Transport of micronutrients (iron, cobalt, nickel, etc.) within biofilms matrixes such as methanogenic granules is of high importance, because these are either essential or toxic for the microorganisms living inside the biofilm. The present study demonstrates quantitative measurements of metal transport inside these biofilms using T1 weighted 3D RARE. It is shown that iron(II)-EDTA diffusion within the granule is independent of direction or the inner structure of the granules. Assuming position dependence of the spin-lattice relaxivity, Fick’s law for diffusion in a sphere can be applied to simulate the diffusion within the methanogenic granules under investigation. A relatively low diffusion coefficient of 2.5*10-11 m2·s-1 was obtained for iron diffusion within the methanogenic granul

As, H., van

Bartacek, J.

Gerkema, E.

Lens, P.

Osuma, B.

Philippi, J.G.M.

Vergeldt, F.J.

English

Name not available

Quantitative NME microscopy of iron transport in methanogenic aggregates

Wageningen Yield 

 Quantitative NMR microscopy of iron transport in methanogenic 
aggregates 
Frank Vergeldt,1 Jan Bartacek,2 Edo Gerkema,1 Bego Osuma,3 John Philippi,1 
Piet Lens,2,4 Henk Van As1 
1 Lab. of Biophysics and Wageningen NMR Centre, Wageningen University, The Netherlands 
2 Sub-dept. of Environmental Technology, Wageningen University, The Netherlands 
3 Chemical and Environmental Engineering Dept., University of Basque, Bilbao, Spain 
4 Pollution Prevention and Control core, UNESCO-IHE, Delft, The Netherlands 
 
Corresponding author: Frank Vergeldt, Lab. of Biophysics, Wageningen University, 6708 HA 
Wageningen, The Netherlands, E-Mail: Frank.Vergeldt@wur.nl 
 
(Submitted to Diffusion Fundamentals on 27 July 2008) 
(Revised and re-submitted to Diffusion Fundamentals on 3 August 2009) 
 
Abstract 
Transport of micronutrients (iron, cobalt, nickel, etc.) within biofilms matrixes such as 
methanogenic granules is of high importance, because these are either essential or toxic for 
the microorganisms living inside the biofilm. The present study demonstrates quantitative 
measurements of metal transport inside these biofilms using T1 weighted 3D RARE. It is 
shown that iron(II)-EDTA diffusion within the granule is independent of direction or the inner 
structure of the granules. Assuming position dependence of the spin-lattice relaxivity, Fick’s 
law for diffusion in a sphere can be applied to simulate the diffusion within the methanogenic 
granules under investigation. A relatively low diffusion coefficient of 2.5*10-11 m2·s-1 was 
obtained for iron diffusion within the methanogenic granule. 
Keywords 
Methanogenic biofilms; Metal transport quantification; MRI microscopy 
1. Introduction 
Up-flow Anaerobic Sludge Bed (UASB) reactors are the most widely applied type of 
anaerobic waste water treatment reactors. The actual purification process takes place in 
methanogenic granular sludge that consist of rigid, well settling microbial aggregates that 
develop by the mutual attachment of bacterial cells in the absence of a carrier material. Heavy 
metal transport into these inhomogeneous, spherical biofilms is an important process both 
from the point of view of toxicity and bioavailability. Detailed experimental data on metal 
transport and biosorption within biofilm matrices is lacking due to the absence of adequate, 
The Open-Access Journal for the Basic Principles of Diffusion Theory, Experiment and Application
© 2009, F. Vergeldt
Diffusion Fundamentals 10 (2009) 31.1 - 31.4 1
 non-invasive analytical tools, hindering refined modeling of these complex processes. In this 
study previous work on methanogenic granular sludge by Lens et al. [1] is extended to 
quantitative MRI microscopy of metal transport. 
2. Materials and methods 
Methanogenic granular sludge was obtained from a full-scale UASB reactor operated at 
30°C treating alcohol distillery wastewater at Nedalco (Bergen op Zoom, The Netherlands). 
The total suspended solids and volatile suspended solids concentration of the wet sludge were 
6.93±0.02% and 6.47±0.03%, respectively. 
The experimental set-up consisted of a glass measuring tube (4 mm i.d.), in which one 
single granule was fixed by glass wool. The iron(II)-EDTA solution circulated through this 
tube with a flow rate of 0.96 mL·min-1. The entire set-up was flushed with argon to avoid 
iron(II) oxidation. The ratio between iron(II) and EDTA was 1.3:1 to avoid changes in the 
granular matrix properties due to complexation and wash out of calcium from the biofilm 
matrix structure [2]. 
All MRI measurements were done at 30±2 °C on a 0.7 T NMR imager. A custom build 
solenoid RF probe was used with an i.d. of 5 mm, which was inductively coupled to avoid 
continuous retuning and matching due to the change in loading by the iron solution. 
Each experiment consisted of a series of T1 weighted 3D RARE measurements using short 
repetitions times till signal intensity inside the granule reached a new equilibrium, after 
applying the iron(II)-EDTA solution. Each series was preceded and followed by T2 mapping 
using 3D MSME and T1 mapping using 3D IR-RARE. All images and maps acquired 
consisted of a matrix of 128×40×20 voxels with a spatial resolution of 0.109×0.109×0.218 
mm3. The repetition time TR for the T1 weighted 3D RARE measurement was 200 ms with an 
effective echo time TE of 4.53 ms. The time demands of the T1, T2 and T1 weighted 3D RARE 
measurements were 160, 80 and 11 minutes, respectively. For further experimental details see 
[2]. 
The experimental data are presented as averaged profiles that were obtained by segmenting 
the granule in layers of one voxel thickness followed by averaging the T1 weighted signal 
intensity per layer. 
The actual iron(II) concentration based on the T1 weighted RARE signal intensity was 
calculated using: 
 
