Canadian Geotechnical Journal

Laboratory studies on ochre formation and removal from

geotextile filters

Journal: Canadian Geotechnical Journal

Manuscript ID cgj-2022-0084.R2

Manuscript Type: Article

Date Submitted by the

13-Jul-2022
Author:

Complete List of Authors: Correia, L.G.C.S.; Federal University of Rio de Janeiro, Dept. of Civil
Engineering; Federal University of Rio de Janeiro, Dept. of Civil
Engineering
Ehrlich, Mauricio; Federal University of Rio de Janeiro, Dept. of Civil
Engineering; Federal University of Rio de Janeiro, Dept. of Civil
Dr

Engineering
Mendonca, M.B.; Federal University of Rio de Janeiro, Dept. of Civil
Engineering
Keim, C.N.; Federal University of Rio de Janeiro
aft

Is the invited manuscript for

consideration in a Special Not applicable (regular submission)
Issue? :

Keyword: Geotextile, clogging, mitigation, ochre

© The Author(s) or their Institution(s)

Page 1 of 28 Canadian Geotechnical Journal

Laboratory studies on ochre formation and removal from geotextile filters

L.G.C.S. Correiaa, M. Ehrlicha,*, M.B. Mendoncab, C.N. Keimc

ABSTRACT

The clogging of drainage systems due to the formation of ochre is considered a major threat to the performance of

filters and drainage systems. Aiming to understand the factors leading to clogging, column tests were conducted

using geotextile filters under three different filter submersion conditions and ferric citrate percolation to stimulate

ochre production. After 76 days, in half of the column tests, the ferric citrate was changed to D-glucose in the

percolation fluid to remove the ochre by the reductive dissolution of iron. The concentrations of dissolved oxygen

(DO), Fe(II), Fe(III), and pH were monitored as a function of time. Ochre-clogged geotextile filters and the effects

of reductive dissolution were observed by scanning electron microscopy coupled to energy-dispersive spectroscopy.

The results indicate that ochre formation decreased substantially in the submerged filters due to the lower availability
Dr

of oxygen for microbial aerobic activities. Percolation with D-glucose led to low DO content, reduced pH in the

percolation fluid, and stimulation of a pre-existing population of iron-reducing bacteria, which reduced the Fe(III) to
aft

soluble Fe(II), reversing clogging in geotextile filters. This fundamental research may indicate a path for a new

procedure for mitigation and removal of ochre of drainage systems.

KEYWORDS: Geotextile, clogging, mitigation, ochre, submersion, dissimilatory iron reduction

aCOPPE, Dept. of Civil Engineering, Federal University of Rio de Janeiro, RJ 21945-970, Brazil, Email:
luizagcorreia@gmail.com
aCOPPE, Dept. of Civil Engineering, Federal University of Rio de Janeiro, RJ 21945-970, Brazil,
Corresponding author, Email: me@coc.ufrj.br
bEscola Politecnica, Dept. of Civil Engineering, Federal University of Rio de Janeiro, RJ 21945-970,
Brazil, Email: mbm@poli.ufrj.br
cInstitute of Microbiology, Federal University of Rio de Janeiro, Brazil, RJ 21941-902,

Email: cnkeim@micro.ufrj.br

*Corresponding author: me@coc.ufrj.br

© The Author(s) or their Institution(s) 1

 Canadian Geotechnical Journal Page 2 of 28

INTRODUCTION

Ochre is a rust-coloured, gelatinous material composed of microorganisms, secreted polymers, and iron

oxyhydroxides (Kuntze 1982). The growth of microorganisms, the secretion of polymeric substances, and the

precipitation of iron minerals lead to the formation of a biofilm that coats the solid surfaces of drainage systems,

reducing the voids available for water seepage. As a result, clogging occurs, compromising the operating efficiency

of the drainage system (Ford 1979; Forrester 1995; Kuntze 1982; Puig et al. 1986; Mendonca et al. 2003; Mendonca

and Ehrlich 2006; Fannin 2010; Koerner and Koerner 2015). Puig et al. (1986) studied two typical cases of drainage

system ochre clogging in road works in France, which revealed the presence of active microorganisms involved in

iron biogeochemistry. Abromeit (2002) described a case of nonwoven geotextile filter clogging, with a significant

reduction of drainage capacity due to ochre formation. Several cases related to earth dams were also reported

(Terzaghi and Leps 1958, Xu et al. 1976), deep horizontal drains (Forrester, 1995) and wells for water supply

(Cullimore and Mansuy 1986, Howsam 1988, Gariboglio and Smith 1993, Forrester 1995). Koerner and Koerner

(2015) cited ochre as the primary threat to the proper performance of filters and drainage systems. In summary,
Dr

biological clogging may occur in both synthetic and granular filters of different drainage systems.

Strategies for the mitigation of ochre clogging have been proposed, such as an elimination of the air-water
aft

interface or producing turbulent flow in the system (Abromeit 2002). To develop feasible measures for the prevention

of ochre clogging in filters, it is important to understand the factors involved in ochre formation. The main route to

the precipitation of iron oxides in nature occurs when dissolved Fe(II) from anoxic environments finds conditions

favourable for oxidation to Fe(III), leading to precipitation. The oxidation state of iron in the environment depends

on several factors, including the dissolved oxygen (DO); pH; chemicals, such as hydrogen sulphide; and

microorganisms. When there is a near-neutral to alkaline pH and saturated DO, dissolved Fe(II) is quickly oxidized

to Fe(III), which readily precipitates as Fe(III) oxyhydroxides. The DO is the main parameter determining the fate of

iron (Kappler and Straub 2005; Melton et al. 2014). Because O2 dissolves poorly in water, its concentration depends

on both sources and sinks (Esteves and Furtado, 2011). Under stagnant conditions, DO is replenished through

diffusion. Diffusion of DO in water obeys Fick's first law (Bear 1972), which describes the movement of molecules

leading to a net transport from regions with a higher concentration to those with a lower concentration. The main DO

sources for aquatic environments and wet soils are the atmosphere and photosynthesis, whereas the main sink is

aerobic biodegradation of organic matter by microorganisms. The surface layer of water is usually saturated with O2

due to transference from the air. Thus, ochre formation is more intense at the air-water interface due to the greater

© The Author(s) or their Institution(s) 2

Page 3 of 28 Canadian Geotechnical Journal

availability of oxygen. Therefore, ochre formation decreases with the depth of the filter in relation to the water surface

and follows the expected reduction of available oxygen below the water surface, as demonstrated by Correia et al.

