1 23

Waste and Biomass Valorization ISSN 1877-2641 Waste Biomass ValorDOI 10.1007/s12649-013-9234-y

In Vitro Testing and CommercializationPotential of Extracted Fulvic Acid fromDredged Sediment from Arid RegionReservoirs

Mitsuteru Irie, Junkyu Han, AtsushiKawachi, Jamila Tarhouni, MohamedKsibi, Kenichi Kashiwagi & Hiroko Isoda

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media Dordrecht. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you

wish to self-archive your work, please use the

accepted author’s version for posting to your

own website or your institution’s repository.

You may further deposit the accepted author’s

version on a funder’s repository at a funder’s

request, provided it is not made publicly

available until 12 months after publication.

ORIGINAL PAPER

In Vitro Testing and Commercialization Potential of ExtractedFulvic Acid from Dredged Sediment from Arid Region Reservoirs

Mitsuteru Irie • Junkyu Han • Atsushi Kawachi •

Jamila Tarhouni • Mohamed Ksibi •

Kenichi Kashiwagi • Hiroko Isoda

Received: 21 December 2012 / Accepted: 22 March 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The surface water resource in arid land is on the

verge of a crisis. The eroded soil deposited in the catchment

area reduces the storage capacity of the reservoir. The

countermeasures, such as dredging and flood water bypass,

are suggested but they are quite costly especially for

developing countries. The authors study the potential of

exploitation of the sediment and its commercialization in

order to reduce the financial burden of sediment dredging

by using the income from sold the products. One of the

possible aspects to utilize is the fulvic acid contained in the

sediment for use as a functional food or medicine. In this

study, fulvic acids were extracted from the sediment sam-

pled from four reservoirs in Tunisia. Elemental analysis and

FT-IR were performed in order to determine the chemical

characteristics of the extracted fulvic acids. The fulvic acids

from the reservoirs had a comparatively low biodegraded

matter than the fulivic acids in other natural water envi-

ronment due to the shorter time of humification. The

functionalities of the extracted fulvic acids on human body

were evaluated using in vitro bioassays. The effect on

energy metabolism and anti-allergic potential of some of

the fulvic acids were confirmed.

Keywords Reservoir sediment � Arid land � Fulvic acid �Bio assay

Introduction

The capacity loss of surface water resources caused by

sedimentation in North African countries reaches 0.5 % of

total storage capacity in Morocco, 0.5 % in Algeria, and

1.0 % in Tunisia per year [1]. Solutions to the sedimenta-

tion problem are indispensable to these arid land countries

which face serious shortage of water. However, the solu-

tions such as dredging and construction of flood water

bypass have not been carried out because they are quite

costly.

In case of Tunisia, although the problem of capacity loss

due to sedimentation have been known for a long time [2, 3],

based on the minutes of the hearing of the Direction Generale

des Barrages et des Grands Travaux Hydrauliques (DGBG/

TH) in the Ministry of Agriculture of Tunisia, which manage

all the reservoirs in Tunisia. However dredging or other

countermeasures have never been done due to economical

reason.

We have previously proposed the exploitation and val-

orization of the sediment which can help shoulder the cost

of dredging or other countermeasure to the sedimentation

problem [4]. For example, because of the clayey charac-

teristics of the sediment, the strength of construction bricks

made from the sediment makes the use of the sediment

economical feasibility as a material for brick industry [5].

Another possibility for its utilization is its use as soil

amelioration material. When the dried pellet of the sedi-

ment was applied to the barley field irrigated with treated

waste water, the heavy metals were captured by the sedi-

ment [6].

M. Irie (&) � J. Han � A. Kawachi � K. Kashiwagi � H. Isoda

Alliance for Research on North Africa, University of Tsukuba,

1-1-1, Tennodai, Tsukuba, Ibaraki 305-8577, Japan

e-mail: irie.mitsuteru.fu@u.tsukuba.ac.jp

J. Tarhouni

National Institute of Agronomy Tunisia (INAT), 43, Ave.

Charles Nicoles, 1082 Tunis, Tunisia

M. Ksibi

National School of Engineering of Sfax (ENIS), University

of Sfax, km 4, Route de Soukra, Sfax, Tunisia

123

Waste Biomass Valor

DOI 10.1007/s12649-013-9234-y

Author's personal copy

One of the possible ways for utilization of sediment is to

use some beneficial compounds from the sediment. It is well

known that the organic matter in sediment is degraded and

humified by microorganisms to become compost, and it

contains some bioactive compounds such as humic sub-

stances (HS). HS are biochemically weathered organic

components formed from decaying plants and animals.

