influence of microbial inoculation and co-composting material on the evolution of humic-like...
TRANSCRIPT
Short communication
Influence of microbial inoculation and co-composting material
on the evolution of humic-like substances during composting
of horticultural wastes
M del Carmen Vargas-Garcıa *, F. Francisca Suarez-Estrella,M Jose Lopez, Joaquın Moreno
Unidad de Microbiologıa, Departamento de Biologıa Aplicada, Universidad de Almerıa, 04120 Almerıa, Spain
Received 25 May 2005; received in revised form 19 January 2006; accepted 20 January 2006
Abstract
Four different raw materials (olive-oil mill, pruning waste, rice straw and almond shell waste) used as additives were mixed with pepper plant
wastes for composting purposes. Three bacterial strains isolated from prior composting processes and identified as Bacillus shackletonni,
Streptomyces thermovulgaris and Ureibacillus thermosphaericus were added to every windrow and their effect on humic-like substances evolution
was studied. In all the cases, an increase in the humification indices (humic to fulvic acids ratio, humification ratio and humification index) was
obtained. On the other hand, inoculation significantly affected the humification indices, and differences due to both raw materials and microbial
inoculants could be observed. When inoculated in almond shell and rice straw heaps respectively, B. shackletonni and U. thermosphaericus induced
higher humification levels than those obtained for other raw material/inoculant combinations. Thus, the improvement of composting processes by
means of inoculation seems to depend on properties of raw materials and microorganisms applied.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Compost; Agricultural wastes; Microbial inoculants; Humification indices
www.elsevier.com/locate/procbio
Process Biochemistry 41 (2006) 1438–1443
1. Introduction
The agriculture practiced in the southeast of Spain (intensive
under plastic agriculture) prevailing in the last years have
favored the progressive impoverishment of soil and the
accumulation of huge amounts of different kind of wastes,
mainly of vegetal nature. Among the different proposals to
lessen the negative effect derived from both aspects, compost-
ing seems to be one of the most interesting, since its influence is
twofold. First, the biotransformation of wastes by means of this
process allows the elimination of potentially dangerous
residues [1–3] and second, compost, the final product obtained,
improves soil quality when it is added as an organic amendment
[4,5]. Benefits of compost addition affect physical properties,
such as bulk density or total porosity, chemical characteristics
as cation exchange capacity, atmosphere or soil pH [6] and
biotic factors, mainly microbial growth [7]. Nevertheless, these
positive effects take place only when the compost applied
* Corresponding author. Tel.: +34 950 015891; fax: +34 950 015476.
E-mail address: [email protected] (M. del Carmen Vargas-Garcıa).
1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.01.011
shows an adequate state of maturity. In this sense, the addition
of products with an incomplete stabilization of their organic
matter fraction causes damage to plant roots, inhibition of seed
germination, suppression of plant growth and nutrient
starvation [8,9].
The lack of a clear definition of maturity makes difficult to
evaluate when compost has reached this stability level. Many
different parameters are involved in this process and some of
them can be related to the changes that take place in this phase.
For a long time, the C/N ratio has been used as an index of
compost maturity [10,11], but there is not a correlation between
its value and the biochemical constitution of the product [12],
so it is not representative of the product composition. Other
parameters have been proposed by different authors to monitor
the composting process, among which the potential to degrade
cellulose [13], the cation exchange capacity [14] or oxygen and
CO2 respirometry [15] can be mentioned. Nature and amount of
humic substances are of interest as well, since they allow
establishing the compost maturity [16,17] on the basis of the
determination of the agronomical value of the final product [5].
Thus, several authors suggest the different alkali extractable
fractions of the organic matter, such as total extractable carbon
M. del Carmen Vargas-Garcıa et al. / Process Biochemistry 41 (2006) 1438–1443 1439
Table 1
Enzymatic activities related to lignocellulose biodegradation observed in
microbial strains used as inoculants
Enzymatic
activity
Strain
B.
shackletonni
U.
thermosphaericus
S.
thermovulgaris
Xylanase � � +
Poly-phenoloxydase � � �Extracellular oxidase + + +
Laccase + + �Tyrosinase � + �Peroxidase + + +
and humic and fulvic acids, as well as the ratio between them,
as the best indicators of compost maturity [18–20]. Therefore,
humification of the organic matter is a key process in
composting and any factor showing influence on it must be
taken into account.
