lnfernarional
PII:
SO964-8305(96)00053-4
Biodeteriorarion & Biodegradafion (1996) 21 I-214
0 1997 Published by Elsevier Science Limited
Printed in Great Britain. All rights reserved
0964-X305/96 $15.00 + 0.00
ELSEVIER
The Study of the Aerobic Bacterial Microbiota
and the Biotoxicity in Various Samples of Olive
Mill Wastewaters (Alpechin) during their
Cornposting Process
M. Monteoliva-SHnchez,”
C. Incerti,” A. Ramos-Cormenzana,”
C. Paredes,’ A. Roigb & J. Cegarra”
“Department of Microbiofogy, University of Granada, 18071 Granada, Spain
hDepartment of Soil and Water Conservation and Organic Waste Management, Centro de Edajologia ,v Biologia Aplicada de1 Segura.
CSIC P.O. Box 4195, 30080 Murcia. Spain
Five different piles were prepared by mixing olive mill wastewater (alpechin) and
alpechin sludge with two bulking agents (cotton waste and maize straw) and two
organic wastes with high content of nitrogen (sewage sludge and poultry
manure), which were composted by the Rutgers static pile composting system in a
pilot plant. The aim of this work was to study the evolution of total nitrogen and
different forms of organic matter and evaluate the variation in the aerobic
bacterial microbiota present and biotoxicity during the composting process.
In piles prepared with alpechin, the use of the maize straw as a bulking agent
reduced the nitrogen losses whereas the use of sewage sludge, instead of poultry
manure, with cotton waste originated the highest degradation of organic matter.
In piles prepared with alpechin sludge a similar evolution of the composting
process was observed. There were not great variations during composting in the
aerobic bacterial microbiota present in the mixtures. However, the pile prepared
with alpechin sludge and maize straw was only one to present bacteria capable of
growing in alpechin, and the toxicity study showed that this was only present in
the starting mixtures. 0 1997 Published by Elsevier Science Limited. All rights
reserved
INTRODUCTION
The expansion
of the agroindustry has grown
enormously over the last decades, this has led to a
higher production of organic wastes. One of these
agroindustrial
organic wastes is the olive mill
wastewater (alpechin). The production
of this
waste in Mediterranean countries is very high,
about 30 millionm3year-‘,
and is generated over
a brief period of the year (November-February).
In spite of the existing laws, alpechin is often
disposed of in the environment, although it is
mainly collected in lagoons with a consequent
pollution linked to odours, insect proliferation
and sludge production.
Composting is considered as one of the most
suitable ways of disposing of unpleasant wastes
and of increasing the amount of organic matter
that can be used to restore and preserve the
environment (Stentiford, 1987). The cornposting
process is a controlled bio-oxidative process that
involves a heterogeneous organic substrate in the
solid state; evolves by passing through
a
thermophilic phase and a temporary release of
phytotoxin; and leads to production of CO*, water
vapour, mineral products and stabilized organic
matter (Zucconi and De Bertoldi, 1987). For this
reason, cornposting is a practical and ecologically
sound way of recycling alpechin, since by means
of this process it is possible to transform alpechin
and alpechin sludge added to a bulking agent into
organic fertilizers or soil amenders with no
phytotoxic effects.
The aim of this work is to study the evolution of
organic matter, organic carbon, total nitrogen,
lignin, a-cellulose and hemicellulose
and to
evaluate the variation in the aerobic bacterial
microbiota present during the cornposting process
M. Monteoliva-Sinchez
212
of alpechin and alpechin sludge mixed with other
organic wastes. The biotoxicity by means of the
‘Microtox’ assay is also investigated.
MATERIALS
AND METHODS
Five different mixtures were prepared
following wet weight percentages:
in the
Pile 1: 65.4% cotton waste+ 34.6% poultry
manure + 1.93 1kg-’ alpechin
Pile 2: 67.9% cotton waste+ 32.1% sewage
sludge + 0.941 kgg’ alpechin
Pile 3: 52.9% maize straw + 47.1% sewage
sludge + 1.77 1kg-’ alpechin
Pile 4: 20% cotton waste + 80% alpechin sludge.