They are found in almost all terrestrial and aquatic envi-

ronments. There are three types of HS, according to their

solubility in water: fulvic acid (FA), humic acid (HA) and

humin. FA consists of a mixture of closely related complex

of aromatic polymers, and chemical, and spectroscopic

analysis have revealed the presence of aromatic rings,

phenolic hydroxyl, keton carbonyl, quinone carbonyl, car-

boxyl, and alkoxyl groups [7]. FA has various useful effects

due to its functional groups. Studies on the physiological

action of FA exerted on the biosystems have been reported.

For example, the antioxidative activity of FA extracted

from peat has been reported [8]. The possible applications

of coal-derived fulvic acid as an antimicrobial [9] and as an

anti-inflammatory substance have been also reported [10].

FA is also contained in waste and its utilization has been

discussed from the point of view of material recycling. The

inhibitory effect of FA extracted from excess sludge on b-

hexosaminidase release has also been reported [11].

Moreover, the Ministry of Health, Labor, and Welfare of

Japan designated FA as a food in 2004.

In this paper, the extraction of FA from the sediment of

four reservoirs in Tunisia was performed and the charac-

teristics of the FA were determined. In addition, the

increase in the intercellular ATP content and the inhibition

of b-hexosaminidase release following treatment with

extracted FA were evaluated using in vitro bioassays.

Materials and Methods

Study Site and Basic Parameters of Sediment Samples

In June 2011, sediment samples were collected from the

bed of four reservoirs by using an Ekman-berge bottom

sampler and core-sampler (RIGO CO., LTD.). The loca-

tions of the four reservoirs in Tunisia are shown in Fig. 1

while the sampling points on each reservoir are shown in

Fig. 2. Bathymetric contours on Fig. 2 are the result of the

bathymetric survey carried out in September 2009 in the

Joumine reservoir and in June 2011 in the other three

reservoirs. The collected samples were packed into plastic

bags in the field, and then brought to Japan. The depths of

the maximum water level of each reservoir at the sampling

points were 9.8–27 m. The reservoir bed at the station No.1

in Joumine reservoir emerges when the water level is at its

lowest in dry season.

In the laboratory, the moisture and ignition loss (IL)

were determined using the following procedure: dried

sample at 105 �C for 24 h was weighed, and placed in a

muffle furnace at 750 �C for 1 h. The ratio of the volatile

loss to the original dried weight is the IL.

Particle size of the sediment samples was also measured.

Small amount of the sample was put into water, containing

beaker and dispersed with supersonic wave. Particle size

was measured using a laser diffraction particle size ana-

lyzer (0.25–350 lm: SALD3000, SHIMADZU, Japan).

Bulk density was measured of the undisturbed samples

of sediment (length: 15–30 cm, diameter: 4 cm) taken

using the core-sampler. The core samples were then divi-

ded at every 10 cm length of the core sampler on the boat

and packed in plastic bottles. The samples in the bottles

were disturbed but the original volume was known (vol-

ume = /4 9 10 cm). In the laboratory, the weight of each

sample was measured after drying at 105 �C for 24 h. The

bulk density was calculated using the dried weight of unit

volume.

The electrical conductivity and pH was measured in a

1:2.5 suspension of soil and distilled water. Exchangeable

cations were measured by using an atomic absorption

spectrophotometer (ASS) after extraction with 0.5 M bar-

ium chloride solution. Soluble cations were measured using

distilled water instead of 0.5 M BaCl2. Heavy metals

concentrations were determined by using ASS after aqua

regia acid digestion.

Fulvic Acid Extraction and Analysis

Fulvic acid (FA) in the sediments was extracted according

to the standard method of International Humic Substance

Society (IHSS). HCl (1 M) was added to dried sediments

until the pH was between 1 and 2. Next, it was shaken for

1 h with HCl (0.1 M) (10 ml/1 g dried sediment), and then

left to stand for 24 h. The supernatant was filtered and

stored (hereafter S1), while the residues were being dis-

persed in a shaker for 4 h with NaOH (0.1 M) (10 ml/1 g

Fig. 1 Location of the observed reservoirs in Tunisia

Waste Biomass Valor

123

Author's personal copy

dried sediment). After the shaking, the samples were cen-

trifuged at 3,500 rpm for 20 min. The supernatant (S2) was

then filtered and mixed with S1 for the next step. The

supernatant (S1 ? S2) was then purified in a column

adsorption resin (XAD-7, Organo Co.), and the fractions

retained by this resin were recovered with NaOH (0.1 M).

