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Bioresource Technology 99 (2008) 1821–1829

Composting and vermicomposting experiences in the treatment

and bioconversion of asphaltens from the Prestige oil spill
Jesús Martı́n-Gil a,*, Luı́s Manuel Navas-Gracia a, Ernesto Gómez-Sobrino a,
Adriana Correa-Guimaraes a, Salvador Hernández-Navarro a, Mercedes Sánchez-Báscones a,
Marı́a del Carmen Ramos-Sánchez b
a
Department of Agricultural and Forestry Engineering, University of Valladolid, Escuela Técnica Superior de Ingenierı́as Agrarias (ETSIIAA),
Avda. de Madrid 57, 34004 Palencia, Spain
b
Laboratory of Microbiology, Hospital Universitario Del Rı́o Hortega, Valladolid, Spain

Received 18 September 2006; received in revised form 20 March 2007; accepted 21 March 2007
Available online 23 May 2007

Abstract

This work illustrates the effectiveness of composting and vermicomposting in degrading fuel-in-water emulsions from oil spills (chapa-
pote), and the isolation of potentially useful microorganisms for its biodegradation. Firstly, an alternative to the biodegradation of
asphaltens from the Prestige oil spill (still present in some chapapote rafts in the Cantabrian coast) by means of the application of com-
posting techniques to a microbial partnership acclimated to fuel-oil is offered. Our aim is that, after a relatively short period of time, the
microorganisms can obtain its source of carbon and energy from asphaltens. The addition of metabolic co-substrates, like cow bed and
potato peelings, allows the fragmentation of complex compounds into smaller structures, susceptible to further degradation. Afterwards,
a maturation of the compost by means of a treatment with earthworms (Eisenia foetida) is necessary. Thus, through the vermicomposting
it will be possible to obtain a valued product, useful in the processes of ground amendment, with little presence of asphaltens and
occluded polycyclic aromatic hydrocarbons, rich in humus, and with an important bacterial flora of Bacillus genera, so that it can be
typical of co-activators and accelerating products in composting processes. Along with this article, we show some parameters that control
the evolution of the compost products (evolved gases, acidity, temperature and humidity); the chemical and microbiological analytical
results; and the germination assays of vermicomposting. Results reveal that by using microorganisms living in either earthworm intes-
tines (Stenotrophomonas maltophilia) or vermiculture substrates (Scedosporium apiospermium), it is possible to degrade and to eliminate
the polycyclic asphaltens into CO2 and H2O, helped by evaporation, dissolution and/or photo-oxidation processes. The obtained end
product has contents of interesting vegetal nutrients and, mainly, it displays very high germination indices.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Prestige oil spill; Eisenia foetida; Bacillus sp.; Stenotrophomonas maltophilia; Scedosporium apiospermium

1. Introduction went to San Vicente de la Barquera and Tagle-Ubiarco, in

By the end of 2002, the Atlantic and Cantabrian shore-
lines of Spain were severely contaminated by the Prestige
heavy fuel oil spill. As a result of this event, some profes-
sors and students of the Escuela Técnica Superior de Ingen-
ierı́as Agrarias (ETSIIAA) of the University of Valladolid
*
Corresponding author. Tel.: +34 79108347; fax: +34 79108440.
E-mail address: montealeku@wanadoo.es (J. Martı́n-Gil).
Cantabria, to carry out controlled experiences of bioreme-
diation on the great spots of emulsion that came to the bea-
ches. Fruit of those experiences, and of the previous
knowledge in composting techniques, are our active lines
of investigation on both biostimulation (by means of the
controlled addition of nutrients) and bioaugmentation pro-
cesses (by means of the use of exogenous microorganisms).
In the case of the Prestige spills and in a first methodolog-
ical stage, we reached the identification of the chemical

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.03.031
1822 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829
components and the microorganisms living in the black
like-mousse emulsions (chapapote) that arrived at the coast
(Martı́n-Gil et al., 2004) and its comparison with those of
other spills. Successively, in a second stage, we boarded
the study of the bioremediation and/or composting pro-
cesses of the tarred remainders gathered in the storage rafts
for the cleaning work.
