ORIGINAL PAPER

Bacterial and fungal taxon changes in soil microbial communitycomposition induced by short-term biochar amendment in redoxidized loam soil

Liao Hu • Lixiang Cao • Renduo Zhang

Received: 13 August 2013 / Accepted: 14 October 2013 / Published online: 18 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract To take full advantage of biochar as a soil

amendment, the objective of this study was to investigate

the effects of biochar addition on soil bacterial and fungal

diversity and community composition. Incubation experi-

ments with a forest soil (a red oxidized loam soil) with and

without biochar amendment were conducted for 96 days.

The culture-independent molecular method was utilized to

analyze soil bacterial and fungal species after the incuba-

tion experiments. Results showed that bacteria and fungi

responded differently to the biochar addition during the

short-term soil incubation. Twenty four and 18 bacterial

genara were observed in the biochar amended and una-

mended soils, respectively, whereas 11 and 8 fungal genera

were observed in the biochar amended and unamended

soils, respectively. Microbial taxa analysis indicated that

the biochar amendment resulted in significant shifts in both

bacterial and fungal taxa during the incubation period. The

shift for bacteria occurred at the genus and phylum levels,

while for fungi only at the genus level. Specific taxa, such

as Actinobacteria of bacteria and Trichoderma and Pae-

cilomyces of fungi, were enriched in the biochar amended

soil. The results reveal a pronounced impact of biochar on

soil microbial community composition and an enrichment

of key bacterial and fungal taxa in the soil during the short

time period.

Keywords Biochar � Bacterial community � Fungal

community � Diversity

Introduction

Biochar is a product of incomplete combustion of biomass

in the pyrolysis process. Biochar amendment to soils has

been shown as a possible strategy of carbon sequestration

to mitigate the climate change (Lehmann 2007a; Lehmann

et al. 2009). Biochar amendment can also improve soil

quality (Chan et al. 2007; Laird 2008; Novak et al. 2009;

Sohi et al. 2010). The enhancement of soil fertility by

biochar is attributable to soil pH increase (van Zwieten

et al. 2010) and cation adsorption of nutrients (Liang et al.

2006). The effects may also be related to changes in the

microbial community composition.

Research on ancient soil management practices, which

are known as pyrogenic carbon-enriched soils in tropical

forests, has shown considerable community composition

differences. In the Terra preta, more diverse bacterial

groups are observed compared to adjacent non-anthropic

soils (Kim et al. 2007; O’Neill et al. 2009; Grossman et al.

2010). Compared to unamended soils, biochar addition

results in lower bacterial diversity in tropical forest soils

(Khodadad et al. 2011) and lower fungal diversity in

temperate region soils (Jin 2010). However, little infor-

mation is available on the soil microbial taxon changes

induced by biochar in subtropical forest soils. Moreover,

short-term and long-term biochar additions may lead to

different bacterial communities (Khodadad et al. 2011).

Greater microbial diversities are found in long-term bio-

char-enriched soils (Kim et al. 2007; O’Neill et al. 2009;

Grossman et al. 2010), whereas lower bacterial diversities

L. Hu � R. Zhang (&)

Guangdong Provincial Key Laboratory of Environmental

Pollution Control and Remediation Technology, School of

Environmental Science and Engineering, Sun Yat-sen

University, Guangzhou 510275, China

e-mail: zhangrd@mail.sysu.edu.cn

L. Cao

School of Life Science, Sun Yat-sen University,

Guangzhou 510275, China

123

World J Microbiol Biotechnol (2014) 30:1085–1092

DOI 10.1007/s11274-013-1528-5

are observed in short-term biochar-amended soils (Jin

2010; Khodadad et al. 2011). The labile substances in

biochar are usually mineralized within a short period of

time (Cheng et al. 2006), which should influence microbial

community greatly in short-term amendment (Steiner et al.

2008). Nevertheless, the effect of short-term biochar

additions on the microbial taxa is poorly understood. Red

oxidized loam soil is a typical type in the subtropical forest

soil. It should be interesting to study how biochar enhances

the fertility of this soil type (Glaser 2007; Novak et al.

2009).

