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Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambie. 29 (Sup. 3) 89-97, 2013
, Katarzyna WROBEL
, Kazimierz WROBEL
, Ricardo NAVARRO
, Margarita TEUTLI-LEÓN
and Francisco RODRÍGUEZ
Subdirección de Investigación y Formación de Talento Humano, Centro de Investigación y Desarrollo
Tecnológico en Electroquímica, S. C., Parque Tecnológico Querétaro, Sanfandila, Pedro Escobedo, C.P. 76703
Querétaro, México
Departamento de Química, Universidad de Guanajuato, L. de Retana No. 5 C.P. 36000 Guanajuato, México
Facultad de Ingeniería, Benemérita Universidad Autónoma de Puebla. Edifcio 108-B Cd Universitaria, 72570
Puebla, México
* Autor responsable;
(Recibido agosto 2011, marzo 2013)
Key words:
fecal bacterias, thermophilic phase, humic substances, size exclusion chromatography
The purpose of this work was to examine the feasibility of using municipal sewage
sludge from San Miguel de Allende city WWTP, located at central Mexico, for compost
production. Experimental piles were prepared with an aerobic digested activated sludge
±rom a municipal WWTP dewatered in a flter press, grass was added as a nitrogen
source and bulking was increased by mixing with either crushed porous volcanic mate-
rial called tezontle (SGT) or wooden sticks wastes collected from municipal gardening
o± green areas (SGW). The specifc composition o± the compost piles was as ±ollows:
30 % municipal sewage sludge, 60 % grass clippings and 10 % small wooden sticks
(compost SGW); 30 % municipal sewage sludge, 60 % grass clippings and 10 %
tezontle (compost SGT); 100 % sewage sludge (control). The e±fcient reduction o±
fecal bacteria was achieved in both piles yielding the USEPA class A compost, yet
better process performance was obtained in SGW. In this pile the thermophilic phase
lasted seven days longer and higher temperatures were reached, for which the desired
removal of coliforms was achieved at two weeks of composting versus three weeks
required in SGT. The analysis of typical spectrophotometric assays used in the compost
characterization, confrmed the decomposition o± organic matter and generation o± hu
mic substances. The fractionation of humic extracts by size exclusion chromatography
with spectrophotometric detection indicated the increase of humic substances amount,
and also the increase of molecular mass with the time of composting process. Overall,
this work demonstrates the feasibility of municipal sewage sludge compost production
for soil fertilization and provides new data about the changes of the molecular mass in
humic fraction during composting.
Palabras clave:
bacterias fecales, fase termofílica, substancias húmicas, cromatografía de exclusión por tamaño
V. Ramírez-Coutiño
et al.
El propósito de este trabajo es examinar la viabilidad de usar lodo residual municipal de
la PTAR en San Miguel de Allende, ciudad localizada en la parte central de México, con
la fnalidad de producir composta. Las pilas experimentales contuvieron lodo residual
proveniente de una planta de tratamiento de aguas residuales de lodos activados que
tuvieron una digestión aerobia y un secado en fltro prensa, el cual Fue mezclado con
césped de corte empleado como fuente de nitrógeno y un material volcánico poroso lla-
mado tezontle (SGT) o desechos de maleza de las áreas verdes municipales (SGW). Las
pilas se prepararon de acuerdo a las siguientes proporciones: 30 % de lodo de la planta
de tratamiento, 60 % de pasto y 10 % de tezontle (composta SGT); 30 % de lodo de la
planta de tratamiento, 60 % de pasto y 10 % de pequeños troncos (composta SGW); 100 %
de lodo de la planta de tratamiento. La reducción efcaz de bacterias Fecales se logró en
ambas pilas, dando lugar a compostas de Clase A de acuerdo con la clasifcación de la
USEPA, la pila que obtuvo
mayor efciencia de reducción de bacterias Fecales Fue la SGW.
En esta pila la fase termofílica duró siete días y se alcanzaron las más altas temperaturas,
provocando la reducción de bacterias fecales deseadas en un periodo de dos semanas
contra tres semanas que requirió la pila SGT. Los análisis típicos espectrofotométricos
Fueron usados para la caracterización de las compostas, confrmando la descomposición de
la materia orgánica y generación de sustancias húmicas. El fraccionamiento de extractos
húmicos por cromatografía de exclusión por tamaño con detección espectrofotométrica
indicó el incremento de su cantidad total y también del incremento de masa molecular con
el tiempo del proceso de compostaje. En conjunto, este trabajo demuestra la viabilidad de
la producción de composta a partir de lodos residuales municipales y proporciona datos
sobre los cambios de la masa molecular de fracciones húmicas durante el compostaje.