(2017).

In micro-oxic environments with a near-neutral pH, the chemical oxidation reaction rate of Fe(II) to Fe(III)

is relatively low. Nevertheless, Fe(II) oxidation to Fe(III), and hence iron oxyhydroxide precipitation, can proceed

by the action of microorganisms, which increases the reaction rate (Kapler and Straub 2005; Weber et al. 2006).

These are known as iron-oxidizing bacteria that colonize micro-oxic environments at the interface between the oxic

and anoxic regions in drainage systems. Iron-oxidizing bacteria oxidize Fe(II) to Fe(III), which readily precipitates

as iron oxyhydroxides in a mass of organic polymers, living cells and organic debris, producing the ochre. Mendonca

and Ehrlich (2006) described two different microbial activities leading to the precipitation of iron oxyhydroxides at

a near-neutral pH. One activity involves conserving energy from the oxidation of Fe(II) to Fe(III), where

microorganisms act as catalysts and greatly increase the Fe(II) to Fe(III) oxidation reaction rate to meet their energy

demands. Another activity is the biodegradation of organometallic complexes by heterotrophic bacteria, where
Dr

organic Fe(III) complexes are degraded and release free Fe(III) in the environment, which promptly precipitates as

ferric minerals (Mendonca and Ehrlich 2006).

aft

In contrast, in anoxic environments, the dissimilatory iron-reducing bacteria perform anaerobic

respiration by reducing Fe(III) to Fe(II) coupled to the oxidation of organics under anoxic conditions. These

microorganisms can use both organometallic Fe(III) complexes and Fe(III) minerals converting them to

soluble Fe(II) and/or Fe(II) minerals (Lovley and Phillips 1988; Weber et al. 2006; Madigan et al. 2016).

In addition, several bacteria can ferment D-glucose and use Fe(III) as an electron sink, reducing to Fe(II)

(Lentini et al. 2012). The present study aims to experimentally verify the efficacy of the promotion of iron-

reducing conditions to unclog geotextile filters coated with ochre. Because Fe(II) is more soluble than

Fe(III), a biological alternative for the removal of ochre from clogged geotextiles can be designed by

stimulating an increase in the population of iron-reducing microorganisms in the drainage system. The

experimental setting consisted of percolating a geotextile filter in a column with ferric citrate and an ochre

crude enrichment culture to cause clogging. This was followed by a replacement of the percolation fluid

with a solution of D-glucose (dextrose), which is a carbohydrate used as a source of energy and carbon by

several heterotrophic microorganisms (Madigan et. al. 2016), to decrease the DO and increase the reductive

© The Author(s) or their Institution(s) 3

 Canadian Geotechnical Journal Page 4 of 28

dissolution of Fe(III) minerals. The D-glucose would promote microbial growth, leading to anoxic

conditions and an emergence of Fe(III)-reducing microorganisms in the ochre that clogs the geotextile. This

approach could reduce Fe(III) to Fe(II) and potentially reverse the process of filter clogging by the

dissolution of Fe(III) minerals. Another advancement of the present work over previous studies (e.g.,

Kuntze 1982; Puig et al. 1986; Mendonca et al. 2003; Mendonca and Ehrlich 2006; Correia et al. 2017) is

the evaluation of the effects of submersion on the dynamics of ochre formation on the filter by monitoring

changes in the pH, DO, and Fe(II) and Fe(III) concentrations.

MATERIALS AND METHODS

In the performed tests, a woven polypropylene geotextile filter 0.4 mm in thickness (ASTM D5199)

with permeability of 0.36 mm/s, permittivity of 0.9 sec-1(ASTM D4491), and filtration opening or apparent

opening size (AOS - ASTM D4751) of 0.8 mm was used.

Samples of ochre were collected from a natural environment to obtain an inoculum of iron-
Dr

precipitating microorganisms for a crude culture, which was used in the tests. The samples were collected

with plastic flasks from the ground surface in a forest area in Rio de Janeiro (Tijuca Forest – coordinates:
aft

22o57’56’’ S, 43o13’33’’ W), where the humidity, temperature, freshwater conditions, and presence of

soluble iron are favourable for the presence of iron-precipitating bacteria (Fig. 1a). A sub-sample was

prepared for light microscopy observation. To promote the enrichment of bacteria that is able to produce

ochre from ferric citrate, an aliquot of 50 mL of the collected material was placed in a 1000 mL Erlenmeyer

flask containing an autoclaved culture medium designed for heterotrophic iron-precipitating bacteria. The

solution contained (NH4)2SO4 0.5 g/L, NaNO3 0.5 g/L, K2HPO4 0.5 g/L, MgSO4·7H2O 0.5 g/L, CaCl2 0.13

g/L, NaCl 9.5 g/L, and 10.0 g/L ferric ammonium citrate [(NH4)xFey(C6H4O7)2]10 in distilled water,

according to APHA (1992). This culture was incubated for three days at 25C and used to inoculate the

column tests described below. An aliquot of 1mL of this culture was inserted into a 10 mL test tube

containing the culture medium (Fig. 1b). The figure shows the test tube after a three-day period containing

a crude culture of iron bacteria (left) and another one with a sterile culture medium (right).