A comparison of the chemical compositions of Prestige
spill (Martı́n-Gil et al., 2004) with those of Exxon-Valdez,
Erica and Baltic Carrier (Bayona, 2002; Le Cedre, 2002;
Oudot, 2002) has led us to deduce (i) that Exxon-Valdez
oil spill was richer in volatile hydrocarbons (and therefore
more easily biodegradable) than the rest of the crude ones;
(ii) that the compositions of the spills from Prestige and
Baltic Carrier (Velando, 2003) were very similar; (iii) that
in comparison with Erika spill the Prestige fuel-oil showed
a smaller concentration in aromatic fractions and a greater
content in sulphur (2.6%); and that (iv) the elevated sulphur
content in the residual fuel-oils, which causes the poisoning
of the catalysts when petroleum distillates are processed,
makes more advisable the use of microorganisms to elimi-
nate sulphur (Yamada et al., 1968).
Chemical compositions of spills change as time passes:
they are enriched in components with more and more com-
plex molecular structures as asphaltens (the most heavy
and polar components in crude oil), resins and paraffins
(Venkateswaran et al., 1995). When microorganisms com-
ing from natural sediments or other sources are present,
its degradation is spontaneous but also slow, and it takes
place in very small proportion. Therefore it is necessary
to accelerate its degradation with the incorporation to the
fuel-oil of different substrates that allow, subsequently,
the introduction of the exogenous microorganisms neces-
sary to initiate the bioremediation processes. Composting
is a self-heating, aerobic solid phase biodegradative process
of organic materials under controlled conditions, which
distinguishes it from natural rotting or putrefaction. From
an etymological point of view, it is a mixture of substrates
that is biodegraded by a mixed microbial community. The
process comprises mesophilic and thermophilic phases
involving numerous microorganisms. In several successive
steps, microbial communities degrade organic substrates
into more stable, humidified forms and inorganic products,
generating heat as a metabolic waste product. It has been
used for a long time for the degradation of organic remain-
ders, and due to its environmental advantages and quick-
ness, it is a suitable way to eliminate oil remainders from
contaminated ground (Levin and Gealt, 1997). Besides
their ability to eliminate polluting agents, the composting
techniques can revalue grounds contaminated by means
of the attainment of an end product of great agronomic
value.
Many evidences exist on the acceleration of the xenobi-
otic organic compound degradation (and inclusively, of
recalcitrant materials) in grounds by means of composting
processes or by the addition of organic materials that
increase the levels of substrate by co-metabolism, co-oxida-
tion or co-transformation mechanisms. In fact, the elimina-
tion by these procedures of polluting agents like
insecticides and herbicides and of the rest of the explosives
like trinitrotoulene has been described (Stevens et al., 1990;
Palmer et al., 1997). In diverse places of the world, the use
of aired composting batteries has also revealed successful
for the treatment of the remainders and marine spills
derived from petroleum (Labrie and Cyr, 1990). Neverthe-
less, the descriptive works on the application of such rem-
edies to the heaviest fractions of petroleum (i.e., the
asphaltenic type molecular conglomerates) are few (Pin-
eda-Flores and Mesta-Howard, 2001).
In spite of the inevitable difficulty of our objective, the
authors of this work try to show that the addition of co-
substrates can be used with advantage to support the co-
metabolic degradation of remainders and/or environmen-
tal polluting agents as recalcitrant as the asphaltens. This
attainment is feasible thanks to the fact that they not only
develop an important microbial activity, but that they
additionally provide the nutrients and the structure neces-
sary to assure the essential ventilation in the composting
process.
2. Methods
The Prestige fuel-oil samples that are the object of the
present study have been provided us by SOGARISA (a
Galician centre for industrial waste treatment) and they
came from storage rafts of the company in Cerceda and
Laracha (A Corunna, Spain).
The analyses of the chemical species in the samples are
carried out with the aid of a mass spectrometer model
Hal 200 from Hyden Analytical. Asphaltene analysis can
be performed after extraction in toluene at room tempera-
ture and later precipitation of the insoluble fraction with
hexane (Long, 1981).