The installation of wastewater treatment plants
(WWTP) is a common strategy to treat municipal
wastewater, yet an undesirable side effect is the gen-
eration of relatively large amounts of sewage sludge
that contain important pollutants such as pathogenic
microorganisms, heavy metals and potentially toxic
organic compounds. Despite obvious health risks
imposed by municipal biosolids (McFarland 2000,
Metcalf and Eddy 2003), this type of waste is attrac-
tive due to the large amount of organic matter and
nutrients, allowing it to be used as a soil amendment
or a fnal cover For municipal solid waste landflls,
only if the sludge has been treated adequately (Gi-
rovich 1996, Grigatti
et al
. 2004).
Composting of sewage sludge has often been
performed by mixing the biosolid with easily de-
gradable organic materials, and formed into piles
under controlled conditions regarding moisture, pH,
oxygenation and carbon/nitrogen ratio, resulting in
an aerobic degradation of organic matter. During the
thermophilic phase, the temperature inside the piles
oscillates between 40 and 60 ºC (Epstein 1997, Le-
et al.
2005, Heinonen
et al.
2006, Bustamante
et al.
2008, Hachicha
et al.
2009), phenomena which
aides in the destruction of pathogens.
At the end of the composting process, a stabilized
organic matter rich in humic substances (HS) is ob-
tained (Spaccini
et al.
2000, Sullivan and Miller 2001).
The term HS refers to a group of heterogeneous and
polyelectrolytic organic compounds with a wide range
of molecular masses (up to 100 kilodaltons (kDa)), that
are considered the main and more stable components
of the soil organic matter. HS contribute to soil fertility
since they act improving structure, oxygenation and
water holding capacity as well as buffer and cationic
exchange capacity (Stevenson 1994). The HS can also
bind and potentially immobilize important xenobiot-
ics as pesticides (Conte
et al.
2005) or heavy metals
(Tipping 2002, Weng
et al.
2002, Halim
et al.
et al.
2002, Wrobel
et al.
Based on the pH dependent solubility, HS are
classifed as humic acids (HA), Fulvic acids (±A)
and humins (HU) (Stevenson 1994, McCarthy 2001,
Sutton and Sposito 2005).
Values of E4/E6 (the ratio of absorbance at
465 and 665 nm), as well as evaluation of func-
tional groups (total acidity, carboxylic and phe-
nolic groups) and fractionation by size exclusion
chromatography have been used to compare the
maturity and stability of organic matter of diverse
origins like peats (Janos 2003, Shirshova
et al.
Fong and Mohamed 2007), compost from municipal
solid waste (Campitelli
et al
. 2006), sewage sludge
et al.
2002, Réveillé
et al.
poultry manure, pig slurry, cotton waste, maize
straw and sweet sorghum bagasse (Sanchez
et al.
2003). In regard to compost, the humifcation pro
cess is studied in order to understand and evaluate
the transformations of organic matter initially pres-
ent in the biosolids (Veeken
et al.
2000). In some
previous works on the compost production from
sewage sludge and solid waste, HA characteristics
have been determined at the beginning and at the
end of the process. According to reported results,
total acidity oF the fnal product increases together
with the amount of phenolic and carboxylic groups
as compared to the initial biosolid (Zbytniewski and
Buszewskie 2005). At the same time, E4/E6 ratios
decrease and the molecular mass of the humic acids
increases slightly (Janos and Zatrepálková 2007).
However, the modifcations oF HA characteristics
during the composting process has not been studied
in detail.
San Miguel de Allende is a touristic city at central
México, with about 140 000 habitants. Considerable
amounts of municipal wastewater are produced and
these are treated in the local WWTP with a ±ow rate
of 70 L/s. The safe disposal of biosolids generated by
the WWTP has become an issue to solve; and the aim
of this work is to examine the feasibility of utilizing
sewage sludge for compost production. Two bulking
materials, necessary for compost aeration were tested,
both of them abundant and available at negligible cost.