The aim of this study was to investigate the effects of

biochar amendment on bacterial and fungal community

structures in a red oxidized loam soil in a subtropical forest

during a short-term incubation. It was hypothesized that

biochar addition affected bacteria and fungi differently in

terms of both diversity and community composition during

the short-term incubation.

Materials and methods

Soil sampling and biochar production

Plant materials for biochar production and bulk soil sam-

ples (10 kg) were collected from the surface and the sur-

face layer without organic litter (0–10 cm), respectively.

The sampling process was conducted in a mixed forest in

the Dinghushan Nature Reserve (23o0902100–23o1103000N,

112o3003900–112o3304100E, 100–700 mH), in Guangdong

Province of South China. Fresh soil samples were put in

sterilized sealing plastic bags and then stored at -80 �C in

the lab for following DNA extraction and experimental

treatments. Determined using the pipette method (Day

1965), the soil texture was loam with 41 % sand, 37 % silt,

and 22 % clay. A mixture of 2 g dried soil (dried at 105 �C

for 24 h) and 5 mL distilled water was shaken for 5 min

and set for 1 h, then was used to measure soil pH with a pH

electrode. The measured soil pH was 3.7.

Biochar was produced using the forest litter collected

from the soil surface. The litter was dried at 40 �C, ground

with a mortar and pestle, and sieved with a 2 mm sieve.

The plant materials were pyrolyzed under N2 in a pipe

furnace with a heating rate of 5 �C min-1 and the final

temperature at 400 �C for 1 h. The biochar particles were

then ground and passed a 250 lm sieve.

Experimental design

Two treatments were set up, including the soil with biochar

addition (denoted by BC) and without biochar addition

(used as the control and denoted by CK). For each treat-

ment, coarse materials were picked out from the soil, and

20 g of soil were added into a column covered with gas

permeable plastic film. For the BC treatment, 1 g of bio-

char (i.e., a moderate application rate of 5 % w/w) was

added and mixed with the soil. Sterilized deionized water

was added to each column to bring the soil to 60 % of the

water holding capacity and the soil moisture was adjusted

every 6–7 days. Triple replicates were set up for each

treatment. The soil columns were incubated at 25 �C in

dark for 96 days. At the end of incubation, samples of the

BC and CK treatments were collected for the following

analyses. To assess the parent microbial phylotypes in the

soil, three original soil samples without incubation (deno-

ted by OS) were used for the following analyses.

DNA extraction

Each soil sample (0.5 g) of the OS, CK, and BC treatments

was frozen in liquid nitrogen and ground into fine powder

in a sterilized and precooled mortar. The powder was then

gently placed into a sterilized 15 mL centrifuge tube. The

total DNA was extracted from the soil powder using a

modified E.Z.N.A.TM Soil DNA kit (Omega Bio-tek., Inc.,

USA) according to the manufacturer’s instructions. Finally,

the DNA extracts were stored in 100 lL of elution buffer at

-20 �C for further studies.

PCR amplification of bacterial and fungal populations

For each soil DNA product, the 16S rRNA V3 genes of

bacteria (hereafter denoted by OSB, CKB, and BCB for the

OS, CK, and BC treatments, respectively) were amplified

using primer 27F and the reverse primer 1492R (Lane

1991). The ITS1-5.8S-ITS2 regions of fungi (hereafter

denoted by OSF, CKF, and BCF for the OS, CK, and BC

treatments, respectively) were amplified with the fungal

specific ITS1F and ITS4 primers (Gardes and Bruns 1993).

The PCR reaction mixture (50 lL) contained 1 9 PCR

buffer (Takara, Dalian, China), 2.5 U Taq DNA polymer-

ase (Takara, Dalian, China), 200 lM dNTP, 0.2 lM of

each primer, 3 mM MgCl2. The reaction conditions

included pre-denaturation at 94 �C for 2 min followed by

30 thermal cycles of denaturation, each at 94 �C for 30 s,

annealing at 56 �C for 30 s, and elongation at 72 �C for

1.5 min. The last step was extended at 72 �C for 5 min.

