**4. Determination of bacterial magnetic moment**

As the magnetosome chain determines a magnetic moment to MTB, let us talk about the different techniques used to estimate that magnetic moment. The first theoretical estimate for the magnetic moment was done counting the contribution of several nanoparticles arranged in a chain [53]. For a magnetosome chain composed of 22 particles of magnetite with every nanoparticle having 1.25 <sup>10</sup><sup>16</sup> cm3 of volume, it is possible to calculate the total magnetic moment as M = nVindMV, where n is the number of particles in the chain, Vind is the volume of each particle (assuming that all are equal), and MV is the magnetic moment per unit volume of the magnetic material. For magnetite, MV = 480 <sup>10</sup><sup>3</sup> Am2 /cm<sup>3</sup> . In this way, a magnetic moment of 1.3 <sup>10</sup><sup>15</sup> Am<sup>2</sup> is obtained. This magnetic moment value means a magnetic to thermal energy rate of about 16 (assuming a temperature of 300 K). This method can be used whenever is possible to observe and count the number of magnetosomes in the chain. This method is not applied in the case of live bacteria and for bigger microorganisms with lots of magnetosomes, as is the case for "*Candidatus* Magnetobacterium bavaricum" and "*Candidatus* Magnetoglobus multicellularis."

nanoparticles, arranged in the proper configuration, to efficiently orient the bacteria in the geomagnetic field direction. A problem with this method is that the Uturn trajectory must be in the focal plane for a good measurement of L, but that is not the case in the general. An alternative is to use only the U-turn time τ because it can be determined well for any U-turn trajectory [57]. The U-turn analysis done by Esquivel and Lins de Barros [56] also assumes that bacteria have spherical geometry, that it is not the general case. When the bacterium is enlarged, as a small cylinder, another approximation must be done. So, assuming that this small cylinder behaves as a set of attached spheres, the contribution to the total torque can be calculated. Doing the experimental analysis in that way, Bahaj et al. [58] calculated a

value of 6.1 <sup>10</sup><sup>16</sup> Am<sup>2</sup> for the magnetotactic spirillum *Magnetospirillum magnetotacticum*. They also calculated the variation of magnetic moment with the

growth time, and observed that it grows from 2.8 <sup>10</sup><sup>16</sup> Am2 at 35 h to 6.5 <sup>10</sup><sup>16</sup> Am2 at 240 h [59]. Another technique widely used to determine the magnetic moment of magnetotactic bacteria is the analysis of the movement in a rotating magnetic field [60]. In that method, a set of four coils (two crossed pairs) is adapted to an optical microscope stage to generate a rotating magnetic field with frequency f. That experimental setup is known as bacteriodrome. The resultant trajectory is a circle, observed clearly by dark-field images. Again, ignoring the flagellar movement and in the low Reynolds number regime, the magnetic torque must be equal to the viscous torque. The magnetic torque depends on the angle among the bacterial magnetic moment and the external magnetic field. That angle increases when the frequency f increases, and its upper limit is 90° meaning that there is a critical value of fc. For values of f higher than fc, the trajectory is not more a circle. The determination of fc permits to calculate the magnetic moment as:

/H, where M is the bacterium magnetic moment, H is the magnetic

, and Pan et al. [61] calculated a value of about

field intensity, c is a shape factor, η is the viscosity, and l is the bacterium length. It is difficult to determine the shape factor, and Petersen et al. [60] proposed an approximated value of 8π. Using this technique, Petersen et al. [60] determined the magnetic moment of magnetotactic bacteria, of natural samples from Southern

1.8 <sup>10</sup><sup>15</sup> Am2 for MYC-1, an uncultivated magnetotactic coccus from China. Other techniques have been used for measuring the magnetic moment of magnetotactic bacteria. Using a SQUID magnetometer, an average magnetic moment of 1.8 0.4 <sup>10</sup><sup>12</sup> emu for bacteria from natural sediments had been determined [62]. This method is interesting because it is a direct measurement and does not need to assume unknown values for parameters from the studied cell. There are two interesting physical techniques involving light for measuring the magnetic moment of magnetic bacteria. One is the analysis of the birefringence arising in a pull of magnetotactic bacteria when in presence of an external magnetic field [63]. The birefringence transforms an input linear polarized light beam in an output elliptically polarized light beam, with a phase shift between the fast and slow components. This phase shift is measured and it depends on the intensity of the external magnetic field and on the magnetic moment. Experiments were done with live and dead bacteria, killed with drops of formalin. The measured values, at normal concentration conditions, for live bacteria were about 1.21 <sup>10</sup><sup>13</sup> emu and for dead bacteria about 1.33 <sup>10</sup><sup>13</sup> emu. Apparently, for dead bacteria, the measured values are higher than for live bacteria. It was assumed that this difference could be an effect of motile behavior in live bacteria and the concept of "effective temperature" Teff was introduced, meaning that live bacteria feels a disorienting thermal energy kTeff higher than the ambient thermal energy in 10–20%. The other technique is the analysis of the light scattered by a pull of magnetic bacteria [64],