For the degradation of the asphaltens a work system of
the type composting–vermicomposting has been designed
(Ndegwa and Thompson, 2001) such as the vermicompo-
sting processes are made after those of composting. The
processes of composting have been carried out in bioreac-
tors of expanded polystyrene foam, of 100 L capacity, able
to be turned around to facilitate the contact with oxygen. A
controlled composter (Kollvick, Multicomp model 2M)
with possibility of forced air supply and provided with sen-
sors was also used in order to control the humidity and
temperature (Delta equipment). As fuel-oil thickeners it
has been used as sawdust, husk of rice, vegetables, seaweed
extracts and potato peels and as natural fertilizer, chemical
fertilizers and/or dung of bovine.
In order to improve the quality of the product and to
reduce the still present asphaltens, the composting process
has been completed by a subsequent vermicomposting pro-
cess. This process consists of using earthworms Eisenia
foetida selected in the ETSIIAA for diverse research pro-
jects. The processes of vermicompost have been made in
three trays for hydroponic culture of 0.40 · 0.50 · 0.12 m3
 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829 1823

in volume and 0.20 m2 in surface area, with Eisenia foetida tics (direct examination) and using a selected taxonomic
earthworms at a density/biomass of 300 g-earthworms/m2. monograph (Hoog and Guarro, 1995).
During a 6 month period, the humidity of the substrate was
maintained between 60% and 80% by means of a manual
sprayer. The controlled parameters have been the following 3. Results and discussion
ones: granulometry, humidity, pH, ashes, organic matter,
total nitrogen, ammonia nitrogen and nitric nitrogen, The chemical composition of the fraction of fuel-oil sub-
C/N ratio, P, Ca, Mg and heavy metals. ject to composting and vermicomposting has been eluci-
The grain size test has been carried out using molecular dated through comparison of changes between the actual
sieves with different mesh size. Representative samples of and initial compositions of the oil spill from Prestige after
compost and vermicomposting have been stored in a free- extraction of hydrocarbons in toluene and subsequent pre-
zer at 4 C after both previous drying in stove at 80 C cipitation of their heavy fraction with hexane. The compar-
and manual desegregation. The geometric size and its stan- ison of the initial chemical composition in 2002 (Martı́n-Gil
dard geometric deviation have been determined from et al., 2004) with most recent of 2005 allows to reach, as a
ASAE, 1986. The metallic elements have been analyzed first result, that the spontaneous environmental conditions
with an atomic absorption Perkin Elmer system and the of maintenance of the fuel-oil have caused a progressive
non-metallic elements (carbon, nitrogen and total sulphur) volatilization and biodegradation of its light fractions
with a Perkin Elmer elementary analyzer. The remaining and an enrichment in resins and asphaltens. In fact, the
determinations also have been made in agreement with analyzed mixtures have changed its composition in high-
the standard procedures. The evolved gas analysis was car- molecular-weight heterocyclic hydrocarbons from 20% to
ried out with the HAL 200 mass spectrometer system from 25% in 2002 to 75% to 80% in 2005 (Table 1). Therefore,
Hyden Analytical previously referred. the samples which have been put under composting pro-
The properties of the end vermicompost that have been cesses fundamentally correspond to heavy fractions of the
assayed included measures of both conductivity and pH fuel-oil (asphaltens), hardly biodegradable with time.