To investigate the ochre production, geotextile filter clogging, and then, the possibility of using an

organic substrate for unclogging, column tests with an upward flow were conducted. Basically, the first
© The Author(s) or their Institution(s) 4
Page 5 of 28 Canadian Geotechnical Journal

part of the tests consisted of the percolation of fluid in the columns and through the geotextile filters to

stimulate the formation of ochre. In each column, the culture medium described above was diluted 1:50 in

tap water and used as the percolating fluid, and then, 50 mL of crude bacterial culture of iron-precipitating

bacteria enriched in the lab was introduced as the inoculum (t=0). The reservoir of the percolating fluid

(Fig.2) was refilled once a week. All the tests were conducted without adjacent soil to evaluate the ochre

formation in geotextile filter in isolation. Note that in the field the ochre formation occurred on the filter,

not in the soil. Thus, the absence of a soil layer in the test may not have directly affected the process of

ochre formation and clogging.

Six filtration column tests were conducted, as described in Table 1. Each column was 50 mm high

and had a 100 mm internal diameter, and was opened to the air. The upward flow was forced by a peristaltic

pump (Fig. 2) under a flow rate of 0.24 L/h. The flow rate was defined aiming to represent field conditions

usually found in drainage systems. Each column was fitted with geotextile filters and placed at different
Dr

submersion depths (L) (Fig. 2). For one pair of columns, the filter was placed at the air-water interface (L=

0 mm), where maximum oxygen saturation occurred. In the other two pairs of columns, the geotextile filters
aft

were submerged at depths (L) of 20 mm and 45 mm. These submersion depths were the same as those

adopted by Correia et al (2017), which were established to produce different DO concentrations. The tests

were performed during a period of 76 days as duplicates. Then, the duplicates became differentiated as D-

glucose was inserted in just one test of each pair to evaluate its effect on ochre removal, whereas the other

test was stopped to analyse the ochre in the geotextiles. During the tests, the concentrations of DO, Fe(II)

and Fe(III), and pH were monitored. The DO and pH were measured daily using electrodes (multiparameter

probe AKSO AK87), whereas the Fe(II) and Fe(Total) concentrations in the input and output fluids were

evaluated once a week using the ortho-phenanthroline method (APHA, 1992). The Fe(III) concentration

was calculated as the difference between the Fe(Total) and Fe(II) concentrations.

The clogging phenomenon was not directly monitored through permittivity measurements during

the tests because very small hydraulic gradients were expected, which would make differences in the

piezometer head readings not accurate. During the tests, the ochre formation dynamics were evaluated

by monitoring the iron concentrations in the fluid, as in Mendonca and Ehrlich (2006).
© The Author(s) or their Institution(s) 5
 Canadian Geotechnical Journal Page 6 of 28

Table 1 shows the program for the performed tests. One column from each depth (L = 0, 20, and 45 mm)

was selected for observations and measurements after 76 days of percolation with ammonium ferric citrate, and the

remaining columns were used in tests with D-glucose (dextrose, C6H12O6, molecular weight 180.16 g/mol). For those

columns, after the period of percolation by diluted ferric citrate medium, the solution containing ferric citrate was

replaced by a culture medium containing D-glucose diluted 1:50 in tap water, which is the same dilution factor used

for the ferric citrate medium. This procedure enabled a comparison of the ochre structures, the elemental

compositions, and the Fe oxidation states before and after D-glucose percolation. The period of percolation with D-

glucose solution was 21 days. The D-glucose was introduced to increase anoxic pockets in the ochre biofilm and

stimulate an increase in the population of iron-reducing microorganisms in the system. These microorganisms reduce

Fe(III) to Fe(II), which is more soluble in water and may reverse the ochre formation process.

To observe the presence of microorganism colonization and evaluate ochre formation on the geotextile filters

at the end of the tests, both light microscopy and scanning electron microscopy (SEM) were used. For the light

microscopy, samples were stained with 2 mg/L DAPI (4′,6-diamidino-2-phenylindole) for 10 minutes before

observation with an Olympus IX71 inverted light microscope equipped with a DP71 CCD camera. DAPI was used
Dr

specifically to highlight microbial cells since it binds to DNA.

aft

For the SEM, the geotextiles were recovered from the columns at the end of the experiments, cut into 10x10

mm square pieces (three samples were produced for each geotextile filter), and fixed in 2.5% glutaraldehyde in 0.1

M PIPES buffer for 2 hours. Then, they were rinsed in the buffer, dehydrated in an ethanol series, and dried in a

critical point dryer (Balzers CPD 020, Liechtenstein). The dried samples were attached to aluminium stubs with

carbon tape and gold coated in a Bal-Tec SCD 050 sputter coater (Balzers, Liechtenstein). They were observed with

SEM (FEI Quanta 250, Eindhoven, the Netherlands) on an instrument equipped with a Noran energy-dispersive

spectroscopy (EDS) system that enabled elemental analysis and mapping.

In addition, samples of the geotextile filters were cut to measure the concentrations of Fe(Total) and Fe(II)

in the material adhered to their surfaces. Three samples of each geotextile filter were cut and maintained in 10 mol/L

hydrochloric acid to dissolve iron minerals from the ochre. After 24 h, the acid samples were diluted ten times in

distilled water and used to quantify the iron concentrations. The iron concentrations were evaluated with a

spectrophotometer (wavelength 562 nm) using the ferrozine method, which is commonly used to determine Fe(II)

and Fe(Total) concentrations in freshwater and marine samples at submicromolar levels (Viollier et al. 2000). Using

© The Author(s) or their Institution(s) 6

Page 7 of 28 Canadian Geotechnical Journal

this procedure, the Fe(II) ion and Fe(Total) concentrations were measured. The Fe(III) concentrations were calculated

as the difference between Fe(Total) and Fe(II).