The performance of the composting process was evalu-
ated in each case, using typical measurements and
assays. Additionally, the evolution of molecular mass
in the humic substances fraction was studied by size
exclusion chromatography with spectrophotometric
detection. Obtained results provided an insight on
the humifcation process and also supply regionally
useful data regarding about the possibility of compost
production from the municipal sewage sludge.
Composting of sewage sludge
The experimental system consisted of three
windrows (piles) (1m
) prepared with the sewage
sludge, grass and two different bulking materials: (T)
tezontle, which is a soft porous material of volcanic
origin, abundant in the region of San Miguel de Al-
lende and (W) small wooden sticks – also abundant
as wastes from municipal green areas gardening.
The sewage sludge was obtained from an activated
sludge unit of the municipal WWTP; it was used
after receiving an aerobic digestion treatment and
thickening on a flter press. The specifc composition
of the compost piles was as follows: 30 % municipal
sewage sludge, 60 % grass clippings and 10 % small
wooden sticks (compost SGW); 30 % municipal sew-
age sludge, 60 % grass clippings and 10 % tezontle
(compost SGT); 100 % sewage sludge (control). The
piles were maintained for a period of three months
mixing the materials every seven days; temperature
inside the piles was measured at several sites and the
reported values are the average of the three succes-
sive measurements.
Biological and chemical analyses
The determination of fecal coliform bacteria
(MPN) was accomplished according to the USEPA
Method 1681 (Fecal Coliforms in Sewage Sludge
(Biosolids) by Multiple-Tube Fermentation using A-1
medium) using culture-specifc media and elevated
temperature to isolate and enumerate fecal coliform
organisms. Obtained compost with the three piles at
different time periods was evaluated for MPN per g
dry mass (dm), in terms oF USEPA classifcation oF
compost (EPA 1994). According to this classifcation,
a class A compost should contains less than 1000
MPN fecal coliforms per g of dm and can be used as
a safe soil amendment for food and non food plants;
class B compost is a safe soil amendment only for
ornamental plants and it cannot contain more than
2 × 10
MPN fecal coliforms per g dm. Samples with
a MPN value higher than 2 × 10
fecal coliforms per g
dm are classifed as class C compost.
The amount of organic matter was assessed by two
techniques, frst as the water-soluble organic carbon
(TOC) and second the total organic matter (OM).
For TOC determination, the aliquot of compost was
suspended in water (1:10 sample: water proportion)
and the mixture was stirred for 2 h, then the sample
was centrifuged at 4000 g (Hermle Z 206A centri-
Fuge) For 20 min and fltered through a 0.45 µm cel
lulose syringe flter. The fltrate was introduced into
a reaction chamber, at 680 ºC, which is packed with a
catalytic oxidizer for carbon oxidation; the generated
was transported by the air ±ow to a non dis
persive infrared analyzer (Shimadzu TOC-5050aM
analyzer). The OM was assessed after ignition of the
dry sample at 550 ºC for an hour in a furnace (Felisa
Fe-361) according to Standard Methods SM 2540G.
The ratio between absorbance measured at 465 nm
and 665 nm was assessed in humic acid extracts. To
do so, three aliquots of each compost sample (10 g)
were extracted with 100 mL of 0.1 M Na
V. Ramírez-Coutiño
et al.
sealed bottles with stirring for 24 h. The extracts
containing humic substances were centrifuged for
20 min at 4000 g, and the supernatants were passed
through a 0.45 µm cellulose syringe flter and acidi
fed to pH=2 with 5 M HCl. To accomplish total HA
precipitation, the samples were maintained at 4 ºC
during 24 h and then centrifuged (20 min, 4000 g);
the pellets were then washed twice with 0.1 M HCl
and with deionized water. The HA were further puri-
fed by stirring with a 0.06 M HCl solution For 12 h,
centrifuging and washing with deionized water. The
HA precipitates were dried at room temperature.
An aliquot of each sample (3 mg) was dissolved in
10 mL of NaHCO
0.05 M adjusted to pH 8.3 with
NaOH 0.02 M. (Chen
et al.
1977), the absorbance
was measured at 465 nm and 665 nm using a spec-
trophotometer HACH DR/4000 and the value of E4/
E6 ratio was calculated.
