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Science of the Total Environment 416 (2012) 476–481

Contents lists available at SciVerse ScienceDirect

Science of the Total Environment


journal homepage: www.elsevier.com/locate/scitotenv

Biochar influences the microbial community structure during manure composting


with agricultural wastes
Keiji Jindo a, Miguel A. Sánchez-Monedero a, Teresa Hernández a, Carlos García a, Toru Furukawa b,
Kazuhiro Matsumoto b, Tomonori Sonoki b, Felipe Bastida a,⁎
a
Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC). Department of Soil Conservation and Waste Management. Campus Universitario de Espinardo, 30100 Murcia, Spain
b
Faculty of Agriculture and Life-Sciences, Hirosaki University, Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The influence of biochar derived from a hardwood tree (Quercus serrate Murray) on the dynamics of the
Received 23 August 2011 microbial community during the composting of poultry manure (PM) and cow manure (CM) was evaluated
Received in revised form 2 December 2011 by phospholipid fatty acid analysis (PLFAs). Changes in the PLFA patterns were related to key composting prop-
Accepted 3 December 2011
erties (C/N ratio, temperature, and bulk density) as the major drivers of the dynamics of the microbial commu-
Available online 5 January 2012
nity. At the beginning of the process, the fungal biomass was significantly greater in PM and CM than in the
Keywords:
respective co-composted materials with biochar (PMB and CMB); this difference declined gradually during the
Biochar process. In contrast, the Gram+ to Gram− ratio was increased by the presence of biochar. After 12 weeks of
Composting composting, factor analysis based on the relative abundances of single PLFAs revealed changes in the microbial
Phospholipid fatty acids community structure which depended on the original organic wastes (CM vs PM).
Microbial community © 2011 Elsevier B.V. All rights reserved.

1. Introduction is based on the biological transformation of the organic wastes under


aerobic conditions, with the participation of a wide range of microbial
The increasing interest in soil carbon sequestration as a strategy to groups (Ishii et al., 2000). The addition of biochar in the initial stages
tackle global warming has stimulated research on the agricultural use of the composting process could induce changes in the microbial com-
of pyrogenic organic materials, such as charcoal, biochar, or activated munity, as observed in biochar-amended soils (Thies and Rillig, 2009)
carbon (Lehmann et al., 2011). The utilization of these materials as and in fire-affected forests, where the charcoal from burning can induce
organic amendments is beneficial in terms of carbon storage and changes in the soil microbial community (Pietikäinen et al., 2000; Wardle
soil fertility (Glaser et al., 2002). The high porosity, cation exchange et al., 2011). Hence, it seems rational to suggest that biochar addition
capacity, and sorption capacity of biochar provide a suitable habitat could exert an effect over the microbial community during composting
for microorganisms, promoting their activity in soil and affecting dif- and thus could influence the performance of the composting process
ferent microbial processes involved in nutrient cycling, organic mat- and also the quality of the end product. According to recent studies
ter (OM) decomposition, greenhouse gas emissions, etc. (Thies and (Hua et al., 2009), biochar as a composting component has shown
Rillig, 2009). Besides its agriculture use, there is increasing interest positive effects, reducing the N losses originating from ammonia vol-
in the utilization of biochar as an alternative strategy for many envi- atilization and enhancing compost quality from a chemical and bio-
ronmental issues such as decontamination of polluted sites (Sun chemical point of view by increasing the degree of humification of
et al., 2011; Park et al., 2011). The effectiveness of the combination the OM (Dias et al., 2010).
of biochar and other organic materials, such as compost, has been Phospholipid fatty acid analysis (PLFAs) is a useful tool for moni-
reported widely in recent years with regard to the remediation of toring the microbial community dynamics (Klamer and Bååth, 1998)
contaminated soils and the improvement of soil nutrient retention and hence can help our understanding of the succession of microbial
and resistance against erosion (Steiner et al., 2008; Verheijen et al., populations. The aim of this work is to study the impact of biochar on
2010; Sizmur et al., in press; Beesley et al., 2011). the microbial community structure during its co-composting with
Composting, a widely-accepted technology for organic waste recy- organic wastes. Changes in the microbial community structure eval-
cling in agriculture, ensures the OM stabilization and sanitization of uated by PLFAs should be related to variations in the C/N ratio, tem-
these wastes (Bernal et al., 1998). This waste treatment technology perature, and bulk density, which are principal indicators of the
composting process. Two different animal wastes (poultry manure
⁎ Corresponding author. Tel.: + 34 968396106; fax: + 34 968396213. and cow manure) were chosen as the starting materials, with rice
E-mail address: fbastida@cebas.csic.es (F. Bastida). husk, and were composted alone or jointly with biochar.