Pile 5: 11.1% maize straw + 88.9% alpechin
sludge
were composted in trapezoidal piles of I-1.5m high
with a 2x3m base in a pilot plant. The Rutgers
static pile composting system was used, involving
on-demand
ventilation
through
temperature
feedback control (Finstein et al., 1985). The timer
was set for 30s ventilation every 15mir-1, and the
ceiling temperature for continuous air blowing was
Table 1. Changes
in the Chemical
Composition
of the Different
Pile
1
Organic matter (%)
Organic carbon (%)
Total nitrogen (%)
C/N
Lignin (%)
a-Cellulose (%)
Hemicellulose (%)
Piles Prepared
with Olive Mill Wastewater
During
time (days)
0
21
49
mature
78.42
40.72
2.71
15.0
25.97
30.42
27.93
69.63
35.95
3.12
11.5
30.73
18.57
19.57
63.13
33.43
3.46
9.7
31.73
10.68
17.93
62.90
33.73
3.47
9.7
31.92
12.34
17.07
42
84
mature
61.47
33.31
3.04
11.0
31.99
13.53
15.96
56.33
30.08
2.99
10.1
31.26
9.55
14.50
56.43
29.37
3.11
9.4
31.76
f 1.37
13.99
28
63
mature
82.95
40.88
2.22
18.4
27.57
25.06
79.68
41.54
3.00
13.8
29. f 1
20.16
74.75
39.43
3.33
11.8
30.91
16.47
80.74
40.54
1.92
21.1
23.26
36.28
22.07
0
3
Organic matter (%)
Organic carbon (%)
Total nitrogen (%)
C/N
Lignin (%)
a-Cellulose (%)
55°C. During this biooxidative phase, the compost
was allowed to mature for a period of 2 months.
The piles were sampled at the beginning, in the
middle and at the end of the biooxidative phase and
after the maturation period.
Organic matter was assayed by loss-on ignition
at 430°C for 24h (Navarro et al., 1993). Total
nitrogen
and organic carbon by automatic
(Navarro
et
al.,
1991).
microanalysis
Concentration
of lignin and a-cellulose were
determined according to the American National
Standards Institute and American Society for
Testing and Materials (1977a, b), holocellulose
according to Browning (1967) and hemicellulose
was calculated by difference between holocellulose
and a-cellulose.
From each sample of the piles, taken in aseptic
conditions, decimal dilutions were obtained and a
count of total aerobic bacteria in Tripticase Soja
Agar medium (TSA), nitrogen tixing bacteria in
the chemically detined Burk N-free medium
(Wilson and Knight,
1952), bacteria
with
cellulolithic activity using mineral media with
cellulose (Paredes et al., 1987) and bacteria
capable of growing in minimal media added of
10 and 30% sterile alpechin as a carbon and
Cornposting
0
2
Organic matter (%)
Organic carbon (%)
Total nitrogen (%)
C/N
Lignin (%)
fx-Cellulose (%)
Hemicellulose (W)
et al.
89.55
47.20
1.52
31.1
27.31
25.48
Cornposting
Aerobic bacterial microbiota in various samples of alpechin
energy source (Moreno
et al., 1987), were
performed.
For the determination
of biotoxicity, lo-’
dilutions were taken of each sample after filtration
and the toxicity was assessed using the luminiscent
marine bacterium Photobacterium phosphoreum in a
Microtox system employing recommended reagents
and procedures (Microbics Corporation,
1991).
Samples were diluted to between 1.5 and 0.5% in
0.9% NaCl and the effective relative concentration
(%) that decreased the bacterial luminescence by
50% (EC&,) was determined after 5min contact, as
were the corresponding toxic units (TU).
RESULTS
AND DISCUSSION
As shown in Table 1, the organic matter content
decreased during the composting process in piles
1, 2 and 3. The decrease was clearly higher in the
second pile. This could be due to a higher
abundance of degradable microbiota of organic
matter in the sewage sludge than in the poultry
manure. Also, the cotton waste was more easily
degradable than the maize straw. The nitrogen
level increased in the three mixtures and this
increase was more relevant in pile 3. Thus, the
maize straw resulted in more efficient retention of
the nitrogen losses during the composting process,
which are mainly due to the volatilization of NH3
(Bishop and Godfrey, 1983). Although the initial
values of C/N ratio were different for each
mixture, at the end of the process the three piles
had similar values (next to 10).
The lignin content
increased
along
the
composting process, showing its resistance to
a-cellulose
whereas
content
degradation,
Table 2. Changes
Composting
in the Chemical
Composition
of the Different
4
Organic matter (%)
Organic carbon (%)
Total nitrogen (%)
C/N
5
Organic matter (%)
Organic carbon (X)
Total nitrogen (%)
C/N
decreased. The highest lignin increase and the
highest cr-cellulose decreased was exhibited in pile
2, whereas the lowest increment in lignin and the
lowest decrease in a-cellulose was in pile 3. These
two results were in agreement
with the
degradation of organic matter. Also, the levels of
hemicellulose
decreased,
following
a similar
pattern in the three mixtures.
Piles prepared with alpechin sludge, piles 4 and
5, followed a similar pattern during composting,
since organic
matter
and organic
carbon
decreased nearly in the same proportion in both
piles (Table 2). The nitrogen increases were also
similar in the two piles. At the end of the
process the C/N ratio in both piles was quite
close as it happened
in the other mixtures
prepared with alpechin.