RESULTS AND DISCUSSION

The sample of ochre collected in the field showed characteristics similar to those described in the

literature, such as its rust colour and gelatinous consistency (Fig.1a). As described before, the collected

material was placed in a culture medium designed for heterotrophic iron-precipitating bacteria. An aliquot

of 1 mL of this enriched material was inserted into a test tube containing the culture medium (Fig. 1b). The

culture medium, which presented a yellow colour initially (test tube on the right), became rust coloured

about three days after inoculation with iron-oxidizing bacteria (test tube on the left). In addition, it formed

a brown ring on the walls of that test tube at the air-water interface, indicating microbial growth and

precipitation of iron oxyhydroxides (Fig. 1b).

Figure 3 shows photomicrographs of the sample of ochre used as inoculum in bright field and
Dr

fluorescence. The rust colour indicates the presence of iron oxides (Fig. 3a, d). The blue fluorescence from

the DAPI shows DNA within the microorganisms (blue rods and cocci) (Fig. 3b), that was distributed within
aft

a mass of exopolymers and iron oxides (Fig. 3c). Some structures resemble the sheaths of microorganisms

of the genus Leptothrix, which are common in iron-rich environments in waterlogged soils and freshwater.

The red fluorescence also indicated the presence of chloroplasts within the microalgae (Fig. 3d-f). The

observations illustrate the diversity of the microorganisms in the ochre that may be responsible for clogging

drainage systems.

Figure 4 shows the variations in the DO and pH as a function of time in the columns during

percolation with ferric citrate followed by D-glucose. The DO and pH of the influent solution were

measured regularly, and the values recorded were about 8 and 7, respectively. The DO values for L = 0

may be super estimated, since the values measured surpassed the solubility of O2 in freshwater at 20oC,

which is about 8 mg/L. For the DO values, no variations were observed in the tests in which the filter was

positioned at the surface. On the other hand, a decrease in the DO over time was verified in the submerged

filters (L = 20 and 45). Low values for the DO (below 1) were observed for most of the time for the ferric

citrate percolation and all the time for the D-glucose percolation. Such low values for the DO indicate an
© The Author(s) or their Institution(s) 7
 Canadian Geotechnical Journal Page 8 of 28

effect from the metabolism of the aerobic microorganisms, including heterotrophs coupling of ferric citrate

degradation to DO consumption, and possibly also aerobic ammonia oxidizers. Ferric citrate is an

organometallic complex whose degradation would release free Fe(III) in the environment that could

promptly precipitate as ferric oxyhydroxides at near-neutral pH values (Mendonca and Ehrlich, 2006). The

process was followed by a period where the values reached an equilibrium at very low DO values, which

suggests that micro-oxic conditions prevailed and anoxic conditions were intermittent. Under anoxic

conditions, microorganisms and chemicals, such as H2S, can convert Fe(III) to Fe(II). H2S in such an

Fe(III)-rich environment could result from the biodegradation of dead cells or secreted materials. Thus, the

environment within the columns could generate Fe(II) from Fe(III) at the end of Fe(III) citrate percolation

and also during D-glucose percolation. Regardless of the submersion depth of the filter, the pH curve

showed values of about 7 during the ferric citrate percolation time period and decreased abruptly from ~7.0

to ~6.5 upon percolation with D-glucose. Part of the percolated D-glucose was possibly fermented, which

produced organic acids that decreased the pH of the medium.

Dr

Figure 5 presents the curves of accumulated mass of Fe(II) and Fe(III) input (Fe(II)AC IN and Fe(III)AC IN)
aft

and output (Fe(II))AC OUTt and Fe(III)AC OUT) versus the percolation time for each test. The accumulated masses of

Fe(II) and Fe(III) were determined considering the iron concentrations in the input and the output and the

accumulated percolation volume calculated for each specific time. The retained mass of Fe (FeR) represents the

accumulated mass of Fe(II) or Fe(III) that remained inside the column considering the mass of iron that entered and

exited it. Notice that Fe(II) and Fe(III) are interchangeable due to oxidation and reduction, and most of the Fe may

be retained inside the filter as Fe(III). The duplicate tests for each submersion depth (L = 0, 20, and 45 mm) did not

exactly reproduce the results for the retained iron mass. This is related to the dynamics of ochre formation, which is

not very reproducible. Nevertheless, a similar trend for the evolution of retained iron as a function of time could be

observed for each duplicate test. In all cases, there was an increase in Fe(II) and Fe(III) retention over time, which

was related to the microbial activities and mineral precipitation, as observed before (Mendonca and Ehrlich, 2006).

Figure 6 shows the results of the retained masses of Fe(II) and Fe(III) as a function of time, as defined by

Eqs. 1 and 2. In Figures 6b, 6d, and 6f, the normalized FeR/FeRmax values were included, where FeRmax is the maximum

measured FeR value in the respective test:

Fe(II)R = Fe(II)AC IN ― Fe(II)AC OUT (1)

© The Author(s) or their Institution(s) 8

Page 9 of 28 Canadian Geotechnical Journal

Fe(III)R = Fe(III)AC IN ― Fe(III)AC OUT (2)

The difference between the Fe(II)R and Fe(III)R values observed in the figures (Figs. 6a, 6c and 6e) is due to

the much higher Fe(III) input concentration in the percolation fluid than that for Fe(II), which would be expected

since we used ferric citrate as the iron source. On the other hand, Fe(II) could be generated from Fe(III) under anoxic

conditions by microbial activities or chemical reduction by H2S generated by microbes. In Figure 6, the results

indicate that after the initial period of time (about 400 hours), Fe(II) and Fe(III) were effectively retained in the

system, indicating growth of the ochre biofilm. Note that the greater Fe(III) retention and ochre formation occurred

in the filters at the air-water interface (L = 0), whereas a higher Fe(II) retention was observed in those that were

submerged (L = 20 and 45).