The resulting alkaline solutions were passed through H ? -

saturated cation exchange resin (AG-MP-50, Bio-Rad Co.).

Then the purified FA was freeze-dried prior to use.

The carbon (C), hydrogen (H) and nitrogen (N) contents

of FA were analyzed using a 2400 CHN elemental analyzer

(Perkin-Elmer). Oxygen content was calculated from the

difference of C, H, and N. Total ash of FA was measured by

drying first at 105 �C for 24 h in dry weighed crucible after

which it was placed in a muffle furnace at 600 �C for 2 h.

Two milligram of the extracted FA was mixed with

100 mg of potassium bromide (KBr), and the mixture was

pressed into a disk. The pellets were then analyzed with an

FT-IR-300 spectrum photometer from 400 to 4000 cm-1

(Jasco).

Cell Culture

Caco-2 cells (passage 35–45) were maintained in Dul-

becco’s modified Eagle’s medium (DMEM, Sigma, St.

Louis, MO) supplemented with 10 % fetal calf serum

(Sigma), 1 % penicillin- streptomycin (Sigma), and 1 %

nonessential amino acids (Cosmo Bio Co. Ltd., Tokyo,

Japan) and incubated in an atmosphere of 5 % CO2 at

37 �C. The cells were passaged at a split ratio of 4–8 every

3 or 4 days. To culture for the measurement of intercellular

ATP content, cells were seeded onto 96 well plates at a

density of 1.0 9 105 cells/ml.

RBL-2H3 cells (passage 5–12) were purchased from

Riken Cell Bank, Japan. The cells were maintained in

MEM supplemented with supplemented with 10 % fetal

calf serum (Sigma), 2 mM L-glutamine, and incubated in

an atmosphere of 5 % CO2 at 37 �C. The cells were pas-

saged at a split ratio of 4–8 every 3 or 4 days. To culture

for the measurement of b-hexosaminidase inhibition assay,

cells were seeded onto 96 well plates at a density of

5.0 9 105 cells/ml.

Measurement of Intracellular ATP Content

ATP was assessed by firefly bioluminescence technique

employed by the luminescence luciferase assay kit (TOYO

Ink, Tokyo, Japan). To determine the increase in intracel-

lular ATP content due to sample treatment, Caco-2 cells

were treated with FA (10, 100 lg/ml) for 6 h. After

treatment with FA, cells were lysed with 100 lL of lysis

buffer (Toyo ink) and placed directly into the luminometer

Fig. 2 Sampling point in each

of the reservoirs

Waste Biomass Valor

123

Author's personal copy

chamber (Powerscan HT; Dainippon Pharmaceutical,

Osaka, Japan). Light emission was recorded after addition

of 100 lL of luciferin-luciferase solution (Toyo ink).

When ATP is the limiting component in a luciferase

reaction, the intensity of light emitted is proportional to the

concentration of ATP in the cytosolic extracts.

b-Hexosaminidase Inhibition Assay

In type I allergy reactions in mast/basophil cells, the binding of

antigens and antibodies is a direct cause of intracellular

organelle flows such as histamine and b-hexosaminidase.

Therefore, in order to determine the inhibitory effect of FA on

the chemical mediator release, b-hexosaminidase inhibition

assays were performed according to the method described by

Kawasaki et al. [12]. For the b-hexosaminidase inhibition

assay at the antigen–antibody binding stage, RBL-2H3 cells

were seeded onto a 96-well plate at 5.0 9 105 cells/mL in

100 lL of medium. The cells were incubated and sensitized

for 24 h at 37 �C and 5 % CO2 with 0.3 lg/mL anti-DNP-IgE.

The cells were then washed twice with PBS to eliminate free

IgE. After incubating the cells at 37 �C for 10 min in 60 lL/

well of a releasing mixture (116.9 mM NaCl, 5.4 mM KCl,

0.8 mM MgSO4.7H2O, 5.6 mM glucose, 25.0 mM HEPES,

2.0 mM CaCl2, and 1.0 mg/mL BSA at pH 7.7) containing

5 lL/well of sample, the cells were exposed to 5 lL/well of

4 lg/mL DNP-BSA in PBS (-), and then incubated again at

37 �C for 1 h. As a positive control, 3 mM of ketotifen was

used. The plates were then put on ice for 10 min to terminate

reactions before 20 lL of supernatant was transferred to

another plate; 80 lL of substrate solution (5 mM 4-nitro-

phenyl N-acetyl-b-D-glucosaminide in a 50 mM C6H8O7

buffer at pH 4.5) was added to the supernatant and incubated at

37 �C for 30 min. Then, 100 lL/well of a stop buffer (0.1 M

NaHCO3/Na2CO3, pH 10) was added and the absorbance at

405 nm was obtained using the multidetection microplate

reader to measure the total activity of b-hexosaminidase. The

percentage inhibition rate of b-hexosaminidase release from

RBL-2H3 cells by sample:

Inhibition rate ð%Þ ¼ 1� T � B

C � B

� �� 100 ð1Þ

Where the test assay (T): contained cell (?), DNP-BSA

(?), and the test sample (?); the blank assay (B): contained

cell (-), DNP-BSA (?), and the test sample (?); and the

control assay (C): contained cell (?), DNP-BSA (?), and

the test sample (-).