M ≈ cη2πfc l

**11**

3

Germany, of about 4 <sup>10</sup><sup>15</sup> Am2

*Biology and Physics of Magnetotactic Bacteria DOI: http://dx.doi.org/10.5772/intechopen.79965*

A statistical analysis of the swimming orientation of magnetotactic bacteria, assuming that they behave as paramagnetic particles, produces the orientation to be equivalent to the average of cosθ (<cosθ>), being θ the angle among the bacterial velocity and the magnetic field. Kalmijn showed that <cosθ> is function of the magnetic to thermal energy ratio [54]: <cosθ> = L (MH/kT) = coth(MH/kT)kT/ MH, where M is the bacterium magnetic moment, H is the magnetic field intensity, k is the Boltzmann constant, T is the absolute temperature, and L(x) is the Langevin function: coth(x)—1/x. For MH/kT ≈ 10, the Langevin function is about 0.9, which means that the bacterial trajectory is well oriented to the magnetic field direction. The analysis of the velocity as function of the magnetic field [54] or of the orientation as function of the magnetic field [55] permits the estimative of the bacterial magnetic moment. Kalmijn stressed the fact that this kind of study is valid only for the orientation of a single bacterium and not for the average orientation of several bacteria [54]. Using this method, it has been shown that "*Candidatus* Magnetoglobus multicellularis" shows values of L(x) lower than 0.9 in the presence of the geomagnetic field. A measuring method for the magnetic moment of individual MTB was developed in [56] and consists in the analysis of the U-turn trajectory, which is the form of the trajectory followed by an MTB when the sense of the external magnetic field vector is inverted. The theoretical analysis assumes that the bacteria and the magnetosome chain forms a rigid body, the bacteria following the movement of the magnetic moment. In the low Reynolds number regime and ignoring the flagellar forces, the sum of the magnetic torque and the viscous torque is equal to zero. From that equation, mathematical expressions are obtained for the time τ and diameter L of the reversal trajectory: L = 8πηR<sup>3</sup> v/(MH) and τ = [8πηR3 / (MH)]ln[2MH/(kT)], where M is the bacterium magnetic moment, H is the magnetic field intensity, k is the Boltzmann constant, T is the absolute temperature, R is the bacterium radius (assuming it is a coccus), v is the velocity, η is the viscosity, and ln is the natural logarithm function. The measurement of those parameters for the U-turn trajectory makes possible to calculate the value of the magnetic moment of magnetotactic bacteria. The experimental measurement of the magnetic moment of bacteria with different sizes and shapes, done by Esquivel and Lins de Barros [56], showed that the magnetic moment can have values from 0.3 <sup>10</sup><sup>15</sup> to <sup>54</sup> <sup>10</sup><sup>15</sup> Am<sup>2</sup> , generating magnetic to thermal energy ratios from 3 to 326. Those results challenge the idea that the magnetosome contains the sufficient magnetic

### *Biology and Physics of Magnetotactic Bacteria DOI: http://dx.doi.org/10.5772/intechopen.79965*

**4. Determination of bacterial magnetic moment**

the magnetic material. For magnetite, MV = 480 <sup>10</sup><sup>3</sup> Am2

multicellularis."