As shown in Table 3, the results make it
apparent that there were not great variations in
the number of colony forming units (CFUg-’
sample) in the different piles tested, in relation to
the total aerobic bacteria, the nitrogen fixers or
the cellulolithic strains. Pile 5 was the only one to
present bacteria capable of growing in alpechin as
sole source of carbon and nitrogen at each
sampling moment.
The biotoxicity studies showed (Table 4) that
this was only present in the samples taken at the
beginning of the composting process for each pile
tested.
ACKNOWLEDGMENTS
This research was supported by the EU Contract
No. EVWA-CT92-0006 Bioremediation of OliveMill Wasters for Use as Fertilizer.
Piles Prepared
Composting
Pile
with Sludge of Olive Mill Wastewater
time (days)
0
91
182
61.18
35.07
1.54
22.8
41.61
24.20
1.80
13.4
37.21
20.84
1.76
11.8
49
91
57.15
30.37
1.39
21.8
43.71
21.69
1.53
14.2
0
61.80
34.65
1.05
33.0
213
mature
34.84
19.95
1.89
10.65
151
37.40
18.78
1.45
13.0
during
M. Monteoiiva-Scinchez et al.
214
Table 3. Number of CFUg-’
Pile
Composting time (days)
1
3
4
5
Total aerobics
1.9x
3.3x
2.5x
4.8x
6.0x
4.9x
1.9x
2.9x
6.5x
2.4x
5.2x
6.8x
2.8x
2.2x
3.6x
2.9x
1.5x
2.4x
6.2x
1.4x
0
21
49
Mature
0
42
84
Mature
0
28
63
Mature
0
91
182
Mature
0
49
91
Mature
2
-,
of Sample of Several Types of Bacteria Found in the Composting Mixtures
Pile
Composting
time (days)
1
0
21
49
Mature
0
42
84
Mature
0
28
63
Mature
0
91
182
Mature
0
49
91
Mature
3
4
5
-,
1.3x106
1.2x106
1.2x lo6
1.7x lo6
4.3x lo5
8.2x lo5
1.5x106
3.4x lo5
2.4x 10’
3.6x 10’
l.lx105
3.7x lo5
2.1x10’
3.5x lo5
7.1x104
2.4x lo5
N2 fixers
Cellulolithic
2.2x lo5
1.1x lo6
3.5x lo5
1.2x lo6
9.2~10~
3.7x lo6
8.6x lo4
8.0x lo4
1.0x lo6
6.2x lo4
2.0x lo6
2.5x lo5
9.3x lo5
4.4x lo6
1.9x lo6
2.7x lo6
4.8x lo6
6.4x lo6
2.6x lo6
2.9x 10’
1.2x105
1.3x lo5
1.2x105
1.1x105
1.5x105
l.0x105
3.3x lo4
6.2x 10’
1.6~10~
1.4x lo5
l.lx105
5.2x lo4
2.2x lo5
1.6x lo5
1.3x lo5
5.5x lo4
1.9x105
1.6x lo5
1.3x105
6.9x lo4
Not detected.
Table 4. Assay of toxicity of the different
composting mixtures by the Microtox test
2
lo9
lo*
10’
10’
IO8
10’
10”
10’
lo6
lo*
lo*
10’
10’
lo8
lo6
10’
lo8
10’
lo7
lo6
Able to grow in
30% alpectin
Able to grow in
10% alpectin
Toxicity (TU)
samples of the
EC5oW)
1233.8
1.9
-
0.08
51.49
815.7
0.12
1496.5
1487.0
0.07
0.07
11 013.6
0.01
Bishop, P. L. and Godfrey, C. (1983) Nitrogen transformation
during sludge cornposting. BioCycle, 24, 34-39
Browning, B. L. (1967) Methods of Wood Chemistry.
Interscience, New York.
Finstein, M. S., Miller, F. C., MacGregor, S. T. and
Psarianos,
K. M. (1985) The Rutgers strategy for
cornposting: process, desing and control. EPA Project
Summary
(EPA/600/S2-851059).
U.S.
EPA,
Washington.
Microbics Corporation (199 1) A Microtox Manual: How to
Run
Toxicity
Test
Using
the
Microtox
Model
500.
Microbics Corporation, Carlsbad.
Moreno, E., Perez, J., Ramos-Cormenzana,
A. and Martinez,
J. (1987) Antimicrobial effect of wastewater form olive oil
extraction plants selecting soil bacteria after incubation
with diluted waste. Microbios, 51, 169-174
Navarro, A. F., Cegarra, J., Roig, A. and Bernal, M. P. (1991)
An automatic microanalysis method for the determination
of organic carbon in wastes. Communications in Soil
Science and Plant Analysis, 22, 2 137-2 144
5464.9
4178.1
0.02
0.02
-
Toxicity not detected.
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American National Standards Institute and American Society
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