Results and Discussion

Soluble Cations and Heavy Metals in the Sediment

Samples

Figure 3 shows the radar chart of soluble cations and heavy

metals content in the sediment samples taken from 7 points

Fig. 3 i Water soluble cations and ii heavy metals in the sediment samples

Waste Biomass Valor

123

Author's personal copy

from each of the 4 reservoirs. The sediments of the 3 res-

ervoirs, except Masri, were sampled at 2 points. The bal-

ance of soluble cation and heavy metal content of the

sediment samples in one reservoir appeared to be the same

while those of other reservoirs showed different tendency.

Sediment in one reservoir showed uniform characteristics

in terms of cations and heavy metals content. Table 1

present the physiochemical parameters of the sediment

samples. The particle size of the sediments at different

points of the reservoir is uniform. It is supposed that these

parameters are related to the condition of the catchment

area and there are no differences due to the transportation

and settled process of the sediment in the reservoirs.

Amount and of Extracted FA

The right column of Table 1 shows the amount of FA which

was extracted from 1 kg sediment and results of elemental

analysis. No FA was extracted from the sediment samples

taken from Mellegue reservoir and the sediment has higher

value of ignition loss. Table 2 shows the correlation coef-

ficient between the physiochemical parameters of sediment

and the FA content of the 7 samples of sediment. TOC and

FA showed higher positive correlation while ignition loss

has negative correlation. There are several reports about the

relationship between TOC and FA content [13, 14]. How-

ever, ignition loss has negative correlation coefficient with

FA though it is used as an indicator of organic content in

water samples. Inorganic carbon which is contained in

calcium carbonate is also volatile. There is a possibility that

the negative correlation between ignition loss and FA is due

to the higher inorganic carbonate content in lower TOC

sediment. Those might be an indicator of the land condition

of the catchment area. The catchment area of Mellegue is

located in the most arid area of the 4 catchments. The area

with thin soil, such as desert or outcrop of limestone, is

common in the catchment area and the discharge of the

calcium carbonate is larger and organic matter supply is

limited. On the other hand, on the other 3 catchments,

farmland and forest are common and calcium carbonate

discharge is regulated and the transportation of organic

matter from the catchment is comparatively larger. Looking

at the water soluble calcium and pH, there is no significant

difference between the catchment areas, but higher EC is

found in the sediment sample of Mellegue, which has higher

ignition loss and lower TOC.

The extraction process of FA was modified because the

amount of FA which was extracted from each sediment

sample was smaller than what was obtained by the previous

studies of FA extraction from other materials which are

between 1 and 10 g/kg (dried sediment) [15, 16, 17, 18].

These amounts are equivalent to about 100–1000 times

than that of what were obtained from this study’s sites. Of

course, the main reason for the low FA content in the

sediment of this study is that samples were obtained from

the water resource reservoirs which can supply portable

water and has lower organic matter inflow while the sedi-

ment samples of previous studies were taken from down-

stream of the rivers and lakes which have high organic

matter inflow. Another reason is that carbonates interfered

with FA extraction [15]. The standard method of IHSS

considers the effect of carbonate. At the first part of the

standard method of IHSS, HCl (1 M) is added to the dried

sediment. The purpose of this process is to convert car-

bonates to CO2. However, on the other hand, the above

discussion pointed out that the sediments have very high

content of carbonate as to increase the values of the igni-

tion loss. Therefore we changed the concentration of HCL

Table 1 Properties of the sediment samples

Dam Point

no.

