Beruflich Dokumente
Kultur Dokumente
15.1
Introduction
ADE sites vary in their size and degree of engineering (Woods and McCann 1999),
and their biochemistry may be influenced by the addition of any of the following:
large amounts of pottery sherds, concentrated organic wastes, charred biomass,
fish bones, shells, various household wastes. In many ADE these additions have
resulted in notably high nutrient concentrations of calcium, phosphorus, and
potassium, and also high levels of black carbon (BC). The latter is thought to play
a key role in greater nutrient retention and stabilization of soil organic matter, as
decomposing residue or within living cells (Glaser et al. 2003; Sombroek et al.
2003), in spite of intense weathering conditions which typically lead to highly
leached soils in the humid tropics (Lehmann et al. 2003). Thus the central role of
the soil microbial community is in this unique soil environment should be traced
to soil amendments such as BC. Black C is not unique to ADE, but occurs
throughout terrestrial and aquatic environments (Schmidt and Noack 2000) as residue from naturally-occurring and human-induced burning. Once created, BC persists over time-scales of millennia and is thought be highly recalcitrant to microbial
degradation (Schmidt and Noack 2000). In soil, BC may enhance soil fertility by
decreasing bulk density, improving moisture retention and increasing pH, and the
surface charge properties of BC are thought to increase cation exchange capacity
(CEC), thereby reducing nutrient leaching (Glaser et al. 2002). The anthropic
addition of BC to ADE may help stabilize inorganic nutrients and thus maintain
soil fertility (Glaser et al. 2003), however, it may also serve directly as a habitat or
as a platform for nutrient exchange for microorganisms (Abu-Salah et al. 1996;
Chitra et al. 1996). High BC additions to soil due to fire events in northern boreal
forest, have been shown to alter soil microbiological community structure and
above ground plant communities (Pietikainen et al. 2000; Zackrisson et al. 1996),
but little other research exists on the impact of BC on below-ground biological
communities. Due to its prevalence in ADE and its unique physical and chemical
characteristics, BC is thought to be central to the biogeochemistry of these soils.
While BC may be central to nutrient cycling in ADE, organic matter fluxes in
soil are a dynamic interaction of chemical and physical factors that affect biological
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processes (Chapin et al. 2002). Soil organic matter (SOM), consisting of both living
cells and dead, colloidal fractions such as humus (Brady and Weil 2002), undergoes
increased turnover rates with high temperatures and moistures typical of the tropics
(Austin and Vitousek 2000; Zhang and Zak 1998). Rapid turnover of SOM together
with environmental extremes leads to the fragile nutrient status of unmodified soils
found adjacent to ADE. While BC may buffer otherwise tenuous soil fertility,
changes in SOM are largely mediated by microbial processes throughout the soil
ecosystem.
Soils are the most complex biological system on Earth (Young and Crawford
2004), containing as many as a million taxa in a 10 g sample (Gans et al. 2005),
including Bacteria, Fungi and Archaea. Traditionally soil microbiologists have
relied on culturing methods to identify soil microorganisms, but only 0.1% of
these may be culturable with known techniques (Torsvik et al. 1990). Cultureindependent methods using direct extraction of DNA followed by genetic analysis
has dramatically enhanced the understanding of soil microbial populations
(Theron and Cloete 2000). With increasing speed of DNA sequencing and computer processing, as well as