 100 ppm of OP (Naled, Terracur-P, coumaphos, malathion, CP). This could be related to the strain ATCC 12837 high tolerance to the pollutant, since decrease in its respiration activity is not observed as a result of exposure to CP.

Tolerance and biodegradation of chlorpyrifos

The tolerance and biodegradation of toxic compounds by Azotobacter spp. have not been fully addressed, especially concerning pesticides. Recently, it has been suggested that strains of this genus can show tolerance to compounds such as CP, and even degrade it (Chennappa et al. 2019). In the present study, even though the strain was exposed to a concentration of 500 ppm of CP, higher than those used in other reports for Azotobacter spp. and other genera (from 10 to 300 ppm) (Maya et al. 2011; Rayu et al. 2017; Akbar and Sultan et al. 2016; Liu et al. 2011: Yang et al. 2005; Abraham and Silambarasan 2016; Shi et al. 2019), the strain ATCC 12837 demonstrated not only tolerance to the compound, but also increased growth and respiration activity.

Commonly, some microorganisms can be tolerant to low concentrations of pesticides such as CP, thanks to primary protective mechanisms mediated by oxidative enzymes as cytochrome p450, peroxidases, and polyphenol oxidases (Abraham and Lambarasan 2018), but high CP concentrations could strongly affect the bacterial growth (Singh et al. 2011) and drastically decrease the number of tolerant organisms at concentrations above 100 ppm (Hernández-Ruíz et al. 2017).

In addition, one of the limiting factors in the complete degradation of CP is usually the generation of secondary metabolites such as TCP. TCP is the main degradation product of CP and tends to be resistant to biodegradation or bactericidal due to its composition, as it contains a pyridinol ring with 3 chlorine atoms (Jabeen et al. 2015). The above limits the number of organisms capable of fully mineralizing the compound (Abraham and Silambarasan 2016). Some strains, such as Pseudomonas sp. and Bacillus megaterium, have been able to degrade CP (100 mg L−1) but not completely TCP (Barman et al. 2014; Zhu et al. 2019). This situation may be reflected with the accumulation of TCP and other intermediates, which prevents the complete elimination of the parent compound (Barman et al. 2014) and may allow further dissipation of contaminants, as e.g. TCP is more soluble than the parent molecule (John and Shaike 2015) and acts as an endocrine disruptor (Fishel 2013).

In our study, A. vinelandii tolerated, grew, and efficiently degrade a high CP concentration in vitro, both in BS and BS2 media, without the accumulation of TCP or formation of other metabolites (DETP or chlorpyrifos oxon). This suggests that, under the conditions tested, the bacteria can completely metabolize CP and use it for growth and energy. However, the mechanisms of CP degradation or the involvement of enzymes associated with its degradation such as organophosphate hydrolases (Li et al. 2007; Barman et al. 2014) have not yet been fully elucidated or reported for Azotobacter spp.

One of the closest genera to Azotobacter that has shown efficiency in CP degradation is Pseudomonas, as it can use it as a carbon source and energy (Gilani et al. 2016); and it has been particularly documented in strains such as ATCC 700113 (Feng et al. 1998). Pseudomonas syringae was able to degrade 99.1% of 100 mg L−1 of CP in 5 d also presenting degradation-associated phosphoesterase enzymatic activity (Zhu et al. 2019). However, the initial concentration is important and another limiting factor in CP degradation. e.g., although Pseudomonas spp. can degrade CP, it decreases its growth or stops degrading TCP at concentrations higher than 200 ppm (Li et al. 2007). In contrast, A. vinelandii ATTCC 12837 strain showed higher tolerance (500 ppm) and degradation efficiency compared to Pseudomonas spp.

On the other hand, Pseudomonas putida is among the most efficient strains in CP degradation (Gilani et al. 2016). Especially when it was developed under optimal growth conditions in glucose supplemented medium (Vijayalakshmi and Usha 2012). In the case of A. vinelandii, a higher percentage of degradation was observed when grown on a nutrient-rich medium (BS medium) (Fig. 7). This is consistent with what was also reported by Gilani et al. (2010), who point out that the degradation of CP in the presence of nutrients increases due to better cell growth by greater availability of easily metabolizable compounds, which allows the pesticide degradation in a co-substrate condition.