Depth from

max. WL. (m)

pH EC (mS/cm) Median

particle size (lm)

CEC

(meq/100 g)

Ignition

loss (%)

TOC

(mg/kg)

FA from

1 kg dried

sediment (mg)

(a) Joumine 1 13.5 7.64 0.366 4.5 30.79 17.5 94.86 28.0

2 25.5 8.24 0.570 4.5 27.26 16.4 117.72 18.0

2 (adding 6 M HCl at the beginning) (71.6)

(b) Sejnane 1 22.3 8.42 0.305 3.9 21.71 10.4 125.64 49.9

2 27.0 8.18 0.310 4.2 24.30 11.5 94.52 49.8

(c) Mellegue 1 19.5 8.6 0.765 3.2 18.14 18.9 54.298 0.0

2 14.6 7.99 0.805 3.3 25.10 18.3 47.56 0.0

(d)Masri 1 9.8 7.8 0.725 4.1 31.53 12.1 97.62 42.8

Table 2 Correlation coefficient between the parameters of the sedi-

ment properties

CEC IL TOC FA

CEC(meq) – -0.10 0.31 0.29

Ignition loss (%) – -0.68 -0.94

TOC (mg/kg) – 0.76

FA (mg) –

Waste Biomass Valor

123

Author's personal copy

to 6 M. As a result, the extracted amount of FA was about

4 times than what was extracted using the original method.

Extracted amounts from the sediment samples were small

with the standard method of IHSS but the extraction rate

can be improved by changing the concentration of HCl at

the first process.

Elemental Analysis and FT-IR

Table 3 presents the results of elemental analysis and

atomic ratio. We compared our data with that of Giovanela

et al. [19] showing the example of atomic ratio of FA and

humic acid samples extracted from lake, estuary and res-

ervoir sediment (Fig. 4). Compared with Giovanela et al.

[19], the FA extracted in this study has higher Nitrogen

content which indicates a lower degree of humification of

FA.

The infrared spectra of FA extracted in this study are

shown in Fig. 5 with the infrared spectrum of FA extracted

from Canadian Sphagnum peat [20] as CP-FA, being

shown for comparison. These infrared spectra were

identified based on the data in the study of Stevenson and

Goh [21]. The infrared spectra had strong absorbance at

3,400, 2,940, 1,720, 1,420, 1,220 and 1,080 cm-1. The

absorbance at 3,400 cm-1 is attributed to an intermolecular

OH stretch, while the absorbance at 2,940 cm-1 is due to

aliphatic C–H stretching vibration which indicates the

presence of methyl and methylene group. The high

absorption at 1,720 cm-1 attributed to the C = O stretch-

ing vibration of COOH group found in the results of all FA.

The results show that the extracted FA had a similar

structure to that of CP-FA. However, there is no significant

band at around 1,620 cm-1 in the results of the extracted

FA as expected in CP-FA. The absorption at the

1,620 cm-1 band is one of the indicators for estimating the

degree of humification. These results show that the

extracted FA had less polycondensation than CP-FA which

suggests that it has been broken down into smaller, more

fulvic subunits by bacterial enzymes, and decarboxyl and

oxidation reaction with time. FA in natural environment

such as the lake, lagoon and estuary also showed a high

absorption value of around 1,620 cm-1 [19]. The sediment

in the reservoirs had shorter time for humification process

than those of FA. These results are consistent with the

results obtained from the elemental analysis and atomic

ratio balance.

Effects of FA on Intracellular ATP Production

To investigate the effect on the activation of energy

metabolism, the levels of ATP, which is the end product of

glycolysis and TCA cycle, were evaluated. ATP is a mul-

tifunctional nucleotide that is most important as a

‘‘molecular currency’’ of intracellular energy transfer. In

this role, ATP transports chemical energy within cells for

metabolism. Intracellular ATP production of the FA-trea-

ted Caco-2 cells was measured by a luciferase reaction

method (Fig. 6). In the extracted FA from the sediment

samples at Joumine1(10 lg/ml)- and that at Joumine2

(10 lg/ml)- treated Caco-2 cells, luminescence was 117

and 110 % higher than in untreated cells (Control),

Table 3 Elemental composition of FAs extracted from the sediments

Dam Point no. Elements Atomic ratio

C H N O H/C N/C O/C Ash (%)

Joumine 1 46.43 5.05 3.51 45.01 1.30 0.065 0.73 38.8

2 40.97 4.78 3.34 50.91 1.39 0.070 0.93 33.3

2 (adding 6 M HCl) 43.94 4.61 3.23 48.22 1.22 0.050 0.76 2.3

Sejnane 1 45.93 4.71 2.7 46.66 1.26 0.051 0.73 30.4

2 46.8 4.94 2.79 45.47 1.36 0.073 0.73 30.6

Masri 1 46.07 5.24 3.9 44.79 1.25 0.063 0.82 5.8

Fig. 4 N/C versus H/C atomic ratios for all HS samples. FA fulvic

acids, HA humic acids. Lake = Peri Lagoon; Sea = Mar Virado

Island and Ubatumirim Beach; Estuary = Ratones Mangrove (Gio-

vanela et al. [19].) and the FAs extracted from the sediment of the

four reservoirs

Waste Biomass Valor

123

Author's personal copy

respectively. From these results, we suggest that FA from

the sediment at Joumine1 and 2 have an activation function

energy metabolism in human intestinal epithelium.