0048-9697/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2011.12.009
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K. Jindo et al. / Science of the Total Environment 416 (2012) 476–481 477

2. Materials and methods Methyl Ester, BAME Mix Cat. No. 47080-U, Supelco, Sigma Aldrich; and
Supelco™ 37 Component FAME Mix Cat. No. 47885-U, Supelco, Sigma
2.1. Biochar preparation Aldrich).
The fatty acids i15:0, a15:0, 15:0, i16:0, i17:0, cy17:0, and cy19:0
Biochar was prepared from the broad-leaved tree Quercus serrate were chosen to represent the bacterial biomass (Frostegard et al.,
Murray, using a Japanese traditional kiln at atmospheric pressure 1993a) and 18:2ω6 was taken to indicate the fungal biomass (Federle
and a temperature range of 400–600 °C. To analyze the physical et al., 1986; Rinnan and Baath, 2009). The ratio of bacterial to fungal
properties of the biochar, we milled and sieved it to less than 0.5 mm PLFAs represents the ratio of the bacterial and fungal biomasses. The
in diameter. The main characteristics of the biochar obtained were: pH Gram+ specific fatty acids i15:0, a15:0, i16:0, and i17:0 and the
(H2O)= 7.23; methylene blue (MB) absorption capacity: 8.3 mg g− 1; Gram− specific fatty acids cy17:0 and cy19:0 were taken as a measure
iodine adsorption capacity: 100 mg g− 1. Compared to activated carbon, of the ratio of the Gram+ and Gram− bacterial biomasses. The ratio of
our biochar showed relatively-low micro and mesoporosity, as evaluat- monounsaturated PLFAs to saturated PLFAs is expressed as mono/sat.
ed by the iodine number method and MB adsorption, respectively All results are given in nmol g− 1. The PLFAs analysis was performed
(Gaspard et al., 2007). in triplicate.