Furthermore, it has been described that, under neutral pH conditions, as our experiment was conducted, CP can be hydrolyzed and follow different biodegradation pathways; and under aerobic conditions, the breaking of the aromatic rings is favored (Jayasri et al. 2014).

Given that in our screening analysis of CP and TCP by UPLC/MS–MS no other intermediate compounds were detectable during in vitro culture development, we show a possible degradation pathway that A. vinelandii ATCC 12837 could follow (Fig. 8). The hydrolysis of CP to TCP, followed by reductive dechlorination of TCP and incorporation of the pyridine ring into the Krebs cycle which completes the degradation of CP and this has also been identified in Pseudomonas (Vijayalakshmi and Usha 2012); or the formation of DETP which is rapidly degraded to ethanol and phosphorothioic acid molecules and can be used as a S, N, and P source for microorganisms (Rokade and Mali 2013; Bose et al. 2021).

Fig. 8
figure 8

Chlorpyrifos biodegradation pathway (own elaboration)

Regarding efficiency, in our study, A vinelandii degraded CP 10 times faster (200 mg L−1 in 4.8 h in BS medium), compared to the bacterium Cupriavidus nantogensis (200 mg L−1 in 48 h) and similarly could tolerate up to 500 mg L−1 (Shi et al. 2019). On the other hand, the fungus Cladosporium cladosporioides degraded only 50 ppm of CP in 5 d and tolerated 500 mgL−1 as well, and although it generated TCP as an intermediate, it degraded rapidly without leading to accumulation; and similar to our results, they did not detect traces of compounds in chromatographic analysis (Chen et al. 2012).

The elimination of CP in the medium suggests that the strain utilizes the pesticide as a carbon source and energy efficiently compared to other strains in addition to being highly tolerant so it could maintain its activities as a PGPR. This has been previously described in Azotobacter salinestris which maintained the highest production of indoleacetic acid (auxin) on medium supplemented with 1 mg tryptophan and CP (1%), indicating that CP did not negatively affect its growth or phytohormone synthesis (Chennappa et al. 2016).

It is worth noting that, decades of research on the effect of pesticides such as CP on the development of Azotobacter spp. generally reported growth impairment, respiratory inhibition, changes in oxygen consumption rate, and no degradation (Mac-Rae and Celo 1974; Omar and Abd-Alla 1992). Recent studies have identified that certain strains have shown greater tolerance to different compounds, particularly to CP (Chennappa et al. 2014a; Farhan et al. 2021). This is attributable, according to some authors, to the fact that rhizospheric microorganisms that have been chronically exposed to pesticides have created resistance and accumulated adaptations to use them as a carbon source and energy (Roy et al. 2020); while, maintaining and even favoring their PGPR activities (Shahgholi and Ahangar 2014; Pant et al. 2016).

This allows that A. vinelandii ATTCC 12837 to be an excellent candidate to be used in CP remediation, both in vitro and in situ, since microorganisms that can degrade pesticides in vitro usually maintain this capacity in soil (Vidya Lakshmi et al. 2009); although considering a decrease in the speed and efficiency of degradation (Deng et al. 2015) due to multiple edaphoclimatic factors that may vary their behavior (Aasfar et al. 2021).

In conclusion, the excessive use of pesticides as CP is related to multiple environmental alterations. Degradation strategies using rhizospheric microorganisms that also favor the development of crops have become more interesting in the search for alternatives that contribute to the reduction of fertilizers and toxic agents. In this context, we proposed a cultivation strategy to evaluate the growth of A. vinelandii ATTC 12837 and the degradation of CP. Our strategy to optimize bacterial growth allowed us to demonstrate that sucrose as a carbon source favored the in vitro development of A. vinelandii ATCC 12837, as well as the degradation of CP. Furthermore, despite the high oxygen consumption rates that are often a limiting step for large-scale production of Azotobacter spp., oxygen-limiting conditions did not affect the growth of ATTC 12837 strain. Also, this is the first time when online respirometric parameters have been estimated in response to the presence of CP for this bacterium. On the other hand, the results demonstrate that the model organism A. vinelandii ATTC 12837 (deeply studied as a PGPR), is also highly tolerant and efficiently degraded chlorpyrifos, without accumulation of toxic secondary metabolites, and with the potential to develop into a promising candidate for improving the productivity of crops in pesticide-contaminated soils.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.