b-hexosaminidase Inhibition Assay

The allergic symptoms, sneezing, runny nose, urticarial,

are induced by chemical mediators such as histamine and

b-hexosaminidase, released from the cell. These chemical

mediators induce vasodilatation, mucous secretion, and

bronchoconstriction. In this study, we assayed the b-hex-

osaminidase-release inhibitory effect of the FA using RBL-

2H3 cells. The b-hexosaminidase release inhibitory effects

of the FA on RBL-2H3 cells are shown in Fig. 7. The FA

extracted from the sediment samples at Sejnane2 and Masri

were did not have any b-hexosaminidase release inhibitory

effects (data not shown). The other FA were found to have

a similar inhibitory effect on b-hexosaminidase release

from RBL-2H3 cells at 10 lg/ml treatment as compared to

Ketotifen treatment. (final concentration; 214 lM,

IC50 = 200–300 lM). This result indicates that the FA

except Sejnane 2 and Masri have anti-allergic effect via the

inhibitory effect on b-hexosaminidase release.

Feasibility of the Exploitation

Two of the basic functionalities of FA extracted from the

sediment on human cell were confirmed by the in vitro bio

assays in this study. Before the industrialization of the FA

extracted from the sediment, its economical feasibility

should be assessed.

First, we compare the resource potential of FA with

vying resource of FA. The recovery rate of FA extracted

from the sediment in this study was less than 0.01 % while

that from CP was 4.8 %, that from solubilized excess

sludge was 3.5 %, and that from the sediments in lakes and

rivers were 1–10 % of the FA content as mentioned in the

previous chapter. The FA extraction rate from the sediment

is far smaller to the other resources. As mentioned above,

higher content of calcium carbonate is one of the reasons

for the reduction of extraction rate. However, if the higher

concentration of HCl is used for the extraction, its cost

would be higher also. In addition, regarding comparatively

lower TOC values of the sediment sample in this study than

that of the other samples in the references shown above,

there is the limit to the improvement of extraction rate.

Indeed, we could find the functionality of the FA extracted

from the sediments but the extraction rate looks far small to

the rate of CP or other resources. However, the excavation

of peat from wetlands, which is one of the main resources

of FA, has recently been severely restricted from the point

of environmental conservation by the Ramsar Convention

[22]. That is why we supposed that the reservoir sediment

can be the alternative resource of FA.

On the other hand, looking at this study from the point

of view of its economical feasibility, the proceeds for

which will help cover the dredging cost, the exploitation of

Fig. 5 FT-IR Spectra of the FAs extracted from the sediment of the

reservoirs

Waste Biomass Valor

123

Author's personal copy

FA from reservoir sediment can be feasible based on the

feasibility study results on Joumine reservoir. The dredging

cost in Tunisia has already been evaluated previously; 4.5

DT (Tunisian Dinar)/m3 equivalent to 2.9 USD/m3 [5]. The

bulk density of the sediment in Joumine reservoir was

0.69 kg/litter, which means that the dredging cost per unit

dry weight of sediment is 0.0042USD/kg.

The market price of FA is unknown but the price of FA

standard sample sold by IHSS at 150USD/100 mg can be

used as a reference. The FA contained in 1 kg of the sed-

iment was 23 mg (average) (Table 1). Therefore, we can

produce FA from 1 kg of the dried sediment which is

equivalent to 35USD.

Another reference is the prices of the supplements

containing FA. They are sold at 2000–5000 JPY (equiva-

lent to 220–550USD) in Japan. The concentration of FA in

the supplement sold in Japan is unknown. According to the

result of the in vitro assay of this study, 10 lg/ml is the

effective concentration that had a positive reaction. FA

may then be used as a supplement or an additive. For

example, a few drops of FA will be added to a cup of water

or tea so that it is diluted 1000 time and adjusted to its

effective concentration, or used as undiluted supplement at

10 mg/ml. Additionally, 1 kg of the dried sediment can be

used as a source of 2.3 ml of undiluted FA supplement

equivalent to 5–13USD. Keeping in mind that the price of

the supplements includes expense in addition to the price of

FA, so that, the actual price of FA in the supplement is

lower. It is reasonable that the price estimated from the

commercial products is cheaper than that of FA standard

sold by IHSS. Compared to the dredging cost, the prices of

these products are quite high.