2.2. Raw materials and the composting process 2.5. Statistical analysis

Composting was carried out at the Kanagi experimental farm of The data were submitted to two-way ANOVA, with treatment and
Hirosaki University, from July to October 2010 (12 weeks). Two mix- composting time as factors, followed by Tukey's significant difference
tures were prepared: cattle manure (CM) (100.9 kg) or poultry ma- as a post hoc test (HSD, honestly significant difference at the 95% con-
nure (PM) with apple pomace (76.8 kg), rice straw (9.7 kg), and fidence interval). In order to determine significant differences among
rice bran (12.7 kg). For the other two treatments, 20 kg of biochar treatments at the same composting time, the data were subjected ad-
(equivalent to 10% of the total weight of the starting mixture) was ditionally to one-way ANOVA. Physico-chemical data and biomass in-
added to each of the mixtures (CMB and PMB, respectively). dicators were subjected to correlation analysis. The factor analyses
The organic waste mixtures were composted in cone-shaped wind- were made using the relative abundances of all identified FAMES at
rows. To aerate and homogenize the organic materials, and thus to pro- each independent time. For the statistical analyses, SPSS v19.0 was
mote composting, the mixtures were turned twice a week in the first used.
week and once a week from the second week onwards. The tempera-
ture of the pile was monitored continuously during the process using 3. Results and discussion
a thermo-recorder (T & D Co. Ltd., Nagano, Japan). To maintain the mi-
crobial activity, the moisture level of the material was kept at around The carbon and nitrogen contents, pH, OM content, and bulk den-
60% by adding water. The composting process lasted approximately sity were influenced significantly by the treatment and composting
100 days for the 4 piles. Three representative samples (triplicate) time (P b 0.05), as demonstrated by the two-way ANOVA. Even
were taken: in the thermophilic phase (the first week of composting, though the additional volume of the final product resulting from
T1), in the mesophilic phase (after 6 weeks of composting, T6), and the biochar addition could be less practical for transport and dispos-
after the maturation (cooling) phase (after 12 weeks of composting, al, the bulk density of the final composts obtained by adding biochar
T12). These samples were collected from different parts of the piles, was lower and may have potential benefits for their application to
air-dried slightly, and sieved to 0.2 mm in order to homogenize the ma- soil.
terial for chemical, physical and microbiological analyses. Samples were The relatively-high C/N ratios of all the initial composting mixtures
stored at 4 °C for PLFA and chemical analysis within the first two days (Table 1) reflect the lignocellulosic nature of the starting materials.
after sampling. For both treatments, the C/N ratio decreased during the composting
process as a consequence of the stabilization of the OM (Benitez et al.,
2.3. Chemical analysis of composting samples 2005). In comparison with the compost without biochar (PM and
CM), higher C/N ratios in the mixtures with biochar (PMB and CMB)
The pH was measured in a 1/10 (w/v) aqueous solution and the
organic matter (OM) concentration by loss on ignition at 550 °C for
Table 1
24 h. The total organic carbon (TOC) and N were determined by an The chemical characteristics of the mixtures at different composting times.
automatic C/N/S analyzer (NA1500, Carlo ERBA). The bulk density
was measured by the method proposed by Blake and Hartge (1986). Composting N C C/N T pH OM BD
time (g kg− 1) (g kg− 1) (°C) (%) (g cm− 3)
The analyses were performed in triplicate.
1 week
PM 0.22d 3.82b 17.7a 57.5 5.8b 79.9b 0.49c
2.4. Phospholipid fatty acid analysis (PLFAs)
PMB 0.18b 5.01d 28.6c 62.0 5.8c 84.9c 0.41a
CM 0.19c 3.65a 18.8b 60.7 6.6c 75.9a 0.47c
Phospholipids were extracted from 2 g of organic materials, using CMB 0.16a 4.82c 30.0d 57.9 7.0a 79.7b 0.42b
a chloroform-methanol extraction based on Bligh and Dyer (1959), 6 weeks
and then fractionated and quantified using the procedure described PM 0.33c 3.00a 9.1a 31.3 8.0b 60.2a 0.72c
PMB 0.25b 4.81c 19.4c 31.0 8.3c 74.4d 0.56a
by Frostegard et al. (1993b) and Bardgett et al. (1996). Phospholipids CM 0.25b 3.00a 12.1b 30.5 7.7a 61.9b 0.60b
were transformed by alkaline methanolysis into fatty acid methyl es- CMB 0.21a 4.40b 21.0d 31.1 7.8a 71.4c 0.57a
ters (FAMEs), which were quantified using a gas chromatograph 12 weeks
(Trace GC Ultra Thermo Scientific) fitted with a 30-m capillary col- PM 0.35c 2.88a 8.3a 21.2 8.2c 55.2a 0.81d
PMB 0.20a 3.92c 19.7c 21.8 7.8b 73.0d 0.57b
umn (Thermo TR-FAME 30 m × 0.25 mm ID × 0.25 μm film), with
CM 0.26b 2.86a 10.9b 20.5 7.7b 59.7b 0.65c
helium as carrier gas. The initial temperature was 150 °C for 0.5 min CMB 0.20a 3.80b 19.0c 21.8 7.6a 68.8c 0.55a
and it was increased to 180 °C at 2 °C min − 1 and then to 240 °C at
T: temperature; OM: organic matter; BD: bulk density. PM: poultry manure compost,
4 °C min − 1. PMB: poultry manure blended with biochar, CM: cow manure compost, CMB: cow
The FAMEs were identified and quantified by comparing their reten- manure blended with biochar. For each treatment, data followed by the same letter
tion times with those of authentic reference compounds (Bacterial Acid are not significantly different according to the HSD test (P ≤ 0.05).
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478 K. Jindo et al. / Science of the Total Environment 416 (2012) 476–481

