Vol.2, No.2, 104-1
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
10 (2011) Agricultural Sciences
Assessment of microbial pools by an innovative
microbiological technique during the co-composting of
olive mill by-products
Teresa Casacchia1, Pietro Toscano1, Adriano Sofo2*, Enzo Perri1
1CRA, Centro di Ricerca per l’Olivicoltura e l’Industria Olearia, c. da Li Rocchi-Vermicelli, Rende, Italy; teresa.casacchia@entecra.it;
pietro.toscano@entecra.it; enzo.perri@entecra.it
2Dipartimento di Scienze dei Sistemi Colturali, Forestali e dell’Ambiente, Università degli Studi della Basilicata, Potenza, Italy;
*Corresponding Author: adriano.sofo@unibas.it
Received 25 January 2011; revised 9 February 2011; accepted 2 March 2011.
Different mixtures of olive pomace (OP), olive
mill wastewater (OMWW) and olive pruning
residues (OPR) were aerobically co-composted
under natural conditions. Compost temperature
showed a sharp increase in the first 40 - 60 days,
followed by a stabilization at 60°C and a decline
after 150 days, whereas compost water content
ranged from 50% - 55% to 25% - 30%. Total and
selective microbial counts were followed
throughout the experiment by means of innova-
tive (IMT) and conventional (CMT) microbiologi-
cal techniques. Pseudomonas spp., anaerobic
bacteria, actinomycetes, and fungi reached lev-
els of 8, 7, 5 and 6 log CF U g–1 compost, respec-
tively, with a slight depression after 30-80 days.
Total and fecal coliforms strongly decreased
during the composting process. The use of IMT
allowed to detect a higher and more stable
growth of microorganisms if compared to CMT.
IMT was demonstrated to be an appropriate and
reliable method for monitoring the microbial
pools during the co-composting process.
Keywords: Composting; Olive Mill Wastewater;
Olive Pomace; P runing Re sidues
The olive pomace (OP), also called ‘sansa vergine’ in
Italian or ‘orujo’ in Spanish, is defined as the residue
that remains after the first oil extraction from olives
(crude olive cake). The OP is a dry material with a 8% -
10% moisture and is composed of ground olive stones
and pulp, with a high lignin, cellulose, and hemicellu-
loses content, and a 3% - 5% oil content, depending on
the olive mill typology (pressure or centrifugation) [1].
This by-product is usually used for residual oil extrac-
tion by solvents, heating, animal feed supplement, or as
an organic amendant for olive grove or other crops soils
[2]. In terms of agronomic value, OP watered with olive
mill wastewater (OMWW), another product of olive
milling, or with other organic material lead to a product
that supplies nutrients to plants and is an efficient
method for the disposal of olive mill residuals [3,4].
According to the Italian law 574/1996, it is possible to
use not-composted OMWW and OP for agronomic pur-
poses, as they are considered simple plant amendants,
with no limitations on the amounts of OP to be applied
to the soil, but the CEE Regulation 91/156 indicates that
composting is one of the methods to recycle and recov-
ery organic wastes.
Olive-mill wastewater (OMWW) is composed by the
own water of the olives (vegetation water) and the water
used in the different stages of oil elaboration [1]. From
an environmental point of view, OMWW is an environ-
mental emergency as it has a considerable polluting or-
ganic load, with a maximum biological oxygen demand
and chemical oxygen demand of about 100 and 220
kg·m–3, respectively, an average concentrations of vola-
tile solids and inorganic matter of 15% and 2%, respec-
tively, and organic matter fraction that includes sugars,
tannins, polyphenols, polyalcohols, pectins and lipids [5].