  1. Aasfar A, Bargaz A, Yaakoubi K, Hilali A, Bennis I, Zeroual Y, Meftah Kadmiri I (2021) Nitrogen-fixing Azotobacter species as potential soil biological enhancers for crop nutrition and yield stability. Front Microbiol.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Abo-amer A, Abu-gharbia M, Soltan E, Abd El-Raheem W (2014) Isolation and molecular characterization of heavy metal-resistant Azotobacter chroococcum from agricultural soil and their potential application in bioremediation. Geomicrobiol J 31:551–561.

    CAS  Article  Google Scholar 

  3. Abraham J, Silambarasan S (2016) Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol using a novel bacterium Ochrobactrum sp. JAS2: a proposal of its metabolic pathway. Pestic Biochem Phys 126:13–21.

    CAS  Article  Google Scholar 

  4. Abraham J, Silambarasan S (2018) Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by fungal consortium isolated from paddy field soil. Environ Eng Manag J 17(3):523–528.

    CAS  Article  Google Scholar 

  5. Akbar S, Sultan S (2016) Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement. Braz J Microbiol 47(3):563–570.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Anderlei T, Büchs J (2001) Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J 7:157–162.

    CAS  Article  PubMed  Google Scholar 

  7. Anderlei T, Zang W, Papaspyrou M, Büchs J (2004) Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochem Eng J 17:187–194.

    CAS  Article  Google Scholar 

  8. Anupama K, Paul S (2009) Ex situ and in situ biodegradation of lindane by Azotobacter chroococcum. J Environ Sci Health - B 45:58–66.

    CAS  Article  Google Scholar 

  9. Askar A, Khudhur M (2013) Effect of some pesticides on growth, nitrogen fixation and nif genes in Azotobacter chroococcum and Azotobacter vinelandii isolated from soil. J Toxicol Environ Health Sci 5:166–171.

    Article  Google Scholar 

  10. Barman D, Haque M, Islam S, Yun H, Kim M (2014) Cloning and expression of ophB gene encoding organophosphorus hydrolase from endophytic Pseudomonas sp. BF1-3 degrades organophosphorus pesticide chlorpyrifos. Ecotoxicol Environ Saf 108:135–141.

    CAS  Article  PubMed  Google Scholar 

  11. Bhosale H, Kadam T, Bobade A (2013) Identification and production of Azotobacter vinelandii and its antifungal activity against Fusarium oxysporum. J Environ Biol 34:177–182

    CAS  PubMed  Google Scholar 

  12. Bose S, Kumar PS, Vo DV, (2021) A review on the microbial degradation of chlorpyrifos and its metabolite TCP. Chemosphere 283:131447.

    CAS  Article  PubMed  Google Scholar 

  13. Castillo J, Casas J, Romero E (2011) Isolation of an endosulfan-degrading bacterium from a coffee farm soil: persistence and inhibitory effect on its biological functions. Sci Total Environ 412–413:20–27.

    CAS  Article  PubMed  Google Scholar 

  14. Castillo T, Heinzle E, Peifer S, Schneider K, Peña MC (2013) Oxygen supply strongly influences metabolic fluxes, the production of poly(3-hydroxybutyrate) and alginate, and the degree of acetylation of alginate in Azotobacter vinelandii. Process Biochem 48:995–1003.

    CAS  Article  Google Scholar 

  15. Castillo T, Flores C, Segura D, Espín G, Sanguino J, Cabrera E, Barreto J, Díaz-Barrera A, Peña C (2017) Production of polyhydroxybutyrate (PHB) of high and ultra-high molecular weight by Azotobacter vinelandii in batch and fed-batch cultures. J Chem Technol Biotechnol 92(7):1809–1816.

    CAS  Article  Google Scholar 

  16. Castillo T, García A, Padilla-Córdova C, Díaz-Barrera A, Peña C (2020) Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers. Electron J Biotechnol 48:36–45.

    CAS  Article  Google Scholar 

  17. Chen S, Liu S, Peng C, Liu H, Hu M, Zhong G (2012) Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by a new fungal strain Cladosporium cladosporioides Hu-01. PLoS ONE 7:1–12.