and a test of compost maturity (a modified phytotoxicity Through experiences of composting of hydrocarbons
test employing seed germination) (Zucconi et al., from the Prestige oil spill (already aged and enriched in
1991a,b). It has been assayed with extracts to different con- asphaltens) with the agrochemical substrates previously
centrations: 10, 5 and 1 g of compost/100 mL of distilled referred and different types of fertilizers and thickening
water, respectively. In the tests, Watercress aleonis (aquatic agents, we have observed that the best results (evolution
watercress) seeds have been used. of the composting process) occur when cow bed and potato
For microbiological study, the samples of chapapote, peelings are used. Table 2 shows the three mixtures which
chapapote composted and chapapote vermicomposted were have been assayed with different substrates in order to
collected in sterilized glass bottles, which were filled up to know the most suitable for bioremediation of the asphal-
overflow with the emulsion, hermetically closed and stored tens. In other studies of ground decontamination via anaer-
at room temperature. The first stage of its processing con- obic (Stevens et al., 1990) it has been verified how the
sisted of its sowing (as in the original state as in sterile
water emulsions), on agar-blood plates and other media Table 1
for bacterial and fungal culture. At 24 h of incubation at Chemical composition of oil spills
37 C, growth of bacterial colonies, and accidentally fungi Saturated and aromatic Resins and
in some samples, were observed. The Vitek-2 from BioMér- hydrocarbons (%) asphaltens (%)
ieux was used in order to identify and analyze bacteria. Prestige (tank, 2002) 79.4 20.6
This system contains microformatted reagent cards for Prestige (fuel-in-water 74.8 24.2
selective biochemical tests. In addition, the Vitek-2 system emulsion, 2002)
Prestige (fuel-in-water 25–20 75–80
automates the front-end sample processing including initial emulsion, 2005–2006)
inoculum dilution, density verification, card filling and Erika 77.8 22.2
carding–sealing steps. The instrument transfers cards to Baltic Carrier 78.8 21.2
the reader-incubator and ejects them into a disposal bin Exxon-Valdez 90.0 10.0
at the completion of testing. The results provided by this
identification system have been correlated by us with basic
characteristics such as macroscospic morphology and Table 2
Gram staining results. Composition (percentage in weight, %) for the three assayed mixtures
For fungal study the samples were inoculated on Sabou- Mixture 1 Mixture 2 Mixture 3
raud agar with chloramphenicol (100 mg/L) and actidione Cow bed: 60 Cow bed: 55 Cow bed: 50
(500-mg/L) (Takashio, 1973). Inoculations were performed Rice husk: 5 Potato peelings: 25 Potato peelings: 33
in separate plates, which were incubated at 30 and 37 C. Seaweed extracts: 5
Pure fungal cultures were identified to genera or species Heavy fuel oil: 20 Heavy fuel oil: 20 Heavy fuel oil: 17
C/N: 55.7 C/N: 32.5 C/N: 21.1
level by their macro- and micromorphological characteris-
1824 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829
addition of non-soluble carbon sources (as starch) supports
the fast reduction of oxygen and favours the performance
of the associations of microorganisms, which degrade
nitroaromatic compounds (any, as stable as herbicide
dinoseb).
In photos 1–4 of Fig. 1, some experiences of evolution of
the process of composting of the fuel-oil in correspondence
with different types of tests are offered. Negative experi-
ences due to both an excessive initial stodgy and to an
excessive humidity (tend to a greater aggregate formation)
were illustrated in photos 1 and 2, respectively. Neverthe-
less, we can observe in photo 3 a compost correctly devel-
oped (it exhibits very few aggregates of big size) and in
photo 4, the evidence of a quasi-end process (it displays
mature parts of darker colour and the appearance of vege-
tation as a result of effectiveness: as very high temperatures
were not reached, the seeds ingested in its day by the cattle
were not killed).
Fig. 1. Follow up of the end product from composting process of fuel-in-
water emulsions from the Prestige oil spill in several assayed mixtures.
Fig. 2. Evolution of the temperature profile corresponding to the mesophilic (under 40 C) and thermophilic (above 40 C) phases of composting process.
Fig. 2 shows the evolution of the temperature corre-
sponding to the mesophilic and thermophilic stages of the
process of composting for three types of mixtures selected
during year 2005. It can be observed that only sample 2
is able to exceed the temperature of 40 C and to carry
out mesophilic, thermophilic and maturation phases. This
result can be referred to the inclusion of potato peelings,
a very easily biodegradable remainder which has been pre-
viously used by J.L González-Hurtado as inoculate in the
composting of wool wastes (personal communication).
The aerobic process, among other advantages, ensures
obtaining high temperatures, remarkably accelerates the
process in comparison to tests conducted in anaerobial
conditions (Stevens et al., 1990), reduces the volume in a
third part with respect to the initial mixture, and eliminates
non-wished scents, and pathogenic and parasitic agents.