Figure 6 shows how the ratio of the retained mass of iron had periods that quickly increased intercalated with

periods that had a very small variation in the retained mass, including a few periods where there was a slight decrease.

Those periods with a small variation may indicate that when the ochre biofilm thickened, some of it was released

from the substrate at different times during the process because of the weakening of its adhesion, which facilitated

its removal by the upward liquid flux. Results reported by Mendonca and Ehrlich (2006) confirmed such behaviour.
Dr

In the process of cell detachment and renewed adhesion, there is an alternation between the sessile (adhered to a solid
aft

surface) and planktonic (floated freely in the aqueous environment) lifestyles of microorganisms (Madigan et al.

2009). Notice that if the percolation of ferric citrate were continued for more than 21 days in Test 1, 3, and 5, the

retained mass of iron (FeR) would increase, following the tendency indicated by the figure and the results presented

by Mendonca and Ehrlich (2006). A marked decrease in Fe(II) and Fe(III) retention was observed after percolation

with D-glucose. Previous work showed that D-glucose enhances the growth of anaerobic microorganisms, including

iron-reducing bacteria, which reduce Fe(III) to Fe(II) (e.g.: Dassonville et al. 2004; Gounou et al. 2010; Parker et al.

2018; Su et al. 2020; Lentini et al. 2012). Bacteria play roles as catalysts in the ochre formation process, as in its

removal. D-glucose favours the presence of iron-reducing bacteria, promoting relatively fast solubilization of retained

iron. Since Fe(II) is the most water-soluble iron form, reduction of Fe(III) to Fe(II) could lead to mineral dissolution

and reverse clogging by ochre. Moreover, the use of D-glucose also decreased the pH (from ~7 to ~6.5), which could

make some Fe(II) generated from Fe(III) soluble, according to the Pourbaix diagram. Notice that this pH decrease

would not be sufficient to remove large amounts of iron from the ochre in the filters, as observed in our experiments

(Pourbaix 1974; Bell et al. 1987; Winklhofer and Petersen 2006).

Table 2 provides the data measured, at the end of the tests, from three geotextile samples randomly selected

for Fe(Total) of the mass of iron retained in the filters per surface area. The results reveal a reasonably uniform

© The Author(s) or their Institution(s) 9

 Canadian Geotechnical Journal Page 10 of 28

distribution of Fe in the filter, indicating, in general, a uniform ochre formation. The average values of the Fe(II),

Fe(III) and Fe(Total) mass of iron retained on the geotextile are presented in Figure 7.

Figure 7a shows a higher iron deposition in the test with geotextile filters at water level L= 0 as a result of

greater oxygen availability. The values measured in the tests with L= 20 mm and L= 45mm were about 20% to 25%

of the amount measured at L=0, respectively. The results may indicate that there is a maximum submersion depth

threshold, beyond which the influence of the submersion depth is insignificant. Indeed, Figure 5 shows that negligible

differences were observed for the DO in the percolating fluid measured in the tests with L= 20 mm and L= 45 mm.

Figure 7b shows a remarkable decrease in the iron retained in the filters after percolation with D-glucose for the three

submersion depths (~ 2 to 3 orders of magnitude). The values of both Fe(II) and Fe(III) represented about 0.5, 0.2

and 0.2% of the values measured in the corresponding tests without percolation of D-glucose for L= 0, L = 20 and L

= 45mm, respectively (Figure 7a). As corresponding tests were performed under similar conditions before the D-

glucose percolation, these results indicate that D-glucose percolation promoted a significant removal of iron from the

ochre. However, results in Figure 6 do not show such large differences in retained iron between the corresponding
Dr

samples percolated or not by D-glucose, as shown in Figure 7. The difference between these results may be due to

the use of distinct methods. Note that at end of the column tests, samples of the geotextile filters were cut for analyses
aft

using the ferrozine method. In the D-glucose percolated filters, it was observed that the ochre biofilm was poorly

adhered to the filters, and some of it may have been lost during handling. That may indicate that the D-glucose

percolation decreased the iron in the ochre biofilm coating of the geotextile filters and also promoted a decrease in

the biofilm adhesion to the substrate. Note that Figure 6 is more reliable for Fe(Total); it shows the evolution of

retention during all the tests. On the other hand, Figure 7 shows direct measurements of Fe(II) and Fe(III) retained in

the filters at the end of the tests.

Table 3 shows the Fe(II)/Fe(Total) and Fe(III)/Fe(Total) ratios for the iron retained in the geotextiles at the

end of the tests with percolation of ferric citrate only and for the tests with ferric citrate followed by D-glucose.

Regarding the geotextile samples with percolation of ferric citrate only, Test 1 (L= 0 mm) showed the lowest ratio

of Fe(II) to Fe(Total) in the ochre, whereas this value more than doubled in the submerged samples related to Tests

3 and 5 (L= 20 and 45 mm, respectively). The same trend was observed for the filters percolated with D-glucose.

The poor aeration in microenvironments within the ochre biofilm in the filters corresponding to the Tests 3 and 5

must have led to Fe(III) reduction to Fe(II) catalysed by anaerobic microorganisms, indicating the influence of

oxygen availability on the minerals deposited in the ochre (Figure 4a). In both cases, there was a maximum

© The Author(s) or their Institution(s) 10

Page 11 of 28 Canadian Geotechnical Journal

submersion depth threshold, beyond which the influence of the submersion depth was insignificant. In all tests with

the percolation of ferric citrate followed by D-glucose (Tests 2, 4, and 6), the Fe(II)/Fe(Total) ratios indicated a higher

proportion of Fe(III) reduced to Fe(II). The consumption of D-glucose by heterotrophic microorganisms may have

increased the degree of anoxia (see Figure 4a), stimulating microorganisms to reduce Fe(III) to Fe(II) in the ochre.