    CAS  Article  Google Scholar 

  18. Chennappa G, Adkar-Purushothama C, Suraj U, Tamilvendan K, Sreenivasa M (2013) Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka, India. World J Microbiol Biotechnol 30:1–7.

    CAS  Article  PubMed  Google Scholar 

  19. Chennappa G, Adkar-Purushothama C, Naik M, Suraj U, Sreenivasa M (2014a) Impact of pesticides on PGPR activity of Azotobacter sp. isolated from pesticide flooded paddy soils. Greener J Agric Sci 4:117–129.

    Article  Google Scholar 

  20. Chennappa G, Adkar-Purushothama CR, Suraj U, Tamilvendan K, Sreenivasa MY (2014b) Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka. India World J Microbiol Biotechnol 30(1):1–7.

    CAS  Article  PubMed  Google Scholar 

  21. Chennappa G, Udaykumar N, Vidya M, Nagaraja H, Amaresh Y, Sreenivasa M (2019) Azotobacter—a natural resource for bioremediation of toxic pesticides in soil ecosystems. New and Future Developments in Microbial Biotechnology and Bioengineering.

  22. Chennappa G, Naik MK, Adkar-Purushothama CR, Amaresh YS, Sreenivasa MY (2016) PGP potential, abiotic stress tolerance, and antifungal activity of Azotobacter strains isolated from paddy soils. Indian J Exp Biol 54(5):322–31

    CAS  PubMed  Google Scholar 

  23. Chennappa G, Naik MK, Amaresh YS, Nagaraj H, Sreenivasa MY (2018a) Azotobacter—a potential bio-fertilizer and bio inoculants for sustainable agriculture. In: Panpatte D (ed) Microorganisms for green revolution Springer Nature, Singapore, pp 78–87.

    Google Scholar 

  24. Chennappa G, Sreenivasa M, Nagaraja H (2018b) Azotobacter salinestris: a novel pesticide-degrading and prominent biocontrol PGPR bacteria. In: Naveen Kumar A (ed) Microorganisms for sustainability 23–43.

  25. Chitara M, Chauhan S, Singh R (2021) Bioremediation of polluted soil by using plant growth-promoting rhizobacteria. Microbial Rejuvenation Pollut Environ.

    Article  Google Scholar 

  26. Chobotarov A, Volkogon M, Voytenko L, Kurdish I (2017) Accumulation of phytohormones by soil bacteria Azotobacter vinelandii and Bacillus subtilis under the influence of nanomaterials. J Microbiol Biotechnol Food Sci 7:271–274.

    CAS  Article  Google Scholar 

  27. Cycoń M, Żmijowska A, Wójcik M, Piotrowska-Seget Z (2013) Biodegradation and bioremediation potential of diazinon-degrading Serratia marcescens to remove other organophosphorus pesticides from soils. J Environ Manage 117:7–16.

    CAS  Article  PubMed  Google Scholar 

  28. Deng S, Chen Y, Wang D, Shi T, Wu X, Ma X, Li X, Hua R, Tang X, Li QX (2015) Rapid biodegradation of organophosphorus pesticides by Stenotrophomonas sp. G1. J Hazard Mater 297:17–24.

    CAS  Article  PubMed  Google Scholar 

  29. Díaz-Barrera A, Peña C, Galindo E (2007) The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii. Appl Microbiol Biotechnol 76:903–910.

    CAS  Article  PubMed  Google Scholar 

  30. Díaz-Barrera A, Aguirre A, Berrios J, Acevedo F (2011) Continuous cultures for alginate production by Azotobacter vinelandii growing at different oxygen uptake rates. Process Biochem 46:1879–1883.

    CAS  Article  Google Scholar 

  31. Díaz-Barrera A, Sanchez-Rosales F, Padilla-Córdova C, Andler R, Peña C (2021) Molecular weight and guluronic/mannuronic ratio of alginate produced by Azotobacter vinelandii at two bioreactor scales under diazotrophic conditions. Bioprocess Biosyst Eng 44:1275–1287.

    CAS  Article  PubMed  Google Scholar 

  32. Dilly O (2001) Microbial respiratory quotient during basal metabolism and after glucose amendment in soils and litter. Soil Biol Biochem 33:117–127.