Total acidity and carboxylic groups in aliquots
HA were measured by titration with barium hydrox-
ide and calcium acetate respectively, according to
conventional methods described by Schnitzer and
Khan (1972). The content of phenolic groups was
calculated as the difference between total acidity and
carboxyl groups.
Size exclusion chromatography of humic fraction
of compost
Humic substances were extracted from compost
samples (10 g) with 100 mL of Na
0.1 M in
sealed bottles with stirring for 24 h. Extracts were
centrifuged for 20 min at 4000 g. Fractionation ac-
cording to the molecular mass was performed using
size exclusion liquid chromatography (SEC) with
DAD detection, as described by Sadi
et al.
The column (750 × 9 mm) was packed with Toyopearl
HW-50± gel; the injection volume was 100 μL, the
mobile phase was 10 mM sodium pyrophosphate (pH
10.3) at a ²ow rate oF 0.5 mL/min; and DAD detection
was carried out in the wavelength range 254 - 600 nm.
The column calibration was performed using bovine
serum albumin (66 kDa), carbonic anhydrase from
erythrocytes (29 kDa), cytochrome c from equine
heart (12.4 kDa) and aprotinin from bovine lung
(6.5 kDa) (r
= 0.9978).
In this work, feasibility of using municipal sewage
sludge in a composting process is evaluated, per-
forming amendment of the biosolids with additional
organic matter and two different bulking materials.
For this purpose, temperature and fecal coliforms
were monitored in the two compost piles contain-
ing sewage sludge with grass and tezontle (SGT) or
wooden sticks (SGW) respectively. A pile containing
sewage sludge without any addition was used as a
control. Structural changes of organic matter were
also characterized.
fgure 1
, a plot of temperature inside the piles
over time is presented; as can be observed similar
changes occurred in the SGW and SGT piles over
six weeks, while environment temperature was
maintained for the control pile.
The initial increase of temperature indicates
microbial activity: in SGW the top value (67 ºC)
was reached at three weeks and in SGT the highest
value was 56 ºC and was registered after two weeks.
Once the thermophilic phase had been completed,
the temperature in both piles gradually decreased,
reaching steady environment temperature about one
week earlier in the SGW as compared to the SGT
pile. Obtained results indicate that higher biological
activity in SGW compost could be consequence of
getting a better oxygenation. The control pile, without
additives, did not show any signifcant variation in
The microbiological analysis of sewage sludge
showed fecal coliforms content higher than 2 × 10
MNP per g, and it was classifed as sludge type C
according to the USEPA regulations. The high con-
centration of fecal coliforms makes impossible the
direct use of the municipal sewage sludge as soil
amendment or fertilizer. The amount of fecal coli-
time, weeks
T (ºC)
Fig. 1.
The evolution of temperature inside the piles during the
frst six weeks oF composting: (
) SGW, (
) SGT,
) control pile. Each point is the mean value obtai-
ned for three successive days and the respective standard
deviation is also marked
forms in SGW and SGT piles was monitored during
the composting process, and the obtained results are
presented in
fgure 2
, indicating that both compost-
ing processes are quite efFcient in the removal of
fecal coliforms.
After two weeks of composting, the SGW com-
post fulFlled the EPA standard for a class A compost
(<1000 MPN/g); while the SGT required three weeks
to reach the desired MPN level. These results are
consistent with temperature proFles registered in
the two piles (
Fig. 1
); higher temperature in SGW
apparently favored removal of coliform bacterias in
a shorter time with respect to SGT, yet in both cases,
the Fnal MPN were below 1 % of the initial values.
The pH values in the SGT and SGW composts
were monitored over the three months of the experi-
ment and the results obtained are shown in
fgure 3
At the beginning, a pH raise was observed for
the two piles, reaching 8.4 at day 14
in SGT and
8.9 at day 21 in SGW. Afterwards, the pH gradu-
ally decreased in both composts to reach neutrality
at the end of the composting process. It has been
reported by several authors that pH increases dur-
ing compost formation; apparently this is due to
the degradation of short chain fatty acids and the
transformation of organic nitrogen to ammonium.
The later descent of pH has been attributed to the
bacteria aided ammonium oxidation (Michel and
Reddy 1998, Sanchez
et al.