In addition, a lack of Fe(III) from the ferric citrate in the D-glucose percolation fluid would have restricted the iron

source for further precipitation of iron minerals in the biofilm, thus decreasing or stopping the formation of new

ochre. The population of aerobic microorganisms that consumed Fe(III) citrate and released Fe(III) that precipitated

as Fe(III) minerals were mainly responsible for the formation of the ochre. These microorganisms would be largely

substituted for microorganisms that are better adapted to metabolize D-glucose.

At the end of the tests, the geotextile samples from Tests 1 and 2 were stained with DAPI and observed under

a fluorescence microscope (Fig. 8). The results showed numerous microbial cells in both the samples percolated with

ferric citrate and those also percolated with D-glucose. The shape and size of predominant cells were distinct in the

two samples, indicating that the ferric citrate and the D-glucose in the percolating fluids led to the development of
Dr

distinct microbiotas. In both cases, the cells were distributed in a thick biofilm, as they were observed for different

focus depths at the microscope.

aft

SEM and EDS were used to observe pieces of the geotextile filters placed on the water surface (L=0) after

the tests were conducted (Figs. 9 and 10). EDS provided a qualitative analysis of the chemical elements present in

the ochre, which were expressed as elemental maps. Figures 9 and 10 show the ochre-bearing areas of the samples

related to Tests 1 and 2, respectively. In the secondary electron images, it was not possible to distinguish microbial

cells amid the mass of EPS and minerals. Nevertheless, microbial cells could be observed using fluorescence

microscopy (Figure 8). Taken together, Figures 8a and b and Figures 9 and 10 suggest that the microorganisms

observed by fluorescence microscopy were enclosed within masses of organic and inorganic materials observed in

the SEM secondary electron images.

The maps in Fig. 9 show carbon concentrated in the polypropylene in the exposed geotextile, which means

that the ochre had less carbon than the geotextile. The secondary electron images show that the sample taken from

Test 2 (Fig. 10) that was percolated with D-glucose was apparently less rough than the sample taken from Test 1

(Fig. 9). In Figure 10, the fact that the geotextile was not exposed resulted in a lower brightness over the whole carbon

map except for some spots in the ochre biofilm, which may correspond to microbial cells. The spotted appearance of

the carbon map indicates that the ochre biofilm surface was dominated by materials low in carbon. In Figures 9 and

© The Author(s) or their Institution(s) 11

 Canadian Geotechnical Journal Page 12 of 28

10, oxygen and iron were co-localized in the ochre, which indicates the presence of iron oxyhydroxides, as expected.

The maps indicate that the main components of the ochre in both cases were iron and oxygen. Even after the iron

removal process promoted by D-glucose, it can be noted that iron and oxygen were still the main components of the

remaining ochre. Despite the significantly smaller iron concentrations observed in the chemical measurements

(Figure 7), SEM-EDS analyses of samples from Tests 3 to 6, which corresponded to different filter depths and

treatments, showed qualitatively similar results (not shown).

CONCLUSIONS

Column tests were performed to simulate ochre formation on geotextile filters. In addition, a new strategy

for ochre mitigation or removal using D-glucose injection was tested. The dynamics of ochre formation and

mitigation were evaluated at three different submersion depths of the filter, which enabled verification of the effect

of oxygen availability on the phenomena of ochre formation and degradation.

The monitoring of the pH, DO, and Fe(II) and Fe (III) concentrations was crucial in advancing an

understanding of the dynamics of ochre formation and removal. The ochre formation was more intense in the filter
Dr

located at the water surface, where oxygen was readily available. The ochre in the samples submerged at depths of
aft

20 and 45 mm, which were exposed to low levels of DO, had a much lower deposition of iron oxyhydroxides in the

filters. Due to a higher availability of oxygen, the microbial aerobic degradation of ferric citrate was faster in the

geotextile at the water surface (L = 0), leading to the deposition of larger amounts of Fe(III) oxyhydroxides. For the

submerged geotextile filters, a remarkable reduction in the DO was observed during the test period, which was related

to the consumption of oxygen by the microorganisms. A decreased DO led to slower citrate degradation by the

aerobic microorganisms, leading to slower Fe(III) release and iron oxide precipitation in the submerged filters. Since

the results for submersion depths L = 20 and L = 45 with a similar percolation routine were equivalent, there must

have been a maximum submersion depth threshold, beyond which the influence of submersion depth was

insignificant. In the performed tests, this depth was lower than 20 mm. The insertion of D-glucose stimulated a pre-

existing population of iron-reducing bacteria, which reduced Fe(III) to Fe(II) in anoxic pockets within the ochre

biofilm, leading to partial dissolution of iron oxyhydroxides and some loss of the ochre structural integrity. This

fundamental research may indicate a path for a new procedure for mitigation and removal of ochre of drainage

systems. Nevertheless, field tests should be performed to assess the feasibility of this remediation strategy.

© The Author(s) or their Institution(s) 12

Page 13 of 28 Canadian Geotechnical Journal

ACKNOWLEDGMENTS

The authors are grateful for the funding for this study provided by the Brazilian Research Council, CNPq, and the

Brazilian Federal Agency for Support and Evaluation of Graduate Education, CAPES.

COMPETING INTERESTS

The authors declare there are no competing interests

DATA AVAILABILITY

Data generated or analysed during this study are provided in full within the published article.

REFERENCES

Abromeit, H.-U., 2002. Revetment damage as a result of geotextile colmation by flocculated ochreous products
and possible repair methods, in: Proc. 7th Int. Conf. on Geosynthetics, Balkema Publisher, Nice, France,
1085–1088.