Once the process of composting finishes, and in order to
optimize the bioremediation degree, a vermicomposting
process has been carried out, for mixture 2, during 6
months. Table 3 shows, to comparative effects, the
chemo-physical properties obtained for compost and
vermicompost.
The granulometric analysis has allowed us to calculate
the particle size of composted and vermicomposted prod-
ucts. Vermicomposted material has 2.3 mm as a geometric
average size and 1.9 mm as a standard geometric deviation.
Therefore, the size of grain of vermicompost is more stable
and homogenous than the one of compost (dimensions
5.1 mm and deviation 3.7 mm). This aim has been attained
due to the fact that Eisenia foetida can consume residual
organic matter very quickly, fragmenting it into smaller
particles, and producing much more degraded faecal
matter.
During the vermicomposting process, nutrients like
nitrogen, potassium, phosphorus, calcium and magnesium
are released and turned by the action of the present micro-
organisms into a more soluble and easy to assimilate forms.
The organic matter is also reduced considerably, which
implies that the composting, followed by vermicomposting
 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829 1825

Table 3 ductivity and pH are accurate for a ground conditioning

Analytical results from the composting and vermicomposting phases (on material. Regarding this aspect, it is noteworthy the high
dry matter)
index of germination in all the assayed extracts, which
Composting After the vermicomposting shows the high agronomic value of the finally obtained
phase process
item, susceptible to be used as a substitute of peat in
Humidity (%) 12.38 58.36 seedbeds.
Ashes (%) 46.98 55.8
Organic matter 53.02 44.2
Fig. 3 shows the evolution of asphaltens composition
(%) along the fuel-oil composting process. By means of a mass
Total N (%) 1.52 1.68 spectrometer Hyden 200 and from studies of thermal
P (%) 1.75 1.32 desorption, we have characterized, either in the aged oil
K (%) 2.0 0.15 fuel or in its degradation products, the presence of the fol-
Ca (%) 3.91 2.18
Mg (%) 1.0 0.40
lowing species: alkanes (n-hexane, C6H14; 2-methylpentane,
Cu (ppm) 32 40.65 C6H14; and phitane, C20H42); cycloalkanes or naphtens
Zn (ppm) 189 310 (cyclopentane, C5H10; cyclohexane, C6H12 and methylcycl-
Fe (ppm) 4486 9874.3 opentane, C6H12); aromatic hydrocarbons (tetraline,
Mn (ppm) 203 225.7 C10H12; acenaphten, C12H10; and methylnaften, C11H10)
Cd (ppm) n.d. n.d.
Cr (ppm) n.d. (25.4)
and polycyclics, such as pyrenes. Other sulphured non-
Pb (ppm) n.d. n.d. hydrocarbon systems, like thiol derivatives: ethanothiol,
Ni (ppm) n.d. n.d. C2H6S or dimethylsulphur, C2H6S; thiophen, C4H4S and
C/N ratio 19.38 14.62 benzothiofen C8H6S (Fig. 3), also appear coming from
the degradation of the asphaltens. In addition, other com-
pounds containing nitrogen like 2-methylpyridin, C6H7N;
Table 4
quinoline, C9H7N and indole, C8H7N; and compounds
Conductivity and pH results for different extracts in the Zucconi test
containing oxygen, like for example, estearic acid,
Extract of 1 g Extract of 5 g Extract of 10 g Blank
C18H36O2; cyclopentane carboxylic acid, C6H10O2; or phe-
Conductivity 247 354 490 nol, C6H6O, have been characterized.