*Microorganisms*

<sup>54</sup> <sup>10</sup><sup>15</sup> Am<sup>2</sup>

**10**

As the magnetosome chain determines a magnetic moment to MTB, let us talk about the different techniques used to estimate that magnetic moment. The first theoretical estimate for the magnetic moment was done counting the contribution of several nanoparticles arranged in a chain [53]. For a magnetosome chain composed of 22 particles of magnetite with every nanoparticle having 1.25 <sup>10</sup><sup>16</sup> cm3 of volume, it is possible to calculate the total magnetic moment as M = nVindMV, where n is the number of particles in the chain, Vind is the volume of each particle (assuming that all are equal), and MV is the magnetic moment per unit volume of

magnetic moment of 1.3 <sup>10</sup><sup>15</sup> Am<sup>2</sup> is obtained. This magnetic moment value means a magnetic to thermal energy rate of about 16 (assuming a temperature of 300 K). This method can be used whenever is possible to observe and count the number of magnetosomes in the chain. This method is not applied in the case of live bacteria and for bigger microorganisms with lots of magnetosomes, as is the case for "*Candidatus* Magnetobacterium bavaricum" and "*Candidatus* Magnetoglobus

A statistical analysis of the swimming orientation of magnetotactic bacteria, assuming that they behave as paramagnetic particles, produces the orientation to be equivalent to the average of cosθ (<cosθ>), being θ the angle among the bacterial velocity and the magnetic field. Kalmijn showed that <cosθ> is function of the magnetic to thermal energy ratio [54]: <cosθ> = L (MH/kT) = coth(MH/kT)kT/ MH, where M is the bacterium magnetic moment, H is the magnetic field intensity, k is the Boltzmann constant, T is the absolute temperature, and L(x) is the Langevin function: coth(x)—1/x. For MH/kT ≈ 10, the Langevin function is about 0.9, which means that the bacterial trajectory is well oriented to the magnetic field direction. The analysis of the velocity as function of the magnetic field [54] or of the orientation as function of the magnetic field [55] permits the estimative of the bacterial magnetic moment. Kalmijn stressed the fact that this kind of study is valid only for the orientation of a single bacterium and not for the average orientation of several

bacteria [54]. Using this method, it has been shown that "*Candidatus*

time τ and diameter L of the reversal trajectory: L = 8πηR<sup>3</sup>

Magnetoglobus multicellularis" shows values of L(x) lower than 0.9 in the presence of the geomagnetic field. A measuring method for the magnetic moment of individual MTB was developed in [56] and consists in the analysis of the U-turn trajectory, which is the form of the trajectory followed by an MTB when the sense of the external magnetic field vector is inverted. The theoretical analysis assumes that the bacteria and the magnetosome chain forms a rigid body, the bacteria following the movement of the magnetic moment. In the low Reynolds number regime and ignoring the flagellar forces, the sum of the magnetic torque and the viscous torque is equal to zero. From that equation, mathematical expressions are obtained for the

(MH)]ln[2MH/(kT)], where M is the bacterium magnetic moment, H is the magnetic field intensity, k is the Boltzmann constant, T is the absolute temperature, R is the bacterium radius (assuming it is a coccus), v is the velocity, η is the viscosity, and ln is the natural logarithm function. The measurement of those parameters for the U-turn trajectory makes possible to calculate the value of the magnetic moment of magnetotactic bacteria. The experimental measurement of the magnetic moment of bacteria with different sizes and shapes, done by Esquivel and Lins de Barros [56], showed that the magnetic moment can have values from 0.3 <sup>10</sup><sup>15</sup> to