APHA, AWWA, WPCF, 1992. Standard Methods for the Examination of Water and Wastewater. 18th Ed., New
York.
Dr

Bear, J., 1972. Dynamics of Fluids in Porous Media. Elsevier, New York.
aft

Bell, P.E., Mills, A.L., Herman, J.S. 1987. Biogeochemical conditions favoring magnetite formation during
anaerobic iron reduction. Appl. Environ. Microbiol. 53, 2610-2616.

Correia, L.G.C.S., Ehrlich, M., Mendonca, M.B. 2017.The effect of submersion in the ochre formation in
geotextile filters. J. Geotext. and Geomembr., 45, 1–7.

Dassonville F., Godon, J.J., Renault, P., Richaume, A., Cambier. P. 2004. Microbial dynamics in an anaerobic
soil slurry amended with glucose, and their dependence on geochemical processes. Soil Biol. Biochem. 36,
1417-1430.

Esteves, F.A., Furtado, A.L.S. 2011. Oxigênio dissolvido. In: Fundamentos de Limnologia, Esteves, F.A. (Ed.),
3rdedition, Editora Interciência, Rio de Janeiro, Brazil. pp. 167-191.

Fannin, R.J. 2010. On the clogging of geotextile filters, in: 9th Int. Conf. on Geosynthetics, Guarujá, Brazil. 401–
412.

Ford, H.W. 1979. Biological clogging of synthetic drain envelopes, in: Proc. 2nd Int. Drainage Workshop.
Washington, D.C.

Forrester, K. 1995. Ochre clogging in subsoil drains, in:D. H. Bell, (Ed.), Landslides. Balkema, Rotterdam, The
Netherlands, 1761–1766.

© The Author(s) or their Institution(s) 13

 Canadian Geotechnical Journal Page 14 of 28

Gariboglio, M. A., and Smith, S. A. 1993. Corrosión e incrustación microbiológica en sistemas de captación y
conducción de agua — Aspectos teóricos y aplicados, Serie Investigaciones Aplicada. Coleccion hidrologia
subterranea, Consejo Federal de Inversiones, Buenos Aires, Argentina (in Spanish).

Gounou, C., Bousserrhine, N., Varrault, G., Mouchel, J-M. 2010. Influence of the Iron-Reducing Bacteria on the
Release of Heavy Metals in Anaerobic River Sediment. Water Air Soil Pollut. 212, 123-139.

Howsan, P. 1988. Considerations for well maintenance and rehabilitation. Developing world water, Grosvenor
Press, Hong Kong, 84—86.

Kappler, A., and Straub, K.L. 2005. Geomicrobiological cycling of iron. Rev. Mineral. Geochem. 59: 85-108.

Koerner, R.M., and Koerner, G.R. 2015. Lessons learned from geotextile filter failures under challenging field
conditions. J. Geotext. Geomembr. 43(1), 272–281.

Kuntze, H.1982. Iron clogging in soils and pipes, analysis and treatment, in: German Assoc. for Water Resources
and Land Improvement, Bulletin 10, Pitman.

Lentini, C. J., Wankel, S. D., and Hansel, C. M. 2012. Enriched iron(III)-reducing bacterial communities are
shaped by carbon substrate and iron oxide mineralogy. Front. Microbiol. 3: 404. doi:
10.3389/fmicb.2012.00404
Dr

Lovley, D.R., Phillips, E.J.P. 1988. Novel mode of microbial energy metabolism: organic carbon oxidation
coupled to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol. 54, 1472-1480.
aft

Madigan, M.T., Martinko, J.M., Dunlap, P.V., and Clark, D.P. 2009. Brock Biology of Microorganisms, 12th ed.,
Pearson Benjamin-Cummings, San Francisco.

Melton, E.D., Swanner, E.D., Behrens, S., Schmidt, C., and Kappler, A. 2014; The interplay of microbially
mediated and abiotic reactions in the biogeochemical Fe cycle. Nature Rev. Microbiol. 12: 797-808.

Mendonca, M.B., and Ehrlich, M. 2006. Column test studies of ochre biofilm formation in geotextile filters. J.
Geotech. Geoenviron. Eng. 132, 1284–1292.

Mendonca, M.B., Ehrlich, M., and Camarota, M.C. 2003. Conditioning factors of iron ochre formation on
geotextile filters. Can. Geotech. J. (ISSN: 0008-3674), 40(6), 1225–1234.

Parker, C.W., Auler, A.S., Barton, M.D., Sasowsky, I.D., Senko, J.M., and Barton, H.A. 2018. Fe(III) reducing
microorganisms from iron ore caves demonstrate fermentative Fe(III) reduction and promote cave
formation. Geomicrobiol. J. 35, 311-322.

Pourbaix, M. 1974. Atlas of electrochemical equilibria in aqueous solutions (2nd ed.). National Association of
Corrosion Engineers. ISBN 9780915567980.

Puig, J., Gouy, J.L., and Labroue, L. 1986. Ferric clogging of drains. in: Proc.3rd Int. Conf. on Geotextiles, v.4,
pp. 1179–1184, Vienna, Austria.

© The Author(s) or their Institution(s) 14

Page 15 of 28 Canadian Geotechnical Journal

Su, C., Zhang, M., Lin, L., Yu, G., Zhong, H., and Chong, Y. 2020. Reduction of iron oxides and microbial
community composition in iron-rich soils with different organic carbon as electron donors. Int. Biodeterior.
Biodegrad. 148, 104881.

Terzaghi, K. and Leps, T. M. 1958. Design and performance of Vermilion Dam, California. J. Soil Mech. Found.
Div., 84(3), 1—30.