(lS/cm)
By fragmentation and biodegradation of linear and
pH 7.74 7.82 7.78
GSN 9.25 9.0 9.0 9.25 branched hydrocarbons, hetero-polyaromatic and aro-
SAL (cm) 7.93 7.72 7.46 5.23 matic of the asphaltens, additional end products were orig-
GI (%) 151.41 134.29 138.71 inated: carbinol, catechol, benzoic acid and succinates. In
GSN, germinated seeds number; SAL, seed average length; GI, germina- the biochemical reactions that lead to these compounds,
tion index. omega, beta and aromatic oxidations take place, respec-
tively (Atlas and Bartha, 2002). A hypothetical structure
process, forms more and more stable products (Edwards of the asphaltens is depicted in Fig. 4, where susceptible
and Bohlen, 1996). regions of fragmentation and biodegradation (Pineda-Flo-
In Table 4, the results of conductivity and pH, and the res and Mesta-Howard, 2001) appear too. Likewise, in
results of the test of Zucconi are shown. The values of con- Fig. 5 some possible mechanisms of biodegradation for

Fig. 3. Mass spectra of aged (2005) fuel-in-water emulsions from the Prestige oil spill. The peaks at m/z: 69 and 75 correspond to benzothiophene.
1826 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829
Fig. 4. Regions susceptible of fragmentation and biodegradation in an asphaltene molecule. 1: photo-oxidation; 2: beta-oxidation; 3: dibenzothiophene
metabolic path; 4: path similar to dibenzothiophene; 5: pyrene path; 6: path similar to benzo(a)pyrenes; 7: similar to carbazols.
Fig. 5. Metabolic route involved in dibenzothiophene biodegradation
(Adapted from van Afferden et al., 1990).
dibenzotiofen and pyrenes are shown (van Afferden et al.,
1990).
The identification of intermediate or final products of
biodegradation of the asphaltens is being the object of
study (using CG–Mass chromatography) by our research
group, in order to identify if they remain in the phases of
composting or vermicomposting. Thus, by the absence in
the mass spectra of the most representative peaks of benzo-
thiophen (m/z: 69, 92, 108 and 135) we have been able to
state its absence in composted products. However, other
analyses of the samples have demonstrated the presence
of asphaltens adsorbed in final humus (10% in the compo-
sting process and 5% during the vermicomposting process).
This feature has also been observed in other studies: Barker
and Bryson (2002) evidenced partial adsorption of polynu-
clear aromatic hydrocarbons in non-accessible sites of the
material to composting, therefore, preventing the corre-
sponding microbial attack.
Alternative physical phenomena to microbial transfor-
mation as adsorption, dispersion, dissolution, evaporation
and photo-oxidation take part in the fuel-oil biodegrada-
tion processes. As time goes by, all these mechanisms con-
tribute effectively to the fuel-oil decay, but we considered
that the most effective physical phenomenon is photo-oxi-
dation. Hence, we have decided to complete the degrada-
tion of the asphaltens adsorbed in the final vermicompost
(5%) by means of solar light and/or UV radiations. The
exposition to this type of light facilitates the conversion
of hydrocarbons into compounds with functional groups
that contain oxygen, such as alcohol, ketones, aldehydes,
carboxylic acids and peroxides.
The microbiota, which was identified in both the com-
posted emulsion and the vermicomposted emulsion, is
given in Table 5. In a previous paper on the microbiota
that colonize the chapapote from the Prestige spill (Mar-
tı́n-Gil et al., 2004) aerobic gram-negative rods (Aeromonas
caviae, Enterobacter amnigenus, Kebsiella pneumoniae and
 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829
Table 5
Bacterial and fungal communities found in fuel-in-water emulsions from
the Prestige’s spill after composting (green compost and vermicompost)
Compost Vermicompost
Bacteria Bacillus spp. Bacillus spp.
Stenotrophomonas maltophilia
Fungi Scopulariopsis spp. Aspergillus niger
Scedosporium apiospermium Scedosporium apiospermium
Proteus mirabilis), anaerobic gram-negative rods (Bacteroi-
des spp.), gram positive rods (Bacillus spp.), gram positive
cocci (Peptostreptococcus spp.) and the non-fermentative
gram-negative rod Shewanella putrefaciens were identified.
Among the species that now have been characterized we
would like to emphasize the presence of both the bacteria
Stenotrophomonas maltophilia and the fungi Aspergillus
niger and Scedosporium apiospermium in the vermicompost,
and the existence of fungi Scopulariopsis in the compost.