    CAS  Article  Google Scholar 

  33. Dilly O (2003) Regulation of the respiratory quotient of soil microbiota by availability of nutrients. FEMS Microbiol Ecol 43(3):375–381.

    CAS  Article  PubMed  Google Scholar 

  34. Farhan M, Ahmad M, Butt KA, ZA, Khan QF, Raza SA, Qayyum H, Wahid A, (2021) Biodegradation of chlorpyrifos using isolates from contaminated agricultural soil, its kinetic studies. Sci Rep 11:10320.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Feng Y, Minard R, Bollag J (1998) Photolytic and microbial degradation of 3, 5, 6-trichloro-2-pyridinol. Environ Toxicol Chem 17:814–819.

    CAS  Article  Google Scholar 

  36. Fishel FM (2013) Epa’s Endocrine Disruptor Screening Program (EDSP). EDIS.

    Article  Google Scholar 

  37. García A, Ferrer P, Albiol J, Castillo T, Segura D, Peña C (2018) Metabolic flux analysis and the NAD(P)H/NAD(P)+ ratios in chemostat cultures of Azotobacter vinelandii. Microb Cell Fact.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Gilani RA, Rafique M, Rehman A, Munis MFH, Rehman SU, Chaudhary HJ (2016) Biodegradation of chlorpyrifos by bacterial genus Pseudomonas. J Basic Microbiol 56(2):105–119.

    CAS  Article  PubMed  Google Scholar 

  39. Gilani S, Ageen M, Shah H, Raza S (2010) Chlorpyrifos degradation in soil and its effect on soil microorganisms. J Anim Plant Sci 20:99–102

    Google Scholar 

  40. Giri K, Rai J (2012) Biodegradation of endosulfan isomers in broth culture and soil microcosm by Pseudomonas fluorescens isolated from soil. Int J Environ Stud 69:729–742.

    CAS  Article  Google Scholar 

  41. Gómez-Pazarín K, Flores C, Castillo T, Büchs J, Galindo E, Peña C (2015) Molecular weight and viscosifying power of alginates produced in Azotobacter vinelandii cultures in shake flasks under low power input. J Chem Technol Biotechnol 91:1485–1492.

    CAS  Article  Google Scholar 

  42. Gurikar C, Naik MK, Sreenivasa MY (2016) Azotobacter: PGPR activities with special reference to effect of pesticides and biodegradation. In: Singh D, Singh H, Prabha R (eds) Microbial inoculants in sustainable agricultural productivity. Springer, New Delhi.

  43. Hernández-Ruíz GM, Álvarez-Orozco NA, Ríos-Osorio LA (2017) Biorremediación de organofosforados por hongos y bacterias en suelos agrícolas: revisión sistemática. Cienc Tecnol Agropecuaria 18(1):139–159

    Article  Google Scholar 

  44. Jabeen H, Iqbal S, Anwar S (2015) Biodegradation of chlorpyrifos and 3, 5, 6-trichloro-2-pyridinol by a novel rhizobial strain Mesorhizobium sp. HN3. Water Environ J.

    Article  Google Scholar 

  45. Jayasri Y, Naidu MD, Mallikarjuna M (2014) Review article microbial degradation of chlorpyrifos. Int J Recent Sci Res 5:1444–1450

    Google Scholar 

  46. John EM, Shaike JM (2015) Chlorpyrifos: pollution and remediation. Environ Chem Lett 13:269–291.

    CAS  Article  Google Scholar 

  47. Kahraman B, Altın A (2020) Evaluation of different approaches for respiratory quotient calculation and effects of nitrogen sources on respiratory quotient values of hydrocarbon bioremediation. Water Air Soil Pollut 231(38):1.

    CAS  Article  Google Scholar 

  48. Klimek J, Ollis D (1980) Extracellular microbial polysaccharides: kinetics of Pseudomonas sp., Azotobacter vinelandii, and Aureobasidium pullulans batch fermentations. Biotechnol Bioeng 22:2321–2342.

    CAS  Article  Google Scholar 

  49. Kumar V, Singh S, Upadhyay N (2019) Effects of organophosphate pesticides on siderophore producing soils microorganisms. Biocatal Agric Biotechnol 21:101359.