TOC values obtained in both, SGW and SGT
composts (
Fig. 4a
) decreased mainly in the Frst
two weeks of the composting process, which is the
time period corresponding to the thermophilic phase
Fig. 1
Our observations support the results, previously
reported, concerning to the microorganism assisted
decomposition of organic matter and the decrease
time, weeks
Fecal coliforms, log (MPN/g)
Fig. 2.
The most probable number of fecal coliform bacteria
(as log(MPN/g), assessed at different time periods of
) SGW, (
) SGT, (
) control
pile as compared to EPA regulations of class A and class
B compost.
02 46 81
time, weeks
Fig. 3.
pH values registered in three piles during composting:
) SGW, (
) SGT, (
) control pile.
TOC (g/kg)
0123 45
Time, weeks
Time, weeks
OM (%)
Fig. 4.
Water-soluble organic carbon TOCs, g/kg (a) and total
organic matter OM, % (b) assayed at different time
periods during six weeks of experiment: (
) SGW,
) SGT, (
) control pile.
V. Ramírez-Coutiño
et al.
in TOC levels (Adani
et al.
1997). At the end of the
process, the amount of TOC found in the two com-
posts corresponded to 7 % and 10 % of the initial
value for SGW and SGT respectively. As shown in
fgure 4b
, the organic matter assay (OM) indicated
a slow decrease, going down to 64 % of the initial
value in SGW (36 % removal) and to 74 % in SGT
(26 % removal).
The measurement results for E4/E6 are presented in
fgure 5
; the initial value found in extract from sludge
(control pile) was lower than those in SGW and SGT
(5.2 versus 5.8 and 5.7 respectively), fact which should
be ascribed to the amendment of biosolid with grass
and supporting materials for compost production.
During the experiment, E4/E6 ratio steadily de-
creased in the extracts from both piles reaching the
value of 4.6 in SGW at three weeks and 4.4 in SGT
at four weeks. The decrease of E4/E6 ratio is related
to the formation and aggregation of humic substances
with the shift toward higher molecular mass, in agree-
ment with the proposed models of the formation of
humic acids (Sanchez
et al.
Total acidity and phenolic groups of the humic
acids are shown in
fgure 6
. In both SGW and SGT
compost, there is an increase in functional groups as
the composting process proceeds.
In the case of total acidity, the value changes from
3.41 to 4.85 meq/g in the SGW compost and from
3.28 to 4.45 meq/g in SGT, reaching typical values
for this parameter for soil samples (Stevenson 1994).
The increment for the phenolic group in composts
SGW and SGT was from 1.2 to 2.09 meq/g and from
1.19 to 1.88 meq/g respectively.
In order to get a further insight on the humiFcation
process during composting, the compost extracts,
obtained at different time periods, were analyzed
by size exclusion chromatography. Typical chro-
matograms of extracts from SGW and SGT piles
obtained at two, six and thirteen weeks are presented in
fgure 7a
In general, the elution proFles registered at the
wavelength of 500 nm indicate two molecular mass
(MM) fractions, in agreement with the results ob-
tained previously in the analysis of municipal waste
compost (Wrobel
et al.
2003). The compounds with
MM > 30 kDa eluted in a form of a broad peak
with retention times lower than 14 min; while the
fraction of compounds with MM < 30 kDa formed
a second broad peak with retention time higher
than 14 min. For all samples analyzed, the total
area under the chromatograms obtained for SGW
was always higher, indicating that a more efFcient
humiFcation process is taking place, in respect to
Time, weeks
Fig. 5.
ProFles of
E4/E6 (the ratio between absorbances measu-
red at 465 nm and 665 nm in HA extract) changes in three
piles during six weeks of composting: (
) SGW, (
) SGT, (
) control pile.
Total acidity, meq/g
Time, weeks
Phenolic groups, meq/g
Time, weeks
Fig. 6.
Total acidity, meq/g (a) and phenolic groups, meq/g
(b) evaluated in three piles over six weeks period:
) SGW, (
) SGT, (
) control pile.
SGT. It can also
be observed that the increase of
chromatographic peaks was more pronounced with
the increasing time of composting for SGW (
Fig. 7a
as compared to SGT (
Fig. 7b
). At the same time,
the amount of humic substance extracted from the
control pile was negligible and did not increased dur-
ing the experiment. These facts and also previously
presented results support the necessity of a biosolid
amendment in the compost production, and indicate
that wooden sticks provide better composting condi-
tions with respect to tezontle.