Microbial activity plays a leading role on transformations
occurring during humification processes [21]. Thus, inocula-
tion with proper microorganisms may activate the biodegrada-
tion of organic matter [22,23] or improve the final
characteristics of the compost [7,24]. When the substrates
composted are of agricultural origin, lignocellulosic sub-
stances constitute an important fraction of the total organic
matter, what potentially makes lignocellulolytic microorgan-
isms the most adequate to conduct the humification process
[25]. Some of the compounds released from lignocellulose
degradation as a consequence of microbial activity, such as
polyphenols, sugar and aminocompounds, seems to promote
the humus formation [26].
According to what has been stated above, this study
proposes the analysis of compost inoculated with lignocellu-
lolytic microorganisms, as well as the establishment of
differences in the evolution of humic-like substances caused
by these microbial strains during composting of green
wastes.
2. Materials and methods
2.1. Compost heaps
Sun-dried and chopped (20–30 mm pieces) pepper plant wastes (PPW) were
mixed with four raw materials (olive-oil mill waste: OMW, pruning waste: PrW,
rice straw: RS and almond shell: AS) subjected to similar treatment and
arranged in trapezoidal heaps (1 m � 1.5 m base; 1 m high) with weights
varying from 400 to 500 kg depending on heap composition. The four raw
materials and pepper plant wastes were mixed in the proportion (v/v) producing
a C/N ratio over 25:
� H
eap OMW: 75% PPW, 16% OMW and 9% AS;� H
eap PrW: 75% PPW, 10% PrW and 15% AS;� H
eap RS: 75% PPW, 10% RS and 15% AS;� H
eap AS: 75% PPW and 25% AS.The heaps were subjected to forced aeration (1100–1200 cm3 s�1 at intervals of
4 h) conducted through three PVC tubes (5 cm diameter) and turning fortnight,
as bio-oxidative phase lasted (45 days). Moisture content and pH were mon-
itored and adjusted to 50% (w/w) and 6.5, respectively throughout the process.
After the bio-oxidative phase, the heaps were maintained in maturation for an
additional period of four and a half months, which prolonged the whole process
for 180 days.
2.2. Microbial inocula
Three microbial strains isolated from composting heaps at different stages
and selected on the basis of their capacity to show enzymatic activities related to
lignocellulose degradation (Table 1) were assayed in relation to their influence
on both the evolution of the composting process and the final characteristics of
the product obtained. Enzymatic activities were determined as described by He
et al. [27] for xylanase activity, and following indications of Rayner and Boddy
[28] for the other activities. Identification of microorganisms was performed by
molecular methods based on the analysis of the sequence obtained from the
direct amplification of the 16s rDNA gene. The sequences used for identifica-
tion and the similarity values were X90640, Z68094, AJ250318 and 99.8%,
99.0%, 95.3%, respectively for Ureibacillus thermosphaericus, Streptomyces
thermovulgaris and Bacillus shackletonni. Inocula were scaled-up as follows:
microorganisms were sequentially cultured in conical 2 L flasks with 750 mL of
medium and 20 L autoclavable propylene recipients with 7.5 L of medium. The
organisms were cultured statically in nutrient broth for 4 days at 40 8C. Each
composting heap was inoculated with sufficient volume of microbial suspension
to reach a proportion of 106 to 107 colony-forming units g�1 (CFU g�1) of
waste. A total of 32 different windrows were set up, 24 of them were inoculated
(four substrates, three inocula and two repeats) and the remaining 8 were used as
uninoculated control heaps (two repeats also).
2.3. Analysis
Samples were extracted at different stages of the composting process (0, 14,
28, 45 and 180 days) and investigated in relation to inoculum persistence (plate
count on nutrient agar incubated at 40 8C for 2 days) and humic-like substances
concentration. Days for sampling were chosen coinciding with turning days to
secure that material was representative of the whole heap and not just super-
ficial. Total organic carbon (TOC) and nitrogen (TN) were determined accord-
ing to modified methods of Mebius [29] and Bremmer and Mulvaney [30],
respectively, while the modified method of Kononova [31] was used for total
humic extracts (THE), humic (HA) and fulvic acids (FA). Humification indices,
humification ratio (HR = THE/oxidizable carbon � 100), humification index
(HI = HA/oxidizable carbon � 100) and CHA/FA (HA/FA ratio), were calculated
according to Iglesias Jimenez and Perez Garcıa [19].