    Article  Google Scholar 

  50. Kumar A, Singh VK, Tripathi V, Singh PP, Singh AK (2018) Chapter 16 - Plant growth-promoting rhizobacteria (PGPR): perspective in agriculture under biotic and abiotic stress. In: Prasad R, Gill SS, Tuteja N (Eds) Crop improvement through microbial biotechnology, Elsevier, 333–342 pp.

  51. Lamy E, Tran TC, Mottelet S, Pauss A, Schoefs O (2013) Relationships of respiratory quotient to microbial biomass and hydrocarbon contaminant degradation during soil bioremediation. Int Biodeter Biodegrad 83:85–91.

    CAS  Article  Google Scholar 

  52. Lenart A (2012) In vitro effects of various xenobiotics on Azotobacter chroococcum strains isolated from soils of southern Poland. J Environ Sci Health B 47:7–12.

    CAS  Article  PubMed  Google Scholar 

  53. Li X, He J, Li S (2007) Isolation of chlorpyrifos degrading bacterium, Sphingomonas sp. strain Dsp-2, and cloning of the mpd gene. Res Microbiol 158:143–149.

    CAS  Article  PubMed  Google Scholar 

  54. Liu ZY, Chen X, Shi Y, Su ZC (2011) Bacterial degradation of chlorpyrifos by Bacillus cereus. Adv Mater Res 356–360:676–680.

    CAS  Article  Google Scholar 

  55. Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Lozano E, Galindo E, Peña CF (2011) Oxygen transfer rate during the production of alginate by Azotobacter vinelandii under oxygen-limited and non-oxygen-limited conditions. Microb Cell Fact.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Mac-Rae IC, Celo JS (1974) The effects of organo-phosphorus pesticides on the respiration of Azotobacter vinelandii. Soil Biol Biochem 6(2):109–111.

    CAS  Article  Google Scholar 

  58. Maya K, Singh RS, Upadhyay SN, Dubey SK (2011) Kinetic analysis reveals bacterial efficacy for biodegradation of chlorpyrifos and its hydrolyzing metabolite TCP. Process Biochem 46(11):2130–2136.

    CAS  Article  Google Scholar 

  59. Menon P, Gopal M, Prasad R (2004) Influence of two insecticides, chlorpyrifos and quinalphos, on arginine ammonification and mineralizable nitrogen in two tropical soil types. J Agric Food Chem 52:7370–7376.

    CAS  Article  PubMed  Google Scholar 

  60. Miller G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428.

    CAS  Article  Google Scholar 

  61. Moneke A, Okpala G, Anyanwu C (2010) Biodegradation of glyphosate herbicide in vitro using bacterial isolates from four rice fields. Afr J Biotechnol 9:4067–4074

    CAS  Google Scholar 

  62. Moral ÇK, Ertesvåg H, Sanin FD (2016) Guluronic acid content as a factor affecting turbidity removal potential of alginate. Environ Sci Pollut Res 23:22568–22576.

    CAS  Article  Google Scholar 

  63. Mousa N, Adham A, Merzah N, Jasim S (2021) Azotobacter spp. bioremediation chemosate. Asian J Water Environ Pollut 18:103–107.

    Article  Google Scholar 

  64. Muttawar AS, Wadhai VS (2014) Isolation of pesticide tolerant Azotobacter species from rhizospheric region of the crop. Int J Res Biosci Agric Techn.

    Article  Google Scholar 

  65. Nagaraja H, Chennappa G, Rakesh S, Naik M, Amaresh Y, Sreenivasa M (2016) Antifungal activity of Azotobacter nigricans against trichothecene-producing Fusarium species associated with cereals. Food Sci Biotechnol 25:1197–1204.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  66. Noar J, Bruno-Bárcena J (2018) Azotobacter vinelandii: the source of 100 years of discoveries and many more to come. Microbiology 164:421–436.

    CAS  Article  PubMed  Google Scholar 

  67. Noguez R, Segura D, Moreno S, Hernandez A, Juarez K, Espín G (2008) Enzyme INtr, NPr, and IIANtr are involved in regulation of the poly-β-hydroxybutyrate biosynthetic genes in Azotobacter vinelandii. J Mol Microbiol Biotechnol 15:244–254.