To confirm repeatability and minimize the PCR bias,

PCR products of the triple replicates for each treatment

were mixed together. The mixed PCR products were

checked by 1 % w/v agarose gel electrophoresis stained

with SYBR-Green I (Sigma, St. Louis, USA), based on the

band sizes of bacteria and fungi about 1.5 kb and 700 bp,

respectively. The bands were excised and purified using the

E.Z.N.A. gel extraction Kit according to the manufac-

turer’s instructions (Omega Bio-tek., Inc., USA).

1086 World J Microbiol Biotechnol (2014) 30:1085–1092

123

Cloning and sequencing

The purified DNA products of each subset were ligated into

the pMD 18-T Vector (Takara, Dalian, China) and the pMD

20-T Vector (Takara, Dalian, China) for fungal and bacterial

fragments, respectively, and transformed into the competent

cells of Escherichia coli DH5a. The recombinants of colo-

nies were identified through the blue-white color selection

on Luria–Bertani (LB) agar plates supplemented with

ampicillin (100 lg mL-1; Sigma, St. Louis, USA), X-gal

(100 lg mL-1; Takara, Dalian, China), and IPTG (0.5 mM;

Takara, Dalian, China). The OSB, OSF, CKB, CKF, BCB,

and BCF clone libraries were separately constructed by

picking 250 white clones randomly in each clone library.

Plasmid DNA was isolated from these selected clones

from each library and was reamplified by the vector primer

pair RVM and M13-47 (Zhang et al. 2011). The PCR

reaction mixture (50 lL) and conditions were used as

above. PCR products of clones containing inserts of the

expected sizes were further analyzed by restriction frag-

ment length polymorphism (RFLP) analysis. PCR aliquots

(each of 10 lL) were characterized by digestion with the

restriction endonucleases HinfI and MspI for fungal frag-

ments and EcoreI and HindI for bacterial fragments. The

digested products were classified based on the agarose gel

electrophoresis. Clones with different restriction patterns

were randomly chosen for sequencing. Sequencing was

performed by Major Biotechnology, Ltd. (Shanghai,

China). The sequences were examined for possible chi-

meras by the program DECIPHER’s Find Chimeras web

tool (http://decipher.cee.wisc.edu/FindChimeras.html) to

remove chimeric sequences.

The sequences data in present study have been deposited

in the GenBank database. Clones from bacteria and fungi

were assigned accession numbers of KF22593–KF226104

and KF225791–KF225935, respectively.

Sequences and phylogenetic analyses

To infer their approximately phylogenetic affiliations, the

bacterial sequences were compared initially with those in

GenBank using EzBioCloud analysis (http://eztaxon-e.

ezbiocloud.net/), and the fungal sequences were com-

pared with those in GenBank using BLAST algorithm

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the National

Center for Biotechnology Information (Altschul et al.

1997). Analyses of the diversity calculation and rarefaction

curves to observed operational taxonomic units (OTUs)

were carried out using the program EstimateS v.8.0 (http://

viceroy.eeb.uconn.edu/estimates/). The library coverage

values were calculated by [1-(n/N)], where n is the

number of OTUs representing a single clone and N is the

number of total OTUs representing the clones in the library

(Good 1953). To quantify bacterial and fungal diversities,

the commonly used diversity indexes, including Shannon-

wiener index, Simpsons index, and Chao I index, were

calculated using the program EstimateS.

Results

Characteristics of libraries

Total DNAs were extracted from the original soil (without

incubation), and unamended soil and amended soil with

biochar after incubation for 96 days. By using the bacte-

rium-specific PCR primer pair 27F-1492R and fungus-

specific primer pair ITS1F-ITS4, six libraries named as

OSB, CKB, BCB, OSF, CKF, and BCF were constructed.

Totally 169 clones for bacteria (53, 45, 71 in OSB, CKB,

and BCB, respectively) and 145 clones for fungi (57, 52, 36

in OSF, CKF and BCF, respectively) were sequenced.