Five replicates were used in all analysis, and data obtained were subjected to
statistical analysis using Statgraphics plus 4.0 software. One way analysis of
variance (ANOVA) was performed to compare the heap mean values for the
different levels of sampling time, substrates and inoculation pattern, and to test
whether there were any significant differences among the means at the 95%
confidence level. In order to determine which means were significantly
(P < 0.05) different from which others, multiple comparison tests (Fishers’s
least significant difference) were used.
3. Results and discussion
Counting data obtained for the different microbial strains
used as inoculants are shown in Table 2. A decrease in the
number of CFUs could be observed in all the cases, being the
beginning of the bio-oxidative phase the time at which this
descend was higher. At the end of this active stage of the
process and along the curing phase, the quantitative presence of
these microorganisms became stabilized or even an increase in
the population density of some of them could be detected. The
establishment of comparisons is rather difficult since available
data in relation to the persistence of inoculants as the process
progress are scarce. Most of the studies regarding to inoculation
in composting processes describe modifications on the process
M. del Carmen Vargas-Garcıa et al. / Process Biochemistry 41 (2006) 1438–14431440
Table 2
Evolution of microbial inoculants in the composting heaps
Time (day) OMW heap PrW heap RS heap AS heap
B. shackletonni
0 6.76 � 0.14 6.85 � 0.29 6.90 � 0.16 6.76 � 0.20
15 5.81 � 0.40 5.95 � 0.22 5.81 � 0.14 5.50 � 0.19
30 6.57 � 0.19 5.31 � 0.16 5.30 � 0.15 5.81 � 0.21
45 6.22 � 0.28 5.37 � 0.14 5.37 � 0.23 5.46 � 0.28
180 6.14 � 0.30 5.82 � 0.23 5.46 � 0.23 5.96 � 0.29
U. thermosphaericus
0 7.01 � 0.07 6.72 � 0.25 6.92 � 0.08 7.01 � 0.11
15 6.26 � 0.36 6.40 � 0.45 6.56 � 0.17 5.98 � 0.37
30 6.12 � 0.11 5.72 � 0.28 5.58 � 0.18 6.05 � 0.23
45 6.03 � 0.28 6.02 � 0.22 5.36 � 0.23 6.02 � 0.21
180 6.06 � 0.20 5.58 � 0.13 6.35 � 0.19 6.02 � 0.09
S. thermovulgaris
0 6.61 � 0.11 6.84 � 0.05 6.86 � 0.14 6.72 � 0.22
15 5.68 � 0.23 5.18 � 0.17 5.09 � 0.15 5.03 � 0.09
30 5.46 � 023 5.07 � 0.12 5.02 � 0.08 5.09 � 0.18
45 5.28 � 0.19 5.18 � 0.16 5.20 � 0.19 5.20 � 0.15
180 5.31 � 0.20 5.37 � 0.22 5.39 � 0.26 5.27 � 0.20
Results are expressed as log10 CFU g�1. OMW: olive-oil mill waste; PrW:
pruning waste; RS: rice straw; AS: almond-shell waste.
evolution and/or in the final product characteristics [32–34],
variations on microbial diversity along the composting process
[22,35,36] or changes in the rhizosphere microbiota when the
compost is applied to soil as amendment [37]. In this study,
microbial population of inoculants, mainly B. shackletonni and
U. thermosphaericus, were just slightly lower than the counts
for general thermophilic microbiota and, even at some stages,
Fig. 1. Temperature evolution during composting of heaps made of different raw m
almond-shell waste. (&) Heaps inoculated with Bacillus shackletonni; (^) heap
Streptomyces thermovulgaris; (*) control heaps. Turning days are signed by arrow
they were very similar (data not shown), what potentially
enables them to compete with the native microbiota and play
and important role on the biotransformations that take place
during composting.
In relation to thermal values (Fig. 1), differences could be
observed both on account raw material and microbial inoculant.