    CAS  Article  PubMed  Google Scholar 

  68. Omar SA, Abd-Alla MH (1992) Effect of pesticides on growth, respiration, and nitrogenase activity of Azotobacter and Azospirillum. World J Microbiol Biotechnol 8:326–328.

    CAS  Article  PubMed  Google Scholar 

  69. Pant R, Pandey P, Kotoky R (2016) Rhizosphere mediated biodegradation of 1, 4-dichlorobenzene by plant growth-promoting rhizobacteria of Jatropha curcas. Ecol Eng 94:50–56.

    Article  Google Scholar 

  70. Peña C, Campos N, Galindo E (1997) Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks. Appl Microbiol Biotechnol 48:510–515.

    Article  Google Scholar 

  71. Peña C, Peter CP, Büchs J, Galindo E (2007) Evolution of the specific power consumption and oxygen transfer rate in alginate-producing cultures of Azotobacter vinelandii conducted in shake flasks. Biochem Eng J 36(2):73–80.

    CAS  Article  Google Scholar 

  72. Peña C, Galindo E, Büchs J (2011) The viscosifying power, degree of acetylation and molecular mass of the alginate produced by Azotobacter vinelandii in shake flasks are determined by the oxygen transfer rate. Process Biochem 46:290–297.

    CAS  Article  Google Scholar 

  73. Plunkett M, Knutson C, Barney B (2020) Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation. Microb Cell Fact.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Praveen Kumar G, Mir Hassan Ahmed S, Desai S, Leo Daniel Amalraj E, Rasul A (2014) In vitro screening for abiotic stress tolerance in potent biocontrol and plant growth-promoting strains of Pseudomonas and Bacillus spp. Int J Bacteriol 2014:1–6.

    Article  Google Scholar 

  75. Rani R, Kumar V (2017) Endosulfan degradation by selected strains of plant growth-promoting rhizobacteria. Bull Environ Contam Toxicol 99:138–145.

    CAS  Article  PubMed  Google Scholar 

  76. Rani R, Kumar V, Gupta P, Chandra A (2019) Effect of endosulfan tolerant bacterial isolates (Delftia lacustris IITISM30 and Klebsiella aerogenes IITISM42) with Helianthus annuus on remediation of endosulfan from contaminated soil. Ecotoxicol Environ Saf 168:315–323.

    CAS  Article  PubMed  Google Scholar 

  77. Rayu S, Nielsen UN, Nazaries L, Singh BK (2017) Isolation and molecular characterization of novel chlorpyrifos and 3,5,6-trichloro-2-pyridinol-degrading bacteria from sugarcane farm soils. Front Microbiol 8:1–16.

    Article  Google Scholar 

  78. Revillas J, Rodelas B, Pozo C, Martínez-Toledo M, González-López J (2000) Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions. J Appl Microbiol 89:486–493.

    CAS  Article  PubMed  Google Scholar 

  79. Rokade KB, Mali GV (2013) Biodegradation of chlorpyrifos by Pseudomonas desmolyticum NCIM 2112. Int J Pharma Bio Sci 4(2):609–616

    CAS  Google Scholar 

  80. Roy T, Das N, Majumdar S (2020) Pesticide tolerant rhizobacteria: paradigm of disease management and plant growth promotion. In: Varma A, Tripathi S, Prasad R (eds) Plant-microbe symbiosis. Cham: Springer, 221–239.

  81. Santos MS, Rondina ABL, Nogueira MA, Hungria M (2020) Compatibility of Azospirillum brasilense with pesticides used for treatment of maize seeds. Int J Microbiol.

    Article  PubMed  PubMed Central  Google Scholar 

  82. Sethi S, Gupta S (2013) Impact of pesticides and biopesticides on soil microbial biomass carbon. Univers J Environ 3(2):326–330

    Google Scholar 

  83. Shahgholi H, Ahangar A (2014) Factors controlling degradation of pesticides in the soil environment: a review. Agric Sci Dev 3(8):273–8

    Google Scholar 

  84. Shahid M, Zaidi A, Ehtram A, Khan M (2019) In vitro investigation to explore the toxicity of different groups of pesticides for an agronomically important rhizosphere isolate Azotobacter vinelandii. Pestic Biochem Phys 157:33–44.