Therefore, a series of studies focused on the degradation
of OMWW and its chemical components [5-8], and
many authors used specific microorganisms for OMWW
treatment [9-13]. Microbiological and physicochemical
parameters were used as indicators to study the kinetic
of OMWW biodegradation, such as chemical oxygen
demand (COD), dissolved organic carbon, counts of
heterotrophs, filamentous fungi, and yeasts, and content
of K, P and N [8-14]. As OMWW does not generally
contain sufficient N and P for an adequate aerobic puri-
fication process, OMWW degradation may be performed
T. Casacchia et al. / Agricultural Sciences 2 (2011) 104-110
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by co-composting, anaerobic digestion or enzymatic
treatment [8-15]. Some authors obtained satisfactory
results, in terms of OMWW degradation and ameliora-
tion of soil physicochemical properties, by adding this
liquid waste to agro-industrial and urban wastes and
monitoring the physicochemical parameters during the
composting process of these matrices [16-18]. Angeli-
daki and Ahring [19] studied a combined anaerobic di-
gestion of OMWW together with manure, household
waste or sewage sludge, and with this method they
managed to degrade OMWW without previous dilution,
without addition of external alkalinity and without addi-
tion of external nitrogen source. Other authors [3] effi-
ciently monitored over four months a compost made of
OP, OMWW and poultry manure, by following tem-
perature, pH, humidity and C/N ratio, in order to ascer-
tain its maturity, and tested its effectiveness in increasing
potato agronomic production. Furthermore, the co-
composting of exhausted olive cake with poultry manure
and sesame shells was recently investigated, and this
process was followed by studying some physicochemical
parameters [4].
Generally, the study on composting process of olive
mill by-products was focused on their physicochemical
aspects. On this basis, the present study was performed
to evaluate if mixtures of olive mill pomace, olive mill
wastewater and olive pruning residues (OPR), without
the adding of any other additive external to olive grove,
can be efficiently composted under “in farm”, non-in
dustrial procedures conditions, based on spontaneous
aerobic degradation by autochthonous microorganisms.
This methods of compost production needs limited re-
sources, low energetic inputs, and uses machinery and
equipment often already present in the farm. As informa-
tion on selective media for the microorganism responsi-
ble for the spontaneous aerobic degradation of compost
deriving from different olive material, and in particular
from OP, is lacking, we have tested an innovative micro-
biological technique (IMT) based on microorganisms
cultivation using a broth extracted from the matrix to be
composted. This method could be used to monitor the
biomass degradation process during OP co-composting.
2.1. Compost Production
The composting trials were carried out in a three-year
period (2006-2008) under “in farm” and open field en-
vironmental conditions located in San Demetrio Corone
(Southern Italy, Calabria Region, 39°34’ N, 16°21’ E).
The matrix for compost production included: olive po-
mace (OP) and olive mill wastewater (OMWW) deriving
from a three-phases olive mill, and rain water, added
with grinded olive branches and leaves deriving from
pruning residues (OPR). OPR were added in order to
give a better structure of the composting matrices.
Depending on the by-products availability in the ex-
perimental field, the matrix composition in the three years
was, respectively:
year 2006 (from 17 March to 4 October): 200 q OP + 8
m3 OMWW + 20 q OPR;
year 2007 (from 14 March to 28 September): 300 q OP
+ 80 q OPR;
year 2008 (from 21 March to 30 September): 400 q OP
+ 5 m3 OMWW + 250 q OPR.
The matrix, uniformly mixed to form a trapezoidal
parallelepiped pile (volume = 52, 60 and 129 m3 in 2006,
2007 and 2008, respectively) placed outdoor in open field,
resulted to have a semi-solid consistency. It was blended
with a mechanical shovel every 7 days, in order to ensure
the aeration and control the warmth of the biomass, and
was subjected to a spontaneous degradation by autoch-
thonous microorganisms. The aeration allowed to reduce
anaerobic fermentation and the consequent high produc-
tion of putrefactive, toxic compounds for aerobic micro-
Compost maturation was followed by weekly sam-
plings to monitor compost temperature (CT) and compost
water content (CWC). The values of CT were measured
by a digital thermometer (model 46908; TR snc, Italy).
The values of CWC were determined from the weight
differences of compost samples before and after drying at
105˚C for 24 h, and expressed as percentages of water on
dry matter. The mean chemical parameters measured at
the end of the composting process in the three years were
the following: humidity = 27.5 ± 1.48% (SD), pH = 7.05
± 0.35, C/N = 24.15 ± 1.06, total organic carbon = 31.4 ±
0.99%, organic matter = 54 ± 1.70%, N = 1.3 ± 0.10%,
P2O5 = 0.7 ± 0.03%, K2O = 1.15 ± 0.10%.
Approximately every 25 days, compost samples (5 kg)
were randomly collected in different parts and depths of
the pile during reversal procedures using sterile gloves,
placed in sterile plastic bags, and stored in a refrigerated
box at 4˚C for 1 h. Before the microbiological analyses,
compost samples were sieved at 4 mm and successively
at 2 mm, to eliminate the roughest fractions.