The above discussion does not include the cost of

extraction and the cost of extraction consists of expenses

for resource and supplies, personnel expense and depreci-

ation cost of extraction processing plant facilities etc. In

addition, it is difficult to estimate the costs for the con-

struction of the extraction plant and personnel expense.

However, the price of the supplies such as reagents and the

resin can be estimated. Table 4 shows the required mate-

rials and their prices for the extraction of FA from 1 kg of

the dried reservoir sediment. The total cost of the supplies

necessary for processing 1 kg of sediment is about 10USD

Fig. 6 Effect of sample on the

intracellular ATP production of

Caco-2 cells. Caco-2 cells were

treated with 10, 100 lg/ml

sample for 6 h. Results

represent one trial (n = 6). Each

bar represents the mean ± SD

(P \ 0.01 **, Student’s t test)

Fig. 7 Inhibitory effect of sample on the b-hexosaminidase release

from RBL-2H3 cells. RBL-2H3 cells were treated with 10 lg/ml

sample. Results represent one trial (n = 6). Statistical different from

the negative control (PBS (-)). Each bar represents the mean ± SD

(P \ 0.01 **, Student’s t test)

Table 4 Cost of the consumables for treating 1 kg of dried sediment

Required

amount for

treating 1 kg

dried sediment

Unit price Price

(1USD =

90JPY)

HCl 2.1 mol (*75 g) 2,200JPY/

1000 g

1.83

NaOH 1.5 mol(*60 g) 8,500JPY/

5000 g

1.13

XAD7 150 ml

(*98 g)/

10times

30,000JPY/

kg

3.27

Cation Exchange

Resins(CEC1.5 meq/

ml)

3 ml(*2.4 g) 68,000JPY/

500 g

3.60

Total cost of consumables 9.83

Waste Biomass Valor

123

Author's personal copy

which is the same as or higher than the product price

(5–13USD) estimated and discussed above.

The amount of FA which is extracted from the sediment

samples in this study is quite small which is the reason why

the extraction cost is higher than the product price. If the

sediment has a higher FA content, the exploitation of

sediment would be feasible because the extraction cost will

be lower than its selling price. The minimum content of FA

in sediment to make it feasible should be at least around a

few hundred mg in 1 kg of sediment.

Conclusion

The FA content of the sediments sampled from the reser-

voirs were investigated for possible utilization. FA was

extracted from the sediments sampled from Joumine res-

ervoir, Sejnane reservoir and Masri reservoir, but it was not

extracted from the sediment sampled from Mellegue res-

ervoir located in the southern most side of the four reser-

voirs. The extraction of FA was obstructed by the presence

of calcium carbonate which is present in high in concen-

tration in the sediment samples. Therefore, the extraction

rate was improved by increasing the concentration of HCl

used at the beginning of the extraction process. The

chemical characteristics of FA, based on the results of FT-

IR and the element analysis were discussed. The FA

extracted from the sediments were relatively at the early

stage of decomposition than that in natural water envi-

ronment shown in the previous studies.

The functionality of FA on human body was also dis-

cussed based on the in vitro bioassay. FA can activate

energy metabolism in human intestinal epithelium and has

anti-allergic ability via inhibitory effect on b-hexosamini-

dase release.

The extraction rate from the sediment in this study was

much lower than when extracted from other sources of FA.

Incidentally, the extraction cost was so expensive that we find

it not feasible. However, we could find the utility or use of FA

from sediment taken from reservoirs which could probably be

commercialized to cover the dredging cost of sediment that

contains more than a few hundred milligram/kg of FA.

Acknowledgments This research was partially supported by the

Ministry of Education, Science, Sports and Culture, Grant-in-Aid for

Scientific Research (B), 2010, 22404009. and JST/JICA, SATREPS.

References

1. Remini, B: La Sedimentation Des Barrages, Universite Saad

Dahlab-Blida. (2006) (in French)

2. Claude, J., Chartier, R.: Mesure de E’envasement dans les rete-

nues de six barrages en Tunisie Campagne de 1975. Cah. Orstom

ser. Hydrol. 14(1), 3–35 (1977). in French

3. Ben Mammou, A., Louati, M.: Evolution temporelle de l’envas-

ement des retenues de barrages de Tunisie. Rev. Sci. de l’Eau

20(2), 201–211 (2007). in French

4. Irie, M., Kawachi, A., Tarhouni, J., Ghrabi, A.: Development of

sedimentation and characteristics of sediment on the reservoir in

Tunisia. J. Jpn. Soc. Civ. Eng. Ser. B1 67(4), I163–I168 (2011)