Gram+/Gram − (Gram-positive to Gram-negative bacteria PLFA ratio); Fung/Bac (fungal to bacterial PLFA ratio); Mono/sat (monounsaturated to saturated PLFA ratio). PM: poultry manure compost, PMB: poultry manure blended with
were maintained until the end of composting process (Table 1). Recal-

ab
b

b
a
a
a
a

a
a
a
a

a
C18:0/C16:0
citrant carbon derived from the added biochar could explain the high
C/N ratio of the PMB and CMB. The initial C/N ratios of the organic ma-

0.19
0.21
0.23
0.22

0.28
0.32
0.27
0.31

0.38
0.27
0.39
0.32
terials affect strongly the microbial community composition (Eiland
et al., 2001). A high C/N ratio for PMB and CMB in the initial phase
might have been related to the lower biomass relative to the mixtures
without biochar (PM and CM) (P b 0.05) (Table 2). This is supported

ab

ab

ab
b
b

b
b
a

a
a
by the negative correlation between the C/N ratio and the bacterial bio-

Mono/Sat
mass (Table 3). A high C/N ratio leads to N limitation of the decomposi-
tion of organic compounds, resulting in low initial decomposition rates

0.24
0.29
0.28
0.23

0.18
0.19
0.16
0.15

0.18
0.18
0.16
0.16
of hemicellulose and cellulose and, hence, probably a lower microbial

biochar, CM: cow manure compost, CMB: cow manure blended with biochar. For each treatment, data followed by the same letter are not significantly different according to the HSD test (P ≤ 0.05).
biomass.
The composting time also played a noticeable role in the commu-

ab

ab
ab
b
b

b
a
a

a
a
a
a
nity structure and biomass. Indeed, it significantly influenced the bac-
terial and fungal biomasses and all the PLFA ratios, as demonstrated

Fung/Bac
by the two-way ANOVA (P b 0.05) (Table 2). The bacterial biomass

0.41
0.50
0.26
0.17

0.07
0.08
0.07
0.07

0.07
0.09
0.07
0.05
showed a general increase after six weeks of composting, compared
to the first week, but had decreased after 12 weeks (except for PMB).
Among the treatments, the mixture without biochar showed a

ab

ab
Gram+/Gram−
greater bacterial biomass (P b 0.05) than mixtures with biochar in

b
a
a

a
c

c
the first week and after six weeks (Table 2). At 12 weeks, the bacterial
biomass was greater in PMB than in PM, but the differences between

21.54
38.14
12.87
15.67

16.20
14.36
16.96
22.05

11.59

16.37
5.37

4.97
CM and CMB were not significant. Generally, both the Gram-positive
and Gram-negative bacterial biomass were significantly higher
(P b 0.05) in PM and CM than in the corresponding PMB and CMB, at
the different sampling times. It is worth pointing out the great ad-

b
Monosaturated

a
a
sorption capacity of biochar surfaces for dissolved organic carbon,
pesticides, hydrocarbons, and heavy metals (Gómez-Brandón et al.,
16.36
11.72
17.95
10.12

15.71
11.21
17.60
10.79

15.33
10.56
10.62
9.92
2011; Beesley et al., 2010). Hence, adsorption of PLFAs by biochar
might have contributed to the lower values of biomass detected in
The microbial biomass (nmol g− 1 soil), the different PLFA groups at the different composting times, and their respective ratios.