S. maltophilia (Xanthomonas maltophilia or Pseudomo-
nas maltophilia) is a bacterial species that resides in the coe-
lomic cavity of the earthworms, and therefore it is strongly
connected with the vermicompost. It showed high efficiency
in the bioremediation of polynuclear aromatic hydrocar-
bons (Lowry and Hurley, 2005).
Aspergillus niger has been included among cellulolytic
microorganisms that help in hastening the manure decom-
position in the vermicompost procedures. It is usually inoc-
ulated in a predecomposition phase because it does not
only improve the quality of product but also reduces stabil-
ization period (Singh and Sharma, 2002).
The genus Scedosporium comprises a group of filamen-
tous fungi found ubiquitously in the environment: soils,
decaying plants matter, polluted water, manure and com-
post. The two major human pathogens within this genus
are S. apiospermum the asexual state of Pseudallescheria
boydii and Scedosporium prolificans. Both histologically
resemble Aspergillus species, with hyphae that are septated
and branching at acute angles. From a clinical view, Sced-
osporium species are known to be largely resistant to tradi-
tional antifungals such as amphotericin B. Nevertheless,
treatment with newer triazoles, such as voriconazole,
appears to be more efficacious. Scedosporium has been
reported as a hyphomycete with the ability to utilize phe-
nol, p-cresol and toluene as carbon and energy source
(Claussen and Schmidt, 1998; Garcı́a-Peña et al., 2001).
The catabolism proceeds via three different dihydroxylated
intermediates (catechol, hydroxyhydroquinone and protoc-
atechuate) which were intradiolically cleaved by the corre-
sponding inducible dioxygenases and further catabolized
via the 3-oxoadipate pathway (Claussen and Schmidt,
1998).
Scopulariopsis sp. is also an ubiquitous fungus, and can
be found on a wide variety of materials including old car-
pets and water-damaged wallpaper. According to Anastasi
et al. (2005) Chrysosporium and Scopulariopsis are the pre-
dominant species in composting. The Scopulariopsis strains
exhibited only endomannanase activity (Mudau and Setati,
1827
2006) when the substrate, as the chapapote, has a marine
origin.
The microbiota of the original samples of fuel-in-oil
emulsion and the resultant in both compost and vermicom-
post only showed a bacterial genus in common: Bacillus sp.
The wide spectrum of Bacillus species isolated (B. subtilis,
B. megaterium, B. licheniformis, B. coagulans, B. polymyxa)
indicates the richness of easily decomposable compounds
in the fresh and processed fuel-in-oil substrate but does
not allow to obtain conclusions on the efficiency of such
species to bioremediation purposes (only Shewanella putre-
faciens has been proved usefulness). On the other hand, an
important feature observed in the processed chapapote is
that the abundance and frequency of occurrence of bacteria
of the genus Bacillus was lower in vermicompost than in
compost. This finding is favourable because Bacillus spe-
cies, which are reported as common components of the
gut-microbiota of Eisenia foetida (Berra et al., 1998), inhi-
bit spore germination and mycelial growth of fungi which
are probably efficient agents in remediation. On the con-
trary, the metabolites from bacteria of the genus Pseudo-
monas, which were isolated from vermicompost, activates
spore germination and mycelial growth of fungi as Asper-
gillus niger (Berra et al., 1998). Thus, we believe that the
presence of this microbiota in our vermicompost is a
unique and remarkable environment where the interaction
between Stenotrophomonas maltophilia and Aspergillus
niger takes place.