5. Irie, M., Kashiwagi, K., Ujiie, K., Nsiri, I., Buguerra, S.,

Tarhouni, J.: Feasibility of exploitation of the sediment in the

reservoirs for the sustainability of surface water resource in

Tunisia. J. Jpn. Soc. Civ. Eng. Ser. G 68(6), II41–II46 (2012)

6. Mtibaa, S., Irie, M., Ksibi, M., Trabelsi, H., Hentati, O., Kallel,

M., Isoda, H.: Soil amendment by sediment from water storage

reservoir as a restoration technique in secondary treated waste-

water irrigated area at El Hajeb region. J. Arid Land Stud. 22(1),

315–318 (2012). Accepted

7. Stevenson F.J.: Humic Substances in soil, sediment and water. In:

Aiken GR, Mcknight DM, Wershaw RL, Maccarthy P(eds),

pp. 13–52. Wiley, New York (1985)

8. Tachibana, Y., Hiribe, S., Tawa, R.: Studies of antioxidative

activity of humic substances in peat (1). Trace Nutr. Res. 23,

104–108 (2004). in Japanese

9. van Rensburg, C.E.J., van Straten, A., Dekker, J.: An in vitro

investigation of the antimicrobial activity of oxifulvic acid.

J. Antimicrob. Chemmother. 46, 835–854 (2000)

10. van Rensburg, C.E.J., Malfeld, S.C.K., Dekker, J.: Topical

application of Oxifulvic acid suppresses the cutaneous immune

response in mice. J. Drug Develop. Res. 53, 29–32 (2001)

11. Motojima, H., Yamada, P., Han, J., Ozaki, M., Shigemori, H.,

Isoda, H.: Properties of fulvic acid extracted from excess sludge

and its inhibiting effect on b-hexosaminidase release. Biosci.

Biotechnol. Biochem. 73(10), 2210–2216 (2009)

12. Kawasaki, M., Toyoda, M., Teshima, R., Sawada, J., Hayashi, T.,

Arisawa, M., Shimizu, M., Morita, N., Inoue, S., Saito, Y.: J.

Food Hyg. Soc. Jpn. 35, 496–503 (1994)

13. Nagao, M.: Hosyaseihaikibutsukenkyu 1–2, 231–242 (1995). in

Japanese

14. Castagnoli, O., Mesmeci, L., Zavattiero, E., Chirico, M.: Water

Air Soil Pollut. 53, 1–12 (1990)

15. Whitby, L.M., Schnitzer, M.: Humic and fulvic acids in sediment

and soil of agricultural watersheds. Can. J. Soil Sci. 58, 167–178

(1978)

16. Moros, J., Hermelo, H.P., Pineiro, M.A., Barrera, B.P., Garrigues,

S., Guardia, M.: Screening of humic and fulvic acids in estuarine

sediments by near-infrared spectrometry. Anal. Bioanal. Chem.

392, 541–549 (2008). doi:10.1007/s00216-008-2279-2

17. Sardessai, S.: Characteristics of humic and fulvic acids in Arabian

sea sediment. Indian J. Marine Sci. 24, 119–127 (1995)

18. Nriagu, O.J., Coker, D.R.: Trace metals in humic and fulvic acids

from lake Ontario sediment. Env. Sci. Tech. 14(4), 443–446

(1980)

19. Giovanela, M., Parlanti, E., Soriano-Sierra, J.E., Soldi, S.M.,

Sierra, M.: Elemental composition, FT-IR spectra and thermal

behaviour of sedimentary fulvic and humic acids from aquatic

and terrestrial environments. Geochem. J. 38, 255–264 (2004)

20. Yamada, P., Isoda, H., Han, J., Talorete, T.P.N., Yamaguchi, T.,

Abe, Y.: Inhibitory effect of fulvic acid extracted from Canadian

Sphagnum peat on chemical mediator release by RBL-2H3 and

KU812 cells. Biosci. Biotechnol. Biochem. 71, 1294–1305

(2007). doi:10.1271/bbb.60702

21. Stevenson, F.J., Goh, K.M.: Infrared spectra of humic acids and

related substances. Geochim. Cosmochim. Acta 35, 471–483

(1971)

22. Matthews, G.V.T.: The Ramsar Convention on Wetlands; Its

History and Development, Luthi, Ramsar (ed.) Ramsar Conven-

tion Bureau, Gland, 122. (1993)

Waste Biomass Valor

123

Author's personal copy