Library coverage values of OSB, CKB, and BCB were

0.81, 0.76, and 0.76, respectively, while the coverage value

of OSF, CKF, and BCF were 0.77, 0.85, and 0.81,

respectively. The coverage value of each library was

Table 1 The proportions (%) of bacterial 16S sequences showing the

highest similarity (B 93 to 99 %) to GenBank database in clone

libraries from the original soil (OSB), unamended soil (CKB), and

biochar amended soil (BCB)

Library 99 % 98 % 97 % 96 % 95 % 94 % B93 %

OSB 4 0 9 17 4 17 50

CKB 6 4 2 9 24 17 38

BCB 7 8 8 4 26 19 27

Table 2 The proportions (%) of fungal 16S sequences, showing the

highest similarity (B 94 to 100 %) to GenBank database in clone

libraries from the original soil (OSF), unamended soil (CKF), and

biochar amended soil (BCF)

Library 100 % 99 % 98 % 97 % 96 % 95 % B94 %

OSF 5 66 4 9 2 2 12

CKF 4 77 2 7 4 4 2

BCF 0 80 6 0 0 3 11

Table 3 Diversity indexes of bacterial and fungal species from the

unamended soil (CKB and CKF, respectively) and the biochar

amended soil (BCB and BCF, respectively)

Index CKB BCB CKF BCF

Shannon-wiener index 2.96 3.36 2.66 2.22

Simpsons index 24.8 35.5 12.4 7.33

Chao I index 29.9 47.4 23.5 18.2

World J Microbiol Biotechnol (2014) 30:1085–1092 1087

123

approximately 0.8, suggesting that the libraries represented

the major bacterial and fungal phyla in the soils. In

the study, the OTUs were defined by [ 97 % sequence

similarity. The proportions of phylotypes under matched

criteria are presented in Tables 1 and 2 for bacterial and

fungal sequences, respectively. In general, fungal sequen-

ces matched higher similarity to the sequences in Genbank

database than bacterial sequences.

Table 4 Taxa of bacterial clone sequences from libraries of the original soil (OSB), unamended soil (CKB), and biochar amended soil (BCB)

Phylum OSB library CKB library BCB library

Speciesa Nb Speciesa Nb Speciesa Nb

Proteobacteria Stella vacuolata 1 Pseudolabrys taiwanensis 6 Pseudolabrys taiwanensis 2

Rhodoplanes elegans 7 Rhodoplanes elegans 4 Rhodoplanes elegans 5

Methylosinus trichosporium 1 Rhodoplanes piscinae 2 Methylocystis echinoides 2

Bradyrhizobium lablabi 1 Bradyrhizobium pachyrhizi 1 Methylosinus trichosporium 1

Shigella flexneri 1 Bradyrhizobium denitrificans 2 Bradyrhizobium japonicum 2

Proteobacteria sp. 4 Bradyrhizobium iriomotense 1

Steroidobacter denitrificans 3 Gamma proteobacterium sp. 2 Bradyrhizobium rifense 1

Stella vacuolata 1 Steroidobacter denitrificans 2 Bradyrhizobium pachyrhizi 2

Rhodopila globiformis 1 Burkholderia graminis 1

Burkholderia tuberum 1

Burkholderia acidipaludis 1

Massilia namucuoensis 4

Nitrobacter vulgaris 1

Acidobacteria Solibacter usitatus 2 Koribacter versatilis 4 Solibacter usitatus 2

Koribacter versatilis 5 Bryocella elongata 1 Acidobacterium capsulatum 2

Acidobacteria sp. 1 5 Edaphobacter aggregans 2 Edaphobacter modestus 1c 5

Acidobacteria sp. 2 1 Edaphobacter modestus 1c 1 Edaphobacter modestus 2c 2

Edaphobacter modestus 2c 2 Edaphobacter aggregans 3

Granulicella arctica 1 Bryocella elongata 1

Telmatobacter bradus 1

Acidipila rosea 3

Actinobacteria Aciditerrimonas ferrireducens 6 Aciditerrimonas ferrireducens 1c 1 Aciditerrimonas ferrireducens 5

Actinoallomurus purpureus 2 Aciditerrimonas ferrireducens 2c 3 Actinoallomurus purpureus 3