    CAS  Article  Google Scholar 

  85. Shi T, Fang L, Qin H, Chen Y, Wu X, Hua R (2019) Rapid biodegradation of the organophosphorus insecticide chlorpyrifos by Cupriavidus nantongensis x1T. Int J Environ Res Publ Health 16(23):4593.

    CAS  Article  Google Scholar 

  86. Singh DP, Khattar JIS, Nadda J, Singh Y, Garg A, Kaur N, Gulati A (2011) Chlorpyrifos degradation by the cyanobacterium Synechocystis sp. strain PUPCCC 64. Environ Sci Pollut Res 18:1351–1359.

    CAS  Article  Google Scholar 

  87. Strobel S, Allen K, Roberts C, Jimenez D, Scher H, Jeoh T (2018) Industrially-scalable microencapsulation of plant beneficial bacteria in dry cross-linked alginate matrix. Ind Biotechnol 14:138–147.

    CAS  Article  Google Scholar 

  88. Sumbul A, Ansari R, Rizvi R, Mahmood I (2020) Azotobacter: a potential bio-fertilizer for soil and plant health management. Saudi J Biol Sci 27:3634–3640.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  89. Tejera N, Lluch C, Martìnez-Toledo M, Gonzàlez-López J (2005) Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere. Plant Soil 270:223–232.

    CAS  Article  Google Scholar 

  90. Then C, Wai O, Elsayed E, Mustapha W, Othman N, Aziz R, Wadaan M, Enshsay H (2016) Comparison between classical and statistical medium optimization approaches for high cell mass production of Azotobacter vinelandii. J Sci Ind Res 75:231–238

    CAS  Google Scholar 

  91. Vidya Lakshmi C, Kumar M, Khanna S (2009) Biodegradation of chlorpyrifos in soil enriched cultures. Curr Microbiol 58:35–38.

    CAS  Article  PubMed  Google Scholar 

  92. Vijayalakshmi P, Usha MS (2012) Degradation of chlorpyrifos by free cells and calcium-alginate immobilized cells of Pseudomonas putida. Adv Appl Sci Res 3:2796–2800

    CAS  Google Scholar 

  93. Walvekar V, Bajaj S, Singh D, Sharma S (2017) Ecotoxicological assessment of pesticides and their combination on rhizospheric microbial community structure and function of Vigna radiata. Environ Sci Pollut R 24:17175–17186.

    CAS  Article  Google Scholar 

  94. Wu X, Xu J, Dong F, Liu X, Zheng Y (2014) Responses of soil microbial community to different concentration of fomesafen. J Hazard Mater 273:155–164.

    CAS  Article  PubMed  Google Scholar 

  95. Yang L, Zhao YH, Zhang BX, Yang CH, Zhang X (2005) Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2- pyridinol degrading bacterium. FEMS Microbiol Lett 251(1):67–73.

    CAS  Article  PubMed  Google Scholar 

  96. Zhu J, Zhao Y, Ruan H (2019) Comparative study on the biodegradation of chlorpyrifos-methyl by Bacillus megaterium CM-Z19 and Pseudomonas syringae CM-Z6. An Acad Bras Cienc 91(3).

Download references


We thank the Instituto de Biotecnología-UNAM CONACYT (277600) and DGAPA-UNAM (AG2002019); and the Laboratorio Nacional para la investigación en Inocuidad Alimentaria CIAD for the facilities provided for the development of the experimental work. We thank Dr. Celia Flores for technical assistance.


Not applicable for that section.

Author information




Conceptualization: VCA. Methodology: CP, BPA, CMV. Investigation: VCA, LDOM, HSL. Writing original draft: VCA. Writing review and editing: OL, CP. Supervision: LDOM. Project administration: VCA, LDOM. Funding acquisition: CP, JBLM, PBB. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Luis Daniel Ortega Martínez.

Ethics declarations

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

Tandem MS conditions.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Conde-Avila, V., Peña, C., Pérez-Armendáriz, B. et al. Growth, respiratory activity and chlorpyrifos biodegradation in cultures of Azotobacter vinelandii ATCC 12837. AMB Expr 11, 177 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Oxygen consumption rate
  • Pesticide degradation
  • Respiratory quotient
  • Rhizobacteria