Viollier, E., Inglett, P.W., Hunter, K., Roychoudhury, A.N., and Van Cappellen, P. 2000. The ferrozine method
revisited: Fe(II)/Fe(III) determination in natural waters. J. Appl. Geochem. 15, 785–790.

Weber, K. A., Achenbach, L. A., and Coates, J. D. 2006, Microorganisms pumping iron: anaerobic microbial iron
oxidation and reduction. Nature Publishing Group 4, 752–763.

Winklhofer, M., and Petersen, N. 2006, Paleomagnetism and magnetic bacteria. In: Magnetoreception and
Magnetosomes in Bacteria; Schüler, D., Ed.; Springer: Berlin/Heidelberg, Germany, 2006; pp. 255-273.

Xu, J. K., Guo, B. J. and Chen, D. C. 1976. Experience in using residual soils for earth dam construction in
Guandong Province, China. Proc., 12th Congrès Int. des Grands Barrages, 821—838.
Dr
aft

© The Author(s) or their Institution(s) 15

 Canadian Geotechnical Journal Page 16 of 28

Table 1. Laboratory tests program.

Test Filter submersion Ferric citrate D-glucose

number depth, L (mm) (76 days) (21 days)

1 x
0
2 x x
3 x
20
4 x x
5 x
45
6 x x
Dr
aft

© The Author(s) or their Institution(s) 16

Page 17 of 28 Canadian Geotechnical Journal

Table 2. Fe(Total) in filters located at different depths below the surface at the end of the tests, calculated from
direct measurements on the ochre.

Total Fe (mg/mm²)

Test 1 17.5 Test 2 14.1

29.7 13.0
(10-3) (10-5)
25.5 14.0

Test 3 4.7 Test 4 1.3

4.4 1.0
(10-3) (10-5)
5.8 1.0

Test 5 5.9 Test 6 1.3

5.8 2.0
(10-3) (10-5)
5.8 1.0
Dr
aft

© The Author(s) or their Institution(s) 17

 Canadian Geotechnical Journal Page 18 of 28

Table 3. Fe(II)/Fe(Total) and Fe(III)/Fe(Total) ratios in filters located at different depths below the surface at the
end of the tests, calculated from direct measurements on the ochre.

Filter submersion Ferric citrate D-glucose Fe(II)/Fe(Total) Fe(III)/Fe(Total)

Test number
depth, L (mm) (76 days) (21 days) (%) (%)

1 x 4.2 95.8
0
2 x x 24.8 75.2
3 x 13.7 86.3
20
4 x x 56.5 43.5
5 x 11.9 88.0
45
6 x x 44.7 55.3
Dr
aft

© The Author(s) or their Institution(s) 18

Page 19 of 28 Canadian Geotechnical Journal

Dr
aft

Figure 1. (a) Location of the ochre collected for this study in Tijuca Forest, Rio de Janeiro, Brazil; (b)Test tubes

containing a three-day crude culture of iron bacteria (left) or sterile with culture medium (right).

© The Author(s) or their Institution(s) 19

 Canadian Geotechnical Journal Page 20 of 28

Dr

Figure 2. Schematic representation of the column tests. Six columns were used, one pair each at 0, 20 and
aft

45 mm of submersion depth (L).

© The Author(s) or their Institution(s) 20

Page 21 of 28 Canadian Geotechnical Journal

Figure 3. Light micrographs of natural ochre samples. (a, d) Bright field; (b, e) fluorescence microscopy;

(c, f) superposition of the bright field and fluorescence images.

Dr
aft

© The Author(s) or their Institution(s) 21

 Canadian Geotechnical Journal Page 22 of 28

Figure 4. Measurements of (a) dissolved oxygen (DO) and (b) pH along time in each experiment, as distinguished
by depth (L) and the presence or absence of D-glucose.
Dr
aft

© The Author(s) or their Institution(s) 22

Page 23 of 28 Canadian Geotechnical Journal

Dr
aft

Figure 5. Input and output curves of accumulated mass of Fe(II) and Fe(III) vs. percolation time: (a-b) L=0; (c-d)

L=20 and; (e-f) L= 45 mm. (a, c, e) Percolation with ferric citrate only and; (b, d, f) percolation with D-glucose

after the period of ferric citrate percolation.

© The Author(s) or their Institution(s) 23

 Canadian Geotechnical Journal Page 24 of 28

Dr
aft

Figure 6. Retained mass of iron (FeR= FeAC IN – FeAC OUT) vs. percolation time for: (a-b) L=0; (c-d) L=20; and (e-f)

L= 45 mm. (a, c, e) FeR, and (b, d, f) normalized FeR curves.

© The Author(s) or their Institution(s) 24

Page 25 of 28 Canadian Geotechnical Journal

Figure 7. Iron mass in geotextile filters at the end of the tests with percolation of (a) ferric citrate only (Tests 1, 3

and 5), and (b) ferric citrate followed by D-glucose (Tests 2, 4 and 6).
Dr
aft

© The Author(s) or their Institution(s) 25

 Canadian Geotechnical Journal Page 26 of 28

Figure 8. Fluorescence photomicrographs of geotextile samples for L = 0 taken at the end of the tests, stained with

DAPI for observation of DNA. (a) Test 1, percolated with ferric citrate; (b) Test 2, percolated with ferric citrate

followed by D-glucose.
Dr
aft

© The Author(s) or their Institution(s) 26

Page 27 of 28 Canadian Geotechnical Journal

Dr
aft

Figure 9. SEM and EDS elemental maps of a representative geotextile sample percolated with ferric citrate for 76

days (Test 1, L=0).

© The Author(s) or their Institution(s) 27

 Canadian Geotechnical Journal Page 28 of 28

Dr
aft

Figure 10. SEM and EDS elemental maps of geotextile samples percolated with ferric citrate followed by D-

glucose (Test 2, L=0).

© The Author(s) or their Institution(s) 28