Conexibacter woesei 4 Conexibacter arvalis 1 Actinoallomurus amamiensis 2

Conexibacter arvalis 3 Mycobacterium cookii 1 Actinoallomurus spadix 1

Solirubrobacter ginsenosidimutans 1 Actinokineospora soli 1 Conexibacter arvalis 1

Mycobacterium kyorinense 1

Streptomyces misakiensis 1

Actinocorallia aurantiaca 1

Planctomycetes Gemmata sp. 1 Planctomycetales sp. 3 Singulisphaera rosea 6

Singulisphaera rosea 2 Gemmata sp. 2 Aquisphaera giovannonii 2

Singulisphaera rosea 1

Pirellula sp. 1

Verrucomicrobia Pedosphaera parvula 1 Pedosphaera parvula 1 Pedosphaera parvula 2

Firmicutes Bacillus fumarioli 1

Bacteroidetes Chitinophaga ginsengisegetis 1

Niastella populi 1

In the OSB, CKB, and BCB libraries, 53, 45 and 71 clones were randomly selected for sequenceing, respectively

The OTUs were defined by [ 97 % sequence similaritya The species in Genbank database are most closely related to the sequenced clones belong to the same OTUb The number of clones matchedc Sequences showed the same most closely related species in Genbank database but belong to the different OTUs

1088 World J Microbiol Biotechnol (2014) 30:1085–1092

123

Bacterial and fungal diversities

For bacteria, the Shannon-wiener index values were dif-

ferent between the biochar-amended and unamended soils

(2.96 and 3.36, respectively). The values of Simpsons

Index and Chao I between the two treatments were also

different (Table 3). For fungi, the Shannon-wiener index

values of CKF and BCF libraries were 2.66 and 2.22,

respectively (Table 3). The index differences between the

two treatments for bacteria were larger than those for fungi.

The CKB library comprised 23 OTUs belonging to 18

genera, while the BCB comprised 35 OTUs belonging to

24 genera (Table 4). The CKF library included 20 OTUs

belonging to 11 genera and the BCF included 14 OTUs

belonging to 8 genera (Table 5). These results suggested

that the diversity of bacteria and fungi in the soil was

affected by the biochar addition differently. According to

rarefaction curves (Fig. 1), the separation of OTU richness

between different libraries was also obvious as the clone

number in each library was greater than about 20. Bacteria

in the biochar-amended soil had greater overall diversity

than in the unamended soil. However, fungi in the biochar-

amended soil had lower diversity than in the control.

Shifts in community composition of bacteria

Besides inducing changes in soil microbial diversity, the

biochar addition also affected the community composition.

Table 5 Taxa of fungal clone sequences from libraries of the original soil (OSF), unamended soil (CKF), and biochar amended soil (BCF)

Plylum OSF library CKF library BCF library

Speciesa Nb Speciesa Nb Speciesa Nb

Chytridiomycota Kochiomyces dichotomus 1

Ascomycota Penicillium sp. 1 Penicillium sp. 1c 2 Penicillium herquei 1

Aspergillus cervinus 1 Penicillium sp. 2c 2 Penicillium sp. 1c 1

Paecilomyces carneus 1 Penicillium pinophilum1c 2 Penicillium sp. 2c 1

Hypocrea koningii 1c 1 Penicillium pinophilum2c 6 Penicillium pinophilum 2

Hypocrea koningii 2c 1 Penicillium adametzii 1 Paecilomyces carneus 4

Chaunopycnis alba 1 Penicillium olsonii 1 Trichoderma sp. 2

Trichoderma gamsii 7 Paecilomyces carneus 1 Trichoderma koningiopsis 1

Pseudallescheria fimeti 1 Aspergillus penicillioides1c 2 Trichoderma gamsii 5

Preussia sp. 1 Aspergillus penicillioides2c 2 Trichoderma viride 1

Helotiales 1 RB-2011 1 Aspergillus sp. 1c 1 Hypocrea sp. 1

Gloeotinia temulenta 4 Aspergillus sp. 2c 1 Cladosporium sp. 2

Chaunopycnis alba 1

Trichoderma gamsii 3

Trichoderma sp. 1

Cladosporium sp. 1c 2

Cladosporium sp. 2c 4

Sordariomycetes sp. 1

Basidiomycota Cryptococcus podzolicus 16 Cryptococcus podzolicus 12 Cryptococcus podzolicus 12