Although temperatures above 50 8C were reached in all the
cases, higher values were detected for those heaps made of
OMW, while lower temperatures were measured in the RS
heaps. As it was expected, a thermal reactivation was promoted
every time turning treatments were applied, especially in the
heaps in which inoculants were present. Therefore, higher
temperatures were maintained in these heaps for a longer time
in almost all the cases.
Data related to humic indices are shown in Tables 3 and 4.
Regardless the nature of composted substrate, most of the heaps
showed an increase in the humic-to-fulvic acid ratio (CHA/FA),
humification ratio (HR) and humification index (HI), being
CHA/FA the index showing a more regular pattern of increase
(Table 3). Nevertheless, some differences could be observed
depending on raw material. Thus, in OMWand RS heaps higher
changes were detected at the beginning of the process, while
CHA/FA values for PrW and AS heaps showed a second increase
between the end of the bio-oxidative phase and the conclusion
of the composting process. This curing stage was also the most
important phase for the evolution of HR and HI, since more
significant variations on these indices could be observed at this
time. These results are consistent with those previously
reported in relation to transformations of organic matter to
humic-like substances [17,38]. In the active initial phase of
aterials. OMW: olive-oil mill waste; PrW: pruning waste; RS: rice straw; AS:
s inoculated with Ureibacillus thermosphaericus; (4) heaps inoculated with
s.
M. del Carmen Vargas-Garcıa et al. / Process Biochemistry 41 (2006) 1438–1443 1441
Table 3
Evolution of humic indices (CHA/FA: HA/FA ratio; HR: humification ratio; HI: humification index) in the different heaps
Time (day) OMW PrW RS AS
CHA/FA HR HI CHA/FA HR HI CHA/FA HR HI CHA/FA HR HI
0 0.58 a 11.43 a 3.55 a 0.46 a 12.15 a,b 3.37 a 0.50 a 11.10 a,b 2.95 a 0.42 a 9.26 a 2.40 a
14 0.72 b 11.09 a 3.89 a 0.59 b 10.49 a 3.36 a 0.71 b 10.37 a 3.65 a 0.54 b 10.13 a,b 3.09 a b
28 0.72 b 11.54 a 3.93 a 0.71 c 11.47 a,b 3.90 a 0.74 b 9.53 a 3.39 a 0.66 c 10.41 a,b 3.66 b,c
45 0.74 b 12.20 a 3.93 a 0.71 c 11.11 a,b 3.81 a 0.76 b 11.11 a,b 3.59 a 0.66 c 10.72 a,b 3.50 b,c
180 0.78 b 13.44 a 4.73 b 0.93 d 13.25 b 4.99 b 0.81 b 12.97 b 4.47 b 0.80 d 11.49 b 4.20 c
180/0 1.34 1.18 1.33 2.02 1.09 1.48 1.62 1.17 1.52 1.90 1.24 1.75
Data are mean values for different inocula. Within a column, means with the same letter are not significantly different (P < 0.05). OMW: olive-oil mill waste; PrW:
pruning waste; RS: rice straw; AS: almond-shell waste.
composting, the most easily available organic matter (simple
carbohydrates and organic acids) is mineralized and humic-type
substances do not change significantly; however, later in the
maturation stage, the formation of these compounds becomes
more notable as a consequence of microbial degradation of
cellulose, hemicellulose and even lignin [39].
These differences in the evolution of humification indices, as
well as the variation on the initial values for these ratios,
promoted the appearance of significant differences between the
four raw materials used. In all the cases, values for the indices
were significantly lower for the AS heaps, while higher ratios
were detected in the OMW heaps, although differences were
significant just for HI index.
The lower specific values for CHA/FA, HR and HI obtained in
these assays when compared to those described by other authors
[18,19,40] may be due to the different nature of raw materials.
However, the increase factor for these ratios (quotient between
final – 180 days – and initial – 0 days – values in Table 3) was in
agreement in most cases with those considered optimal for
maturity (1.7 for CHA/FA and 1.34 for HI according to Iglesias
Jimenez and Perez Garcıa [19]). Thus, the values obtained for
this increase factor for CHA/FAwere 2.02 (PrW Heaps), 1.90 (AS
Heaps), 1.62 (RS Heaps) and 1.34 (OMW Heaps), while values
of 1.75 (AS Heaps), 1.51 (RS Heaps), 1.48 (PrW Heaps) and
1.33 (OMW Heaps) were observed for the HI ratio.