the composts with biochar. An alternative explanation for the low


biomass might be the lower availability of nutrients due to their ad-

ab
b

b
a

a
c
sorption to biochar in the composting piles. However, we can still
use the ratios between the different groups of PLFAs and multifactori-
Saturated

al analysis of the relative abundance of PLFAs in order to evaluate the


67.26
40.41
65.13
44.84

88.60
59.11

72.62

55.33
84.28
67.76
65.58
108.83
dynamics of the microbial community structure, as discussed below.
The ratio of fungi to bacteria was higher at the first sampling time
(T1) than at 6 or 12 weeks. There were no significant differences
(P b 0.05) between the treatments with and without biochar (Table 2).
ab
ab
b

b
a

a
c

However, in the first week, the effect of the differing origins of the or-
ganic materials on this ratio was noticeable: both PM and PMB showed
Bacteria

significantly-higher ratios (P b 0.05) than CM and CMB. At 12 weeks, the


30.57
15.36
27.61
19.74

52.08
32.45
60.90
42.13

32.54
43.91
42.95
39.69

highest value was for PMB (P b 0.05). In addition, the pattern of PLFAs
during the composting showed certain parallelism with the findings
of other authors. For instance, the stearic acid to palmitic acid ratio
ab

ab

(C18:0/C16:0) has been proposed as an indicator of the stability and


b
b

b
a

a
a
c

maturity of the final compost (Barje et al., 2008); this ratio tended to in-
crease with the time of composting.
Fungi

7.57
7.02
3.33

3.55
2.47
4.23
2.79

2.13
3.93
2.90
2.18
12.40

Due to the recalcitrant character of biochar produced at high tem-


peratures, it might be reasonable to consider a potential negative stress
in the microbial community generated by the addition of biochar to
the composting pile. The monounsaturated to saturated PLFAs ratio
d
b

b
a

a
c

is used as an indicator of physiological or nutritional stress in micro-


Gram−

bial communities (Bossio and Scow, 1995) and it is lower in microbi-


1.36
0.39
2.00
1.18

3.03
2.11
3.40
1.88

5.13
3.49
7.18
2.29

al communities that inhabit environments where organic carbon


and/or nutrients are limiting (Gómez-Brandón et al., 2011). In our
study, the highest monounsaturated to saturated ratio occurred in
mature PMB compost (T12) and, generally, no significant differences
ab
b

b
a

a
c

(P b 0.05) were observed in this ratio among treatments through the


composting process, with and without biochar (Table 2). Thus, con-
Gram+

trary to our previous statement, these results indicate that biochar


29.21
14.97
25.61
18.55

49.05
30.34
57.50
40.25

27.40
40.42
35.76
37.40

does not induce nutritional stress in the microbial community.


Temperature was used as a key parameter to monitor the com-
12 weeks

posting process (Table 1). All four composting piles showed the typ-
6 weeks
1 week
Table 2

CMB

CMB

CMB
PMB

PMB

PMB

ical trend, characterised by an initial thermophilic phase after the


CM

CM

CM
PM

PM

PM

fourth day, with temperatures above 60 °C for two weeks, and then
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K. Jindo et al. / Science of the Total Environment 416 (2012) 476–481 479

Table 3
Correlations between the different physical, chemical, and biomass variables.

OM pH C/N ratio C N Density Gram + Gram − Fungi Bacteria Saturated

pH − 0.74⁎⁎
C/N ratio 0.89⁎⁎ − 0.65⁎⁎
C 0.86⁎⁎ − 0.43⁎⁎ 0.92⁎⁎
N − 0.84⁎⁎ 0.67⁎⁎ − 0.88⁎⁎ − 0.72⁎⁎
Density − 0.94⁎⁎ 0.78⁎⁎ − 0.89⁎⁎ − 0.77⁎⁎ 0.94⁎⁎
Gram + − 0.61⁎⁎ 0.60⁎⁎ − 0.62⁎⁎ − 0.58⁎⁎ 0.41⁎ 0.50⁎
Gram − − 0.82⁎⁎ 0.57⁎⁎ − 0.75⁎⁎ − 0.75⁎⁎ 0.62⁎⁎ 0.74⁎⁎ 0.39⁎
Fungi 0.57⁎⁎ − 0.68⁎⁎ NS NS NS − 0.51⁎⁎ NS − 0.43⁎⁎
Bacteria − 0.69⁎⁎ 0.65⁎⁎ − 0.68⁎⁎ − 0.65⁎⁎ 0.47⁎⁎ 0.58⁎⁎ 0.99⁎⁎ 0.51⁎⁎ NS
Saturated − 0.49⁎ 0.45⁎⁎ − 0.57⁎⁎ − 0.57⁎⁎ NS 0.38⁎⁎ 0.96⁎⁎ 0.33⁎ NS 0.94⁎⁎
Monounsaturated NS NS NS NS NS NS 0.44⁎⁎ NS 0.54⁎⁎ 0.40⁎ 0.65⁎⁎