On the other hand, in
fgures 7a
a shift
of the chromatographic signals toward lower reten-
tion times can be also observed, which points to the
increase of HA molecular mass during compost-
ing. For a more clear presentation of this effect, in
fgures 8a
the changes of MM values ver-
sus composting time is plotted for SGW and SGT
respectively. Since the chromatographic peaks were
broad, tentative MM values at the peak maxima were
taken for this purpose.
At frst it can be noted that more drastic changes oF
molecular mass occurred in the fraction of MM > 30
kDa (
Fig. 8a
) as compared to MM < 30 kDa (
Fig. 8b
during the composting process both in, SGW and in
SGT. Furthermore, as it is shown in
fgure 8a
, for
two weeks a similar increase of high MM occurred
in the two compost piles (about 37 % with respect to
the initial value); at longer periods of time a steady,
slow increase of MM occurred in SGW, while in SGT
Time, min
a) SGW
2 weeks
6 weeks
13 weeks
Absorbance, mAU
Time, min
b) SGT
2 weeks
6 weeks
13 weeks
Absorbance, mAU
Fig. 7.
Typical size exclusion chromatograms (detection at 500 nm)
obtained for humic fractions extracted at different time
periods of composting: (a) control pile (b) SGW and (c)
SGT piles. (two weeks (
); six weeks (-------); 13
weeks (-------
Fig. 8.
Changes of the molecular mass of humic extracts during
the composting process: (
) SGW, (
) SGT.
Tentative MM value corresponding to the mass at the
maximum of broad chromatographic peak is presented
in each case.
(a) humic substances with MM > 30 kDa;
(b) humic substances with MM < 30 kDa
Time, weeks
Molecular weight, kDa
Time, weeks
Molecular weight, kDa
V. Ramírez-Coutiño
et al.
there was a tendency toward lowering MM in this
fraction of humic substances.
More pronounced differences between the two
composts can be observed for the fraction of com-
pounds with MM < 30 kDa (
Fig. 8b
). In particular,
the increase of molecular mass was apparently de-
layed in SGW as compared to SGT. Furthermore,
the profles oF MM evolution in the two HA Fractions
corresponding to SGW suggest that the aggregation
of small HA molecules would contribute to the
progress oF humifcation, in agreement with data
previously reported (Jouraiphy
et al.
2005, Amir
et al.
An evaluation of the composting process using
municipal sewage sludge from the treatment plant
located in San Miguel de Allende, city located at
central Mexico was done in this work. Obtained
results revealed the necessity of adding sludge
amendments for increasing bulking properties; in
particular, it can be aFfrmed that in respect to includ
ing tezontle (SGT), a better process performance
was obtained using wooden sticks wastes (SGW) as
a bulking agent. In the thermophilic phase, higher
temperatures were reached in SGW with respect
to SGT (about 10 ºC difference) and elevated tem-
peratures were maintained for longer time periods
(about one week diFFerence). Even though eFfcient
reduction of fecal coliforms was achieved in both
systems, a shorter time was needed for SGW to
accomplish USEPA class A compost requirements
(two weeks in SGW versus three weeks in SGT).
The results of spectrophotometric assays typically
used in the compost characterization, confrmed the
decomposition of organic matter and generation
of humic substances, as it is shown by the gradual
decrease of TOC, OM
and E4/E6 ratio over the time
of composting, as well as an increase of total acidity,
phenolic and carboxyl groups. The fractionation of
humic extracts by size exclusion chromatography
indicated the increase of their total amount and also
the increase of HA molecular mass with the time of
composting process. The results obtained for SGW
seems to confrm that the aggregation oF smaller
molecules contributes to the humifcation process.
Overall, this work demonstrates the feasibility of
municipal sewage sludge compost production for
soil fertilization use,
and it provided new data on
the evolution of the molecular mass in HA fraction
during composting.
The authors thank the Council for Science and
Technology oF Chiapas (COCyTECH) For fnancial
support of this work (Grant CHIS-2005-C03-078).
V.R. also acknowledges CONACyT for a graduate
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