In relation to the effect of inoculation on humification
during composting processes, a general influence of this
factor was found (Table 4). Nevertheless, when specific trials
were observed, some differences could be noted, mainly for
CHA/FA. Thus, the addition of inoculants in PrW heaps did not
produce a significant increase on CHA/FA when compared to
Table 4
Humic indices (CHA/FA: HA/FA ratio; HR: humification ratio; HI: humification in
composting processes
Inoculant OMW PrW
CHA/FA HR HI CHA/FA HR
Control 0.58 a 9.68 a 2.93 a 0.70 b 8.80 a
B. shackletonni 0.71 b 12.55 b 4.23 b 0.66 a,b 12.83 b
U. thermosphaericus 0.75 b 12.85 b 4.29 b 0.76 b 12.94 b
S. thermovulgaris 0.79 b 12.70 b 4.58 b 0.59 a 12.21 b
Data are mean values for different times. Within a column, means with the same let
pruning waste; RS: rice straw; AS: almond-shell waste.
control heaps, while just the action of U. thermosphaericus
inoculum led to a value of this ratio significantly higher than
that obtained in uninoculated RS heaps. In all the other cases,
and regardless of raw materials and inocula, the presence
of external microorganisms improved humification indices
significantly.
The influence of each microorganism used as inoculant was
also dependent on raw material. Thus, no differences could be
established between B. shackletonni, U. thermosphaericus and
S. thermovulgaris when they were added to OMW and PrW
heaps, except in the values of CHA/FA of the latest. However, the
action of U. thermosphaericus on RS heaps was more efficient
than that observed for the other inoculants, and a similar effect
was obtained for Bacillus on AS heaps.
The use of inoculants to speed up the composting process or
to obtain better final compost has been a controversial subject
for a long time and, in fact, contradictory results have been
described by different authors [22–24,32,41–45,]. Neverthe-
less, such controversy should not be surprising if we consider
the complexity of the biological events that take place and the
many factors the process depends on. One of the most
influential factors is the raw material used for composting, since
qualitative and quantitative chemical composition and, there-
fore, microbiological activity, relies on it. Given the strong
variations between substrates in that matter [46], it is not
difficult to understand the different ability of microorganisms
used as inoculants to biotransform the organic matter on each
material, as it has been stated by other authors [47]. Thus, a
prior study may be necessary to know the suitability of every
microbial species or even strain for a specific substrate when a
composting process is to be improved.
dex) in the different heaps according to the microbial inoculum used in the
RS AS
HI CHA/FA HR HI CHA/FA HR HI
2.87 a 0.66 a 8.43 a 2.51 a 0.49 a 8.40 a 2.33 a
4.26 b 0.61 a 11.30 b 3.51 b 0.76 c 11.20 b 4.22 c
4.50 b 0.88 b 11.71 b 4.52 c 0.61 b 11.47 b 3.53 b,c
3.91 b 0.66 a 12.57 b 3.91 b,c 0.62 b 10.54 b 3.40 b
ter are not significantly different (P < 0.05). OMW: olive-oil mill waste; PrW:
M. del Carmen Vargas-Garcıa et al. / Process Biochemistry 41 (2006) 1438–14431442
4. Conclusion
The results described here suggest that inoculation in
composting processes can be a useful tool to increase the
humification degree in the final product and, therefore, to
improve the agricultural quality of compost by achieving a
higher stabilization and maturity levels. In order to reach the
best results, particularly when lignocellulosic wastes are
involved, the microorganisms considered for inoculation must
be selected on the basis of their ability to speed up the
biodegradation of raw materials at initial stages and this early
decomposition will favor a faster and more efficient humifica-
tion. This is an important result since works concerning the
capacity of microbial inoculants to improve composting
process and compost are scarce and contradictory.
Acknowledgement
This research has been funded by the Spanish ‘‘Ministerio de
Ciencia y Tecnologıa’’ (CICYT project no. AGL2001-2815).
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