1 Bulk
R 1,Fe(II) 0
1[Fe(II)] · ln 1 (1/ )
·
⎡ ⎤⎛ ⎞= − − −⎢ ⎥⎜ ⎟⎢ ⎥⎝ ⎠⎣ ⎦
S T
T R A
 (1) 
with TR the repetition time, R1,Fe(II) the spin-lattice relaxivity for iron(II)-EDTA, and S the 
signal intensity from the T1 weighted 3D RARE measurements, A0 proton density from 3D T2 
mapping and T1 from 3D T1 mapping, both prior to iron(II)-EDTA addition. In this equation 
the effect of T2 on the signal intensity S is neglected, because TE is much shorted than the 
shortest T2 present. This assumption is justified in Fig. 1A where no short T2’s are visible. 
Fractions with very short T2 could be present but are invisible to the experiment and therefore 
do not affect the results. 
© 2009, F. Vergeldt
Diffusion Fundamentals 10 (2009) 31.1 - 31.4 2
 3. Results 
4. Discussion 
This study demonstrates a method for quantitative investigation of iron diffusion within an 
anaerobic granule. A complete methodology was developed including the experimental set-
up, an optimized MRI sequence (3D RARE), image analysis and calculation of the increase of 
the iron concentration during time. The basic concepts were adopted from pioneering work by 
Nestle et al. [3]. 
It is shown that iron(II)-EDTA diffusion within the granule is independent of direction or 
the inner structure of the granules [2]. The final iron concentration after equilibration with the 
bulk solution, as measured by MRI, was not distributed equally over the granule, but ranged 
from 50 up to 115% of the concentration in the bulk solution (1.75 mM iron(II)-EDTA) (see 
Fig. 2B). One major assumption is that the spin-lattice relaxivity of iron(II)-EDTA in solution 
is representative for the relaxivity inside the granule matrix. An alternative approach is to use 
the T1 maps before and after exposure to the iron(II)-EDTA solution to calculate spin-lattice 
relaxivity assuming that everywhere inside the granule the final iron(II)-EDTA concentration 
equals 1.75 mM (see Fig. 2C). This results in a position dependence of the spin-lattice 
relaxivity (see Fig. 2D). 
One indication that this 
assumption is reasonable is 
given by simulating iron 
transport into a sphere using 
Fick’s law for diffusion [4]. 
The results are well 
described by a diffusion 
coefficient of 2.5*10-11 m2·s-1 
(see Fig. 3). 
5. Conclusions 
MRI microscopy is feasible to quantify metal transport in biofilms. 
Fig. 3: Comparison of the measured iron 
concentration (black lines with points) and 
the theoretical values obtained applying the 
Fick’s law for diffusion into a sphere (gray 
wire net). 
 
A B
C D
Fig. 1: Cross section of the methanogenic 
granule prior (+) and after () 15h exposure 
to a 1.75 mM iron(II)-EDTA solution. (A) 
Effect on T2, (B) T1, and (D) proton density 
A0. (C) The signal intensity S of a T1 
weighted 3D RARE measurement with a 
repetition time of 200 ms shows a clear effect 
of the presence of iron(II)-EDTA. 
Fig. 2: Effect of iron(II)-EDTA penetration 
into a single methanogenic granule. 
(A) Averaged T1 weighted signal intensity 
per layer of the granule. (B) [iron(II)-EDTA] 
calculated using Eq. (1). (C) [iron(II)-EDTA] 
calculated under the assumption that the spin-
latice relaxivity R1,Fe(II) is position dependent. 
(D) Average T1 profile prior (--+--) and upon 
(–◊–) the exposure of the granule to the 1.75 
mM iron(II)-EDTA solution and the 
distribution of the spin-latice relaxivity 
R1,Fe(II) (•••+•••). 
A B 
C D 
© 2009, F. Vergeldt
Diffusion Fundamentals 10 (2009) 31.1 - 31.4 3
 References 
[1] P. Lens, R Gasteri, F. Vergeldt, A. van Aelst, A. Pisabarro, H. Van As, Diffusional 
properties of methanogenic granular sludge: H-1 NMR characterization. Appl. Biochem. 
Biotechnol. 69 (2003) 6644-6649. 
[1] J. Bartacek, F. Vergeldt, E. Gerkema, P. Jenicek, P. Lens, H. Van As, Magnetic 
resonance microscopy of iron transport in methanogenic granules, JMR, accepted, 2009. 
[3] N. Nestle, R. Kimmich, NMR microscopy of heavy metal absorption in calcium alginate 
beads. Appl. Biochem. Biotechnol. 56 (1996) 9-17. 
[4] J. Bartacek, F. Vergeldt, E. Gerkema, P. Jenicek, H. Van As, P. Lens, Quantitative 
analysis of iron transport within methanogenic granules using MRI microscopy, in 
preparation, 2008. 
© 2009, F. Vergeldt
Diffusion Fundamentals 10 (2009) 31.1 - 31.4 4


As, Diffusional properties of methanogenic granular sludge: H-1 NMR characterization.

As, Magnetic resonance microscopy of iron transport in methanogenic granules,

NMR microscopy of heavy metal absorption in calcium alginate beads.

Quantitative analysis of iron transport within methanogenic granules using MRI microscopy, in preparation,

van As, H.

NARCIS 

Quantitative NME microscopy of iron transport in methanogenic aggregates

Abstract

Similar works

Full text

Available Versions

Name not available

Wageningen Yield

NARCIS