results challenge the idea that the magnetosome contains the sufficient magnetic

, generating magnetic to thermal energy ratios from 3 to 326. Those

/cm<sup>3</sup>

. In this way, a

v/(MH) and τ = [8πηR3

/

nanoparticles, arranged in the proper configuration, to efficiently orient the bacteria in the geomagnetic field direction. A problem with this method is that the Uturn trajectory must be in the focal plane for a good measurement of L, but that is not the case in the general. An alternative is to use only the U-turn time τ because it can be determined well for any U-turn trajectory [57]. The U-turn analysis done by Esquivel and Lins de Barros [56] also assumes that bacteria have spherical geometry, that it is not the general case. When the bacterium is enlarged, as a small cylinder, another approximation must be done. So, assuming that this small cylinder behaves as a set of attached spheres, the contribution to the total torque can be calculated. Doing the experimental analysis in that way, Bahaj et al. [58] calculated a value of 6.1 <sup>10</sup><sup>16</sup> Am<sup>2</sup> for the magnetotactic spirillum *Magnetospirillum magnetotacticum*. They also calculated the variation of magnetic moment with the growth time, and observed that it grows from 2.8 <sup>10</sup><sup>16</sup> Am2 at 35 h to 6.5 <sup>10</sup><sup>16</sup> Am2 at 240 h [59]. Another technique widely used to determine the magnetic moment of magnetotactic bacteria is the analysis of the movement in a rotating magnetic field [60]. In that method, a set of four coils (two crossed pairs) is adapted to an optical microscope stage to generate a rotating magnetic field with frequency f. That experimental setup is known as bacteriodrome. The resultant trajectory is a circle, observed clearly by dark-field images. Again, ignoring the flagellar movement and in the low Reynolds number regime, the magnetic torque must be equal to the viscous torque. The magnetic torque depends on the angle among the bacterial magnetic moment and the external magnetic field. That angle increases when the frequency f increases, and its upper limit is 90° meaning that there is a critical value of fc. For values of f higher than fc, the trajectory is not more a circle. The determination of fc permits to calculate the magnetic moment as: M ≈ cη2πfc l 3 /H, where M is the bacterium magnetic moment, H is the magnetic field intensity, c is a shape factor, η is the viscosity, and l is the bacterium length. It is difficult to determine the shape factor, and Petersen et al. [60] proposed an approximated value of 8π. Using this technique, Petersen et al. [60] determined the magnetic moment of magnetotactic bacteria, of natural samples from Southern Germany, of about 4 <sup>10</sup><sup>15</sup> Am2 , and Pan et al. [61] calculated a value of about 1.8 <sup>10</sup><sup>15</sup> Am2 for MYC-1, an uncultivated magnetotactic coccus from China.

Other techniques have been used for measuring the magnetic moment of magnetotactic bacteria. Using a SQUID magnetometer, an average magnetic moment of 1.8 0.4 <sup>10</sup><sup>12</sup> emu for bacteria from natural sediments had been determined [62]. This method is interesting because it is a direct measurement and does not need to assume unknown values for parameters from the studied cell. There are two interesting physical techniques involving light for measuring the magnetic moment of magnetic bacteria. One is the analysis of the birefringence arising in a pull of magnetotactic bacteria when in presence of an external magnetic field [63]. The birefringence transforms an input linear polarized light beam in an output elliptically polarized light beam, with a phase shift between the fast and slow components. This phase shift is measured and it depends on the intensity of the external magnetic field and on the magnetic moment. Experiments were done with live and dead bacteria, killed with drops of formalin. The measured values, at normal concentration conditions, for live bacteria were about 1.21 <sup>10</sup><sup>13</sup> emu and for dead bacteria about 1.33 <sup>10</sup><sup>13</sup> emu. Apparently, for dead bacteria, the measured values are higher than for live bacteria. It was assumed that this difference could be an effect of motile behavior in live bacteria and the concept of "effective temperature" Teff was introduced, meaning that live bacteria feels a disorienting thermal energy kTeff higher than the ambient thermal energy in 10–20%. The other technique is the analysis of the light scattered by a pull of magnetic bacteria [64],

based on the fact that the presence of an external magnetic field determines an angular distribution in the orientation of bacteria. This angular distribution affects the structure factor in the scattered light intensity. With this method, the average length and average magnetic moment can be determined. For two different cultures of *Aquaspirillum magnetotacticum* were determined values of (2.2 0.2) <sup>10</sup><sup>13</sup> emu and (4.3 0.5) <sup>10</sup><sup>13</sup> emu, which are in good agreement with the value obtained by electron microscopy, or about 4.4 <sup>10</sup><sup>13</sup> emu. Using a similar experimental approach in [65] was determined the magnetic moment of a wild-type *Magnetospirillum gryphiswaldense* strain and obtained a value of about 25.3 (1.6) <sup>10</sup><sup>13</sup> emu. Other methods found in literature are based basically in the analysis of the bacterial body rotation caused by the magnetic torque and in the analysis of the equation magnetic torque = viscous torque. For example, in Ref. [66], it was measured the magnetic moment of single *Magnetospirillum gryphiswaldense* cells using magnetic tweezers, observing and analyzing the rotation of the bacterial body after a magnetic field reversion. They observed that the measured magnetic moment has a dependence on the magnetic field intensity, as occurs in magnetization measurements of magnetic materials, starting from a remanence magnetization at zero magnetic field and progressively increasing until the magnetization saturates at higher magnetic fields. They measured for 6 mT < H <sup>&</sup>lt; 23 mT a magnetic moment of 2.4 (1.1) <sup>10</sup><sup>13</sup> emu and for 90 mT <sup>&</sup>lt; <sup>H</sup> <sup>&</sup>lt; 130 mT a magnetic moment of 7.7 (3.4) <sup>10</sup><sup>13</sup> emu. **Table 2** resumes the magnetic moment measured with the different techniques, remembering that 1 emu = 10<sup>3</sup> Am<sup>2</sup> . It can be observed that the magnetic moment obtained by the direct measurement from the magnetosome chain is always bigger than that obtained from indirect physical methods. In the study by Zahn et al. [66], this fact is explained identifying the direct measurement in the magnetosome chain as the saturation magnetization, that is only observed for higher magnetic fields.