The effectiveness obtained in our vermicomposting expe-
riences is evident (the percentage of nitrogen, phosphorous
and potassium increased while pH and total organic car-
bon declined as a function of the vermicomposting period;
and the shape of the samples changed from asphaltenic to
tobacco-like) but such efficiency should not be assigned
only to the bacterial action of Stenotrophomonas malto-
philia. Some S. maltophilia strains are known to be effective
without apparent association to other factors (e.g., the
GB1 strain that was isolated from a Nigerian well in the
biodegradation of weathered heavy crude oil reported by
Okoh et al., 2000); and another one that seems to carry dib-
enzothiophene desulphurization genes (Duarte et al., 2001),
but these are isolated cases. Moreover, the S. maltophilia
potential to degrade diesel fuel has been questioned in
one case (Saadoun, 2002) and in another one, presumable
antagonistic effects to compost have been reported (Litter-
ick and Harrier, 2004). Consequently, we hypothesized that
the bacterial action of S. maltophilia is in conjunction with
Eisenia foetida and fungi. Certainly, it could be an error to
reduce the Eisenia foetida just to be either a simple vector
or a simple digester. The work of Bilej et al. (1995) and
Bolotetskii and Kodolova (2002), enhancing the biochemi-
cal actions of Eisenia foetida, suggest specific actions.
As far as concerns to the fungi found in the composted
samples, they must be related to the respective composting
processes. The presence of Scopulariopsis sp. in composted
samples is an expected feature (Anastasi et al., 2005) like so
it is their absence from our vermicomposted samples.
1828 J. Martı́n-Gil et al. / Bioresource Technology 99 (2008) 1821–1829
Moreover, Scopulariopsis seemed to be effective in inhibi-
tion of vermicompost (Hu et al., 2003) and now is used
to control pests (Padmanaban and Sathiamoorthy, 2004;
DARE/ICAR, 2004). Scedosporium apiospermum has been
reported in compost and self-heating organic materials at
mesophilic and thermophilic temperatures (Ryckeboer
et al., 2003) and it has been included among the keratolytic
fungi found in soil heavily contaminated with petroleum
during bioremediation (Ulfig et al., 2003).
An intriguing result of our work has been the finding of
Aspergillus niger in our vermicomposted samples. It was
shown that the earthworm Eisenia foetida preferred dark-
coloured fungi (Cl. cladosporioides, Al. alternata) as a food
but also has been reported that the substrata with the addi-
tion of mycelium of Aspergillus niger were not attractive for
Eisenia (Marfenina and Ishchenko, 1997). On the other
hand, the presence in the vermicompost of Aspergillus niger
cannot be attributed neither to the substrate (because in the
original fuel-in-water emulsion from the Prestige oil spill
no fungi was characterized) nor to its harbouring by the
intestines of Eisenia foetida (only A. fumigatus, Mucor cir-
cinelloides F. circinelloides and P. expansum seemed domi-
nant in the intestines of earthworms) (Pizl and Nováková,
2003). Thus, in order to justify the presence of this oppor-
tunistic microfungi we suggest biotic factors as the special
Pseudomonas–Aspergillus niger interactions above related
or the vermicomposting conditions (characterized by high
temperatures and neutral pH) that make its growth
favourable.
Finally, we think that the results in chapapote vermi-
composting are a consequence of the combined action of
Eisenia foetida and microorganisms living in both earth-
worm intestines (S. maltophilia) and vermiculture sub-
strates (Scedosporium apiospermium). Such action should
be referred to the enzymatic action of peroxidases, leuci-
neaminopeptidases, phosphoglucomutases and octanolde-
hydro-genases from the reported biological consortium
(Claussen and Schmidt, 1998; Bolotetskii and Kodolova,
2002).
4. Conclusions
The most important conclusion of this work is that the
application of composting techniques, followed by vermi-
composting techniques, constitutes an effective alternative
for the treatment and elimination of the heaviest fractions
of petroleum (resins and asphaltens). The obtained end
product has contents of interesting vegetal nutrients and,
mainly, it displays very high germination indices.
Additionally, on the basis of the obtained results, we can
also conclude with other authors that the microbial part-
nerships are susceptible to become acclimated to petroleum
and inclusively to degrade their more recalcitrant products.
Using substrates that act co-metabolically, it is possible to
degrade and to eliminate the polycyclic asphaltens and vol-
atile hydrocarbons into CO2 and H2O, helped by the evap-
oration, dissolution and/or photo-oxidation processes.
Acknowledgement
Authors gratefully acknowledge Dr. Juliana L. Gonzá-
lez-Hurtado from the Department of Agroforestal Sciences
of the University of Valladolid for their helpful discussions.
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