Thelephoraceae sp. 2 Wallemia sp. 5 Cryptococcus sp. 2

Pseudozyma sp. 3 Sterigmatomyces sp. 2 Amanita spissacea 1

Leucocoprinus birnbaumii 1

Mortierellomycotina Mortierella humilis 2

Mortierella kuhlmanii 8

Mortierella sp. 2

Mortierella hyalina 1

Others Fungi sp. 1

In the OSF, CKF, and BCF libraries, 57, 52, and 36 clones were randomly selected for sequenceing, respectively

The OTUs were defined by [ 97 % sequence similaritya The species in Genbank database are most closely related to the sequenced clones belong to the same OTUb The number of clones matchedc Sequences showed the same most closely related species in Genbank database but belong to the different OTUs

World J Microbiol Biotechnol (2014) 30:1085–1092 1089

123

For bacteria, both CKB and BCB comprised of Proteo-

bacteria, Acidobacteria, Actinobacteria, Planctomycetes,

and Verrucomicrobia. These phyla were all detected in

OSB, indicating that the major taxa in the treatments were

mainly related to the parent bacterial community compo-

sition in the forest soil. Bacteroidetes and Firmicutes were

only detected in BCB (Table 4). The proportion of Ac-

tinobacteria in BCB was 5.6 % higher than that in CKB,

whereas Proteobacteria and Planctomycetes were detected

with lower percentages (Fig. 2a). Although the proportion

of Proteobacteria decreased in the soil amended with

biochar, more taxa of Proteobacteria was detected in the

BCB library.

Shifts in community composition of fungi

Differences between fungal communities were found

mainly at the levels of genera and species (Fig. 2b). The

OSF library comprised of five phyla, but only Ascomycota

and Basidiomycota of these phyla were detected in CKF

and BCF, which suggested that fungal growth of other

phylotypes might not adapt to the incubation conditions.

Compared to the control, the proportion of Basidiomycota

in the biochar-amended soil increased slightly. At the

genus level, Cryptococcus, Talaromyces, Cladosporium,

Trichoderma, Penicillium, and Paecilomyces were detected

in the CKF and BCF libraries. The proportion of Tricho-

derma in BCF was 14.5 % higher than that in CKF, and the

proportion of Paecilomyces in BCF was 9.2 % higher than

that in CKF.

Discussion

The results supported our hypothesis that biochar addition

affected bacteria and fungi differently in terms of diversity

and community composition during the short-term incu-

bation. After 3 months of biochar application, the overall

diversity of bacteria prominently increased. The results

were consistent with previous long-term studies in biochar-

enriched Terra preta soils (Kim et al. 2007; O’Neill et al.

2009). However, the diversity of fungi declined in the

biochar amended soil. The different diversity results sug-

gested that bacteria were more adaptive than fungi to the

Fig. 1 Rarefaction analyses of (a) bacterial sequences from the

unamend (CKB) and biochar amended soils (BCB), (b) fungal

sequences from the unamend (CKF) and biochar amended soils (BCF)

Fig. 2 Proportions of (a) bacterial phyla in the unamended (CKB)

and biochar amended soils (BCB), (b) fungal phyla in the unamended

(CKF) and biochar amended soils (BCF)

1090 World J Microbiol Biotechnol (2014) 30:1085–1092

123

change of soil environment induced by biochar (Lehamann

et al. 2011). Another possible reason for the different

results was that bacteria might be more readily to utilize

the nutrients and mineral elements by sorbing to biochar

surface or colonizing in biochar pores (Liang et al. 2008;

Thies and Rillig 2009). Both bacterial and fungal com-

munity structures altered markedly due to the biochar

amendment, with fungi mainly at the genus level and

bacteria at both genus and phylum levels. This may indi-

cate a higher sensitivity of bacteria to biochar in this soil

type. In our study, the bacterial diversity was different from

the previous study of forest soils amended with oak or

grass biochar (Khodadad et al. 2011). Possible reasons for

the inconsistent results might be related to the different

treatments of soils and the types of biochar (Lehmann

2007b), or to the diverse parent bacterial communities

between the two studies (Lehamann et al. 2011).