**5. The movement of magnetotactic bacteria**

*Biology and Physics of Magnetotactic Bacteria DOI: http://dx.doi.org/10.5772/intechopen.79965*

only in cultured MTB.

[19, 68, 71].

**13**

Several experimental observations show that magnetotaxis functions together with aerotaxis, determining the so-called magneto-aerotaxis [8, 67]. Basically, two different behaviors have been identified in magneto-aerotaxis: polar magnetotaxis, that consists in the North-seeking or South-seeking behaviors in the search for the better oxygen concentrations; and axial magnetotaxis, in that case, MTB move in the magnetic field direction but without preferential sense. MTB from natural samples always present polar magnetotaxis. Axial magnetotaxis has been observed

MTB are easily identified because of their response to the inversion of the local magnetic field direction: after the inversion bacteria swim following the new magnetic field direction. It can be stated that magnetic field inversions stimulate MTB to swim, making them a model for the study of microorganism swimming. Bacteria swim in the low Reynolds number regime, where viscous forces and torques act to null the resultant force and torque [68]. In that regime, microorganisms swim following an helical trajectory [69] whose parameterization in Cartesian coordinates (x, y, z), considering the helix axis as the z axis, can be written as (Rcos(ωt), Rsin (ωt), Vt), where R is the helix radius, V is the axial velocity, and ω = 2π*f* being *f* the helix frequency. In the case of magnetotactic microorganisms, the helical trajectory

of the multicellular magnetotactic prokaryote "*Candidatus* Magnetoglobus

multicellularis" has been studied for two different applied magnetic fields (3.9 and 20 Oe) [70] and for magnetic fields from 0.9 to 32 Oe [55]. Those studies show that for spherical multicellular magnetotactic prokaryotes, the axial velocity V is about 90 μm/s, the radius R is about 8 μm for lower magnetic fields, and the helix

frequency *f* is about 1.1 Hz. For uncultured magnetotactic coccus, the helical movement has been studied recently (data not published), in the presence of magnetic fields of about 0.7 Oe, and the helical parameters measured were: axial velocity of about 90 μm/s, radius of about 2.5 μm, and helix frequency of about 1 Hz. For other magnetotactic microorganisms, it has been observed that the 2D trajectory is similar to the projection of a 3D helix in the microscope focal plane (for example, see

For the theoretical study of microorganisms, motion in the low Reynolds number regime is necessary to know all the forces and torques acting on the microorganism. Nogueira and Lins de Barros [68] developed a model in that regime, considering a spherical MTB with a single flagellum and a magnetosome chain aligned to the flagellum line. The equations to be considered are **F**flagella + **F**viscous = 0 and **τ**flagella + **τ**viscous + **τ**magnetic + **τ**body = 0. Using the appropriate expressions for the forces and torques in that model, they were able to calculate numerically the temporal evolution of the center of mass coordinates (x, y, z) and of the Euler's angles for the rigid body (θ, ϕ, ψ), being the trajectory similar to a cylindrical helix. In the other hand, Refs. [72, 73] studied the motion of nonspherical MTB, to include the effect of the bacterial body geometry on the viscous forces. Also, Yang et al. [73] studied MTB with two flagellar bundles. To do that, they calculated numerically the motion using the second Newton's law, considering all the forces and torques and calculating the appropriate inertial terms for the geometrical body form. They also studied the effect of the relative inclination λ between the magnetosome chain and the flagella. Those studies showed that when λ 6¼ 0, the velocity decreases when the magnetic field increases, effect also observed experimentally in the work by Pan et al. [74] when studying the circular movement of the MYC-1 strain. In that case, it was measured the velocity in the circular trajectory obtained in a bacteriodrome as function of the applied magnetic field, in the hope to obtain a growing Langevin curve as predicted by Kalmijn [54]. But they observed that the velocity decreases as