2.2. Microbiological Analysis
The screening of microbic pools has been carried out
at different steps of composting process. Two methods
were utilized: an innovative microbiological technique
(IMT) here applied for the first time, and a conventional
microbiological technique (CMT) by methods usually
adopted in soil analysis [20].
For CMT, a 10 g aliquot of compost was mixed to 90
ml of sterile peptoned water (triptone 10 g·L–1 and so-
dium chloride 5 g·L–1; pH 7.2) and diluted up to 108 in a
T. Casacchia et al. / Agricultural Sciences 2 (2011) 104-110
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
physiological solution. Microbial community has been
assessed through plate-counter and expressed as col-
ony-forming units (CFU) per mL. Serial dilutions, using
peptoned water as diluent, were prepared to obtain CFU
counts in the range of 30 - 300 per plate and 100 µl of the
corresponding decimal dilutions were plated in triplicate
the following specific nutritive media: Plate Count Agar
(PCA; Oxoid Lim., Hampshire, UK) for total cultural
bacteria; Yeast Dextrose Agar (YPD: 1% yeast extract, 2%
peptone, 2% glucose) supplemented with 150 ppm
chloramphenicol (Sigma-Aldrich, MO, USA) for moulds
and yeast; Pseudomonas Agar Base medium (Oxoid) with
the addition of Pseudomonas C-N supplement (Oxoid) for
Pseudomonas spp.; PCA under anaerobic conditions for
anaerobic bacteria; Violet Red Bile Agar (VRBA; Merck
and Co. Inc., NJ, USA) for total coliforms; and Slanetz
and Bartley medium (Merck) for fecal coliforms.
All plates were incubated at 25˚C, as this temperature
is adequate for mesophilic microorganisms [20], with the
exception of fecal coliforms, that were incubated at 44˚C.
Microbial counting took place after 24, 36 and 48 h.
For IMT, a 10-g aliquot of compost was mixed to 90
ml of sterilized olive matrix broth (OMB), extracted
from the matrix. The OMB was obtained by melting 1
kg of compost matrix in 3 L of sterile de-mineralized
water. The 1:3 ratio was chosen on the basis of various
trials carried out in our laboratory. The solution was fil-
tered with a cheesecloth, and successively sterilized at
121˚C for 20 min, in order to obtain the final OMB. For
the microbiological analyses of each compost sample,
the OMB used was obtained from that specific sample.
The values of pH in the OMBs used ranged from 6.7 to
7.2, with a mean of 7.0 ± 0.2 (SD). For IMT, OMB was
utilized as a diluents both for scalar dilutions and sub-
strate preparation. The serial dilutions procedures and
the growth media used were the same of CMT. Each
measurement was replicated three times and the mean
values (± SD) were calculated.
2.3. Statistical Analysis
Data were treated by analysis of variance (ANOVA)
using the SAS software (SAS Institute, NC, USA) in
order to detect significant differences (PROC GLM).
A new approach in fruit orchard management is im-
posed by environmental emergencies, such as soil deg-
radation, water shortage and greenhouse effect [21]. In
particular, in olive groves a positive influence of sus-
tainable orchard management, including pruning residues
re-use and compost application, on soil biochemical cha-
racteristics and soil microbial diversity was recently ob-
served [22,23]. Among the agronomic sustainable prac-
tices, the input of soil organic matter as compost is one of
the most important factor affecting soil fertility, in terms
of enhancement of soil permeability and water retention,
better endowment and availability of nutrients for plants,
higher CO2 uptake and carbon fixation, and reduction of
soil erosion [23-26].
In all the three years of the experiment, the CT values
of the matrices showed a similar trend, with a sharp in-
crease in the first 40 - 60 days followed by a stabilization
(Figure 1). This period, characterized by high values of
CT, corresponds to the so called active composting time
(ACT), the phase of bio-oxidation, during which the de-
gradation processes of labile organic components facili-
tated by pile aeration occurs, with high heat release and
oxygen consumption [2,15].