Most of bacterial sequences in both biochar-amended

and unamended soils matched those in the databases with

\ 97 % similarity, indicating that these soils contained

taxonomically novel bacterial phylotypes. The proportion

of bacterial sequences with [ 97 % similarity was higher

in BCB than that of CKB, and the proportion of sequences

with \ 93 % similarity was lower in BCB. These results

showed that the biochar treatment might not serve a good

habitat to novel bacterial population in the soil but sup-

ported the growth of original bacterial groups. On the

contrary, it was found that most of sequences in OSF, CKF,

and BCF were with[ 98 % similarity, and the proportions

of sequences with\ 94 % similarity in OSF and BCF were

12 and 11 %, respectively, higher than that in CKF. The

result indicated that the biochar addition had some

advantageous effects to ensure the growth of novel fungal

phylotypes in the soil.

Taxa analysis of bacteria showed that both relative

abundance and diversity of Actinobacteria increased with

the biochar treatment, which was consistent with the

studies on char layers of forest soils (Baath et al. 1995),

Terra preta (O’Neill et al. 2009), and soils amended with

pyrogenic carbon (Khodadad et al. 2011). These results

demonstrated that Actinobacteria was the representative

species in recalcitrant carbon-rich soils. This is the first

report that the phylum of Bacteroidetes, which is com-

monly found in animal centeric canals and able to degrade

polysaccharose and cellulose, was detected in the soil

amended with biochar. Fungal taxa were altered mainly at

the genus level. In contrast to the findings of Jin (2010),

Zygomycota was not detected in this study. The difference

might be attributable to the different parent populations in

the two soil types or the different incubation time periods

(long vs. short). Both Trichoderma and Paecilomyces

showed high proportions in the biochar amended soil.

These two taxa of fungi have been widely studied and

applied in biological control to promote plant growth and

enhance soil quality. Therefore, the biochar-induced

enhancement of such fungal groups may show its potential

effects in biological control. As very little is known about

changes of specific soil microbes to biochar addition

(Graber et al. 2010), to take full advantage of biochar as a

soil amendment, further research is necessary to focus on

biochar influences on targeted microbial groups with spe-

cific soil functions.

Acknowledgments This work was partly supported by grants from

the Chinese National Natural Science Foundation (Nos. 51039007

and 51179212).

References

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,

Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new

generation of protein database search programs. Nucleic Acids

Res 25:3389–3402

Baath E, Frostegard AF, Pennanen T, Fritze H (1995) Microbial

community structure and pH response in relation to soil organic

matter quality in wood-ash fertilized, clear-cut or burned coniferous

forest soils. Soil Biol Biochem 27:229–240

Chan KY, Van Zwieten L, Meszaros I, Downie A, Joseph S (2007)

Agronomic values of green waste biochar as a soil amendment.

Aust J Soil Res 45:629–634

Cheng CH, Lehmann J, Thies JE, Burton SD, Engelhard MH (2006)

Oxidation of black carbon by biotic and abiotic processes. Org

Geochem 37:1477–1488

Day PR (1965) Particle fractionation and particle-size analysis. In:

Black CA, Evans DD, White JL, Ensminger LE, Clark FE (eds)

Methods of soil analysis, part 1, physical and mineralogical

properties, including statistics of measurement and sampling.

ASA-SSSA, Madison, pp 545–567

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity of

basidiomycetes: application to the identification of mycorrhizae

and rusts. Mol Ecol 2(2):113–118

Glaser B (2007) Prehistorically modified soils of central Amazonia: a

model for sustainable agriculture in the twenty-first century.