**Table 2.**

*Magnetic moment value for MTB using different physical techniques.*

based on the fact that the presence of an external magnetic field determines an angular distribution in the orientation of bacteria. This angular distribution affects the structure factor in the scattered light intensity. With this method, the average length and average magnetic moment can be determined. For two different cultures of *Aquaspirillum magnetotacticum* were determined values of (2.2 0.2) <sup>10</sup><sup>13</sup> emu and (4.3 0.5) <sup>10</sup><sup>13</sup> emu, which are in good agreement with the value obtained by electron microscopy, or about 4.4 <sup>10</sup><sup>13</sup> emu. Using a similar experimental approach in [65] was determined the magnetic moment of a wild-type *Magnetos-*

(1.6) <sup>10</sup><sup>13</sup> emu. Other methods found in literature are based basically in the analysis of the bacterial body rotation caused by the magnetic torque and in the analysis of the equation magnetic torque = viscous torque. For example, in Ref.

*gryphiswaldense* cells using magnetic tweezers, observing and analyzing the rotation of the bacterial body after a magnetic field reversion. They observed that the measured magnetic moment has a dependence on the magnetic field intensity, as occurs in magnetization measurements of magnetic materials, starting from a remanence magnetization at zero magnetic field and progressively increasing until the magnetization saturates at higher magnetic fields. They measured for 6 mT < H <sup>&</sup>lt; 23 mT a magnetic moment of 2.4 (1.1) <sup>10</sup><sup>13</sup> emu and for 90 mT <sup>&</sup>lt; <sup>H</sup> <sup>&</sup>lt; 130 mT a magnetic moment of 7.7 (3.4) <sup>10</sup><sup>13</sup> emu. **Table 2** resumes the magnetic moment measured with the different techniques, remembering that

. It can be observed that the magnetic moment obtained by the

Magnetotactic spirillum MS1 1.3 <sup>10</sup><sup>15</sup> [53]

Natural samples <sup>4</sup> <sup>10</sup><sup>15</sup> [60]

Uncultivated coccus MYC-1 1.8 <sup>10</sup><sup>15</sup> [61]

**(Am<sup>2</sup> )**

0.3 <sup>10</sup><sup>15</sup> to <sup>54</sup> <sup>10</sup><sup>15</sup>

(dead) 4.3 <sup>10</sup><sup>16</sup>

2.4 1.1 <sup>10</sup><sup>16</sup> (high H) 7.7 3.4 <sup>10</sup><sup>16</sup> **References**

[56]

[64]

[66]

*pirillum gryphiswaldense* strain and obtained a value of about 25.3

1 emu = 10<sup>3</sup> Am<sup>2</sup>

*Microorganisms*

Electron microscopy

Rotating magnetic

Rotating magnetic

field

field

**Table 2.**

**12**

[66], it was measured the magnetic moment of single *Magnetospirillum*

direct measurement from the magnetosome chain is always bigger than that obtained from indirect physical methods. In the study by Zahn et al. [66], this fact is explained identifying the direct measurement in the magnetosome chain as the saturation magnetization, that is only observed for higher magnetic fields.

**Technique Organism Magnetic moment**

marine water

Light scattering *Aquaspirillum magnetotacticum* (live) 2.2 <sup>10</sup><sup>16</sup>

Magnetic tweezers *Magnetospirillum gryphiswaldense* (low H)

*Magnetic moment value for MTB using different physical techniques.*

U-turn modified *Magnetospirillum magnetotacticum* 6.1 <sup>10</sup><sup>16</sup> [58] SQUID Fresh water uncultured bacteria 1.8 <sup>10</sup><sup>15</sup> [62]

Light scattering *Magnetospirillum gryphiswaldense* 25.3 1.6 <sup>10</sup><sup>16</sup> [65] Birefringence *Aquaspirillum magnetotacticum* 1.21 <sup>10</sup><sup>16</sup> [63]

U-turn analysis Several microorganisms from fresh to