During the ACT, CT remained quite stable for about
40-50 d, reaching maximum values of about 60˚C (Fig-
ure 1). These high temperatures allows compost hy-
gienization during the ACT and thus are an important
requisite for the following utilization of the compost. The
bio-oxidative phase was considered finished after 140,
160 and 155 days in 2006, 2007, 2008, respectively, when
CT started to decrease and reheating did not occur (Figu
Figure 1. Compost temperature (black circles) and compost
water content (white circles) during the year (a) 2006, (b) 2007
and (c) 2008. The values represent the averages (± SD) of 10
independent replicates.
T. Casacchia et al. / Agricultural Sciences 2 (2011) 104-110
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
re 1). In fact, during the following maturation time
(curing), degradation processes and heat production fin-
ish, whereas the biosynthesis of humic compounds occurs
by fungi [24]. As for CT, the values of CWC showed
similar trends in the three years, starting from values of
50-55% and reaching values of about 25% - 30% at the
end of curing, with a progressive and gradual decrease
throughout the degradation process (Figure 1).
While some authors used OMWW as a substrate for the
production of enzymes and organic compounds by mi-
croorganisms [27,28], the microbiological application of
sterilized compost extracts were never studied. The use of
IMT allowed to detect a higher growth of microorganisms
if compared to CMT (Figur e 2), likely due to the fact that
OMB is richer of nutrients if compared to peptoned water,
and its compositions is similar to that usually experienced
by the microorganisms living in the compost. Another
Figure 2. (a) Total cultural bacteria, (b) Actinomycetes, and (c)
yeasts and moulds counts (read after a 36-h incubation) using
the innovative microbiological technique (IMT; dashed lines) or
the conventional microbiological technique (CMT, continuous
lines). Experimental period: 2006 (black circles), 2007 (white
circles) and 2008 (triangles). The values represent the averages
(± SD) of three independent replicates. All the IMT and the
respective CMT resulted to be significantly different (P 0.05).
advantage is that the cultures based on OMB showed
more stable and comparable values both throughout the
experimental period and between the three years (Figure
2), a basic requisite for their use as bio-indicators of the
composting degradation process. The trends of microbial
number detected by IMT (Figures 2 and 3), and in par-
ticular the kinetics of total and fecal coliforms (Figure
3(c,d)), indicate that this technique could be also adopted
for olive grove soils, and urban and industrial sludge, two
Figure 3 . (a) Pseudomonas spp., (b) anaerobic bacteria, (c) total
coliforms, and (d) fecal coliforms counts (read after a 36-h
incubation) using the innovative microbiological technique
(IMT; dashed lines) or the conventional microbiological tech-
nique (CMT, continuous lines). Experimental period: 2006
(black circles), 2007 (white circles) and 2008 (triangles). The
values represent the averages (± SD) of three independent rep-
licates. All the IMT values and the respective CMT values
resulted to be significantly different (P 0.05), with the excep-
tion of those with the asterisk on the top.
Total culturable bacteria
(log CFU g–1 compost)
(log CFU g–1 compost)
(log CFU g–1 compost)
Pseudomonas spp.
(log CFU g–1 compost)
Anaerobic ba cteria
(log CFU g–1 compost)
Total coliforms
(log CFU g–1 compost)
Fecal coliforms
(log CFU g–1 compost)
T. Casacchia et al. / Agricultural Sciences 2 (2011) 104-110
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matrices that host microorganisms living in conditions
similar to those studied in this paper [3,8].
Total cultural bacterial counts determined by IMT did
not significantly differ during the three years (Figure
2(a)), so highlighting that bacterial growth is not inhib-
ited by the different percentages of OMWW, a by-product
with a very high concentration of polyphenols [1,4]. Ac-
tinomycetes (e.g., Streptomyces), fungi produce a number
of enzymes that help degrade organic plant material, such
as lignin and chitin, and so are abundant in soils rich of
organic matter [20]. Furthermore, actinomycetes are able
to use root exudates as carbon source, supply roots with
easily assimilable nitrates, and are involved in the sup-
pressive action of the soil [23]. Actinomycetes (Figure
2(b)), and fungi (Figure 2(c)), counted by IMT, reached
levels of about 5 and 6 log CFU g–1 compost, respectively.