Philos T R Soc B 362:187–196

Good IJ (1953) The population frequencies of species and the

estimation of population parameters. Biometrika 40:237–262

Graber ER, Harel YM, Kolton M, Cytryn E, Silber A, David DR,

Tsechansky L, Borenshtein M, Elad Y (2010) Biochar impact on

development and productivity of pepper and tomato grown in

fertigated soilless media. Plant Soil 337:481–496

Grossman JM, O’Neill BE, Tsai SM, Liang B, Neves E, Lehmann J,

Thies JE (2010) Amazonian anthrosols support similar microbial

communities that differ distinctly from those extant in adjacent,

unmodified soils of the same mineralogy. Microb Ecol 60:192–205

Jin H (2010) Characterization of microbial life colonizing biochar and

biochar-amended soils. Ph.D. Dissertation, Cornell University

Khodadad CLM, Zimmerman AR, Green SJ, Uthandi S, Foster JS

(2011) Taxa-specific changes in soil microbial community

composition induced by pyrogenic carbon amendments. Soil

Biol Biochem 43:385–392

Kim JS, Sparovek S, Longo RM, De Melo WJ, Crowley D (2007)

Bacterial diversity of terra preta and pristine forest soil from the

Western Amazon. Soil Biol Biochem 39:648–690

Laird DA (2008) The charcoal vision: a win–win-win scenario for

simultaneously producing bioenergy, permanently sequestering

World J Microbiol Biotechnol (2014) 30:1085–1092 1091

123

carbon, while improving soil and water quality. Agron J 100:

178–181

Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E,

Goodfellow M (eds) Nucleic acid techniques in bacterial

systematics. Wiley, Chichester, pp 115–175

Lehamann J, Rilling MC, Thies J, Masiello CA, Hockaday WC,

Crowley D (2011) Biochar effects on soil biota-A review. Soil

Biol Biochem 43:1812–1836

Lehmann J (2007a) A handful of carbon. Nature 447:143–144

Lehmann J (2007b) Bio-energy in the black. Front Ecol Environ

5:381–387

Lehmann J, Czimczik C, Laird D, Sohi S (2009) Stability of biochar in

the soil. In: Lehmann J, Joseph S (eds) Biochar for environ-

mental management: science and technology. Earthscan,

London, pp 183–205

Liang B, Lehmann J, Solomon D, Kinyangi J, Grossman J, O’Neill B,

Skjemstad JO, Thies J, Luizao FJ, Petersen J, Neves EG (2006)

Black carbon increases cation exchange capacity in soils. Soil

Sci Soc Am J 70:1719–1730

Liang B, Lehmann J, Solomon D, Sohi S, Thies JE, Skjemstad JO,

Luizao FJ, Engelhard MH, Neves EG, Wirick S (2008) Stability

of biomass-derived black carbon in soils. Geochim Cosmochim

Ac 72:6069–6078

Novak JM, Busscher WJ, Laird DL, Ahmedna M, Watts DW,

Niandou MAS (2009) Impact of biochar amendment on fertility

of a southeastern coastal plain soil. Soil Sci 174:105–112

O’Neill B, Grossman J, Tsai MT, Gomes JE, Lehmann J, Peterson J,

Neves E, Thies JE (2009) Bacterial community composition in

Brazilian Anthrosols and adjacent soils characterized using

culturing and molecular identification. Microb Ecol 58:23–35

Sohi S, Krull E, Lopez-Capel E, Bol R (2010) A review of biochar

and its use and function in soil. Adv Agron 105:47–82

Steiner C, Das KC, Garcia M, Forster B, Zech W (2008) Charcoal and

smoke extract stimulate the soil microbial community in a highly

weathered xanthic ferralsol. Pedobiologia 51:359–366

Thies JE, Rillig M (2009) Characteristics of biochar: biological properties.

In: Lehmann J, Joseph S (eds) Biochar for environmental manage-

ment: science and technology. Earthscan, London, pp 85–105

Van Zwieten L, Kimber S, Morris S, Chan KY, Downie A, Rust J,

Joseph S, Cowie A (2010) Effects of biochar from slow pyrolysis

of papermill waste on agronomic performance and soil fertility.

Plant Soil 327:235–246

Zhang W, Wu C, Mai K, Chen Q, Xu W (2011) Molecular cloning,

characterization and expression analysis of heat shock protein 90

from Pacific abalone, Haliotis discus hannai Ino in response to

dietary selenium. Fish Shellfish Immun 30:280–286

1092 World J Microbiol Biotechnol (2014) 30:1085–1092

123