N = 36; OM: organic matter; Gram+: Gram-positive bacteria; Gram −:Gram-negative bacteria.
⁎ Significant at P b 0.05.
⁎⁎ Significant at P b 0.01.

a stabilization period of approximately five weeks (3 weeks of 50–40 °C them are thermotolerant in the composting process (Beffa et al., 1996a,
plus 2 weeks of 40–30 °C) during which the mesophilic temperatures b).
slowly decreased to values similar to ambient (mature compost). The Biochar improves aeration (due to its high nano-porosity and
use of biochar did not affect the temperatures of the CMB and PMB large surface area) (Downie et al., 2009) and water retention in soil
piles during the thermophilic phase (Table 1). Nevertheless, the Gram- (Glaser et al., 2009). Thus, apart from the chemical impact of biochar,
positive to Gram-negative ratio after 12 weeks of composting was its physical properties may also explain the changes observed in the
increased by the presence of biochar. These results suggest a notable microbial community structure. The presence of biochar significantly
role of Gram-positive bacteria at the end of composting with biochar. decreased (P b 0.05) the bulk density inside the compost piles, due to
Normally, Gram-positive bacteria constitute the strictly-thermophilic its high porosity (Table 1), which may have benefits for the applica-
bacteria that proliferate especially during the high-temperature phase tion of compost to soil (Kato and Miura, 2008; Leconte et al., 2011).
(Amir et al., 2010); in particular, Bacillus spp. dominate the microbial Variation in the bulk density may alter the natural aeration of the
community in the thermophilic phase (Steger et al., 2005). Gram- composting pile (Brodie et al., 2000) and influence microbial coloni-
negative bacteria are less resistant to high temperatures and few of zation and its biomass, as indicated by the positive correlation of

1 week 8 weeks
2.00 2.00

1.50 1.50 PMB


1.00 CM 1.00
PM
0.50 CMB 0.50
Factor 1 (39.90%)
Factor 1 (37.4%)
0.00 0.00
1.50 1.00 0.50 0.00 0.50 1.00 1.50 2.00 3.00 2.00 1.00 0.00 1.00 2.00
0.50
Factor 2 (29.4%)
Factor 2 (11.6%)

0.50

1.00 1.00 PM
CMB CM
PMB 1.50 1.50

12 weeks
1.50

PM and CM 1.00

0.50
Factor 1 (55.1%)
0.00
1.00 0.50 0.00 0.50 1.00 1.50 2.00
0.50 PMB
Factor 2 (24.0%)

1.00

1.50
CMB
2.00

Fig. 1. Distribution of the treatments after a factor analysis using all the individual PLFAs analyzed at different composting times.
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480 K. Jindo et al. / Science of the Total Environment 416 (2012) 476–481

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We are very grateful for the financial support by the Japanese 2008;99:3372–80.
Society for the Promotion of Science and the CSIC programs for bi- Klamer M, Bååth E. Microbial community dynamics during composting of straw mate-
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lateral projects. The financial support from the SENECA Foundation 9-20.
and the GOGO fund are also appreciated. We express our deep grat- Hua L, Wu W, Liu Y, McBride MB, Chen Y. Reduction of nitrogen loss and Cu and Zn mo-
itude to Mr. Sasaki for making possible the pyrolysis process and for bility during sludge composting with bamboo charcoal amendment. Environ Sci
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JAE-Doc CSIC Program and the Marie Curie Reintegration Grant denaturing gradient gel electrophoresis analysis. J Appl Microbiol 2000;89:768–77.
(DYNOMIWAS, PERG07-GA-2010-263897) for financial support. Leconte MC, Mazzarino MJ, Satti P, Crego MP. Nitrogen and phosphorus release from
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