The use of specific cultural media allowed the isolation
of important physiological groups of microorganisms
related to compost maturation (Figure 3). Pseudomonas
spp. and anaerobic bacteria (e.g., Bacillus spp.) are the
main decomposers of complex polymers, such as ligno-
celluloses and chitin, but they also have proteolytic en-
zymes and are of key-importance important for the pro-
duction of assimilable nitrogen in soils [20]. The ob-
served trends of microbial counts using IMT, show that
Pseudomonas spp. (Figure 3(a)) and anaerobic bacteria
(Figur e 3(b)) reached levels of about 8 and 7 log CFU g–1
compost, respectively. The slight decrease of the micro-
bial numbers of these two groups during the first 30-40
days (Figure 3(a,b)) was likely due to the increase of
temperature [1]. Moreover, this depression started earlier
if compared to Actinomycetes, and yeasts and moulds,
that declined during the first 70-80 days (Figure 2(b,c)).
The high number of Pseudomonas spp. observed
throughout the composting process (Figure 3(a)) indi-
cates that these microorganisms are affected by the or-
ganic content of the matrix and are able to initiate their
oxidative biodegradation without being particularly in-
hibited by polyphenols. The presence of anaerobic bac-
teria (Figure 3b) indicates that, considering the size of
the compost pile and the high microbial activity, with the
consequent strong oxygen demand, mechanical aeration
did not allow the complete removal of anaerobic degra-
dation processes.
On the other hand, the use of compost in agriculture is
often associated with health risks because of the possible
presence of human pathogens, enteric in origin, such as
bacteria, viruses, protozoa and helminthes [25]. The
number of total and fecal coliforms strongly decreased
during compost maturation (Figure 3(c,d)), so attesting
the efficiency of degradation process and the good hygi-
enic conditions of the matrix. In fact, coliform bacteria are
of key importance because the low number or the absence
Figure 4. Total cultural bacteria counts read after an incubation
time of 12 h (black columns), 24 h (white columns) and 36 h
(grey columns) by using (a) the innovative microbiological
technique (IMT) or (b) the conventional microbiological tech-
nique (CMT). The values represent the three-years averages (±
SD) of three independent replicates. All the values obtained by
12-h incubation time were significantly different (P 0.05)
from those of 24-h and 36-h incubation times, whereas no sig-
nificant differences (P 0.05) were found between the values of
24-h and 36-h incubation times.
of this group of micro-organisms indicates compost heal-
thiness and safety [25]. Notwithstanding the widespread
use as amendants of olive mill residues and by-product in
Mediterranean countries, legislative limits for compost
deriving from these matrices are lacking. The only micro-
biological threshold values for compost application in
agriculture, present in the EC Regulation 1774/2002, are
1000 CFU g–1 compost for both Escherichia coli and
Enterococcus spp., and 0 CFU g–1 compost for Salmo-
nella spp. Thus, we suggest that the use of total and fecal
coliforms could be efficacious bio-indicators, more spe-
cific and less variable than the microbial groups listed in
EC Regulation 1774/2002.
Our investigation also focused on the more appropriate
incubation time before colonies counting. In Figure 4 we
reported the averages total cultural bacterial counts calcu-
lated from the values of the three years, determined by IMT
(Figure 4(a)) and CMT (Figure 4(b)). For both CMT and
IMT, a counting time of 36 h was the most reliable, as total
and specific microbial counts ranged between 4 and 9 log
CFU g–1 compost. In fact, the range of microbial number
after 24 h was lower (between 0 and 2 log CFU g–1 com-
post), whereas after 48 h it did not vary significantly from
the that obtained after a 36-h incubation.
Total culturable bacteria (log CFU g–1 compost)
T. Casacchia et al. / Agricultural Sciences 2 (2011) 104-110
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Our experiment demonstrated that sterilized olive ma-
trix broth can represent an efficacious diluent to be used
for monitoring the microbial pools during the co-com-
posting of olive mill by-products. The results also show
that the aerobic stabilization of olive pomace, suitably
co-composted with olive mill wastewater and/or other
crop by-products, is a real sustainable agronomical prac-
tice, that allows to obtain a low-cost stabilized organic
amendant, in line with the legislative parameters of mixed
composted amendants (Italian law 748/1984). The ap-
plication of this product could recovery, maintain or in-
crease soil fertility, with environmental benefits and
positive influence on crop yields.
Authors thanks to Mr. Carlo Priori, owner of composting site, for his
hospitality and support during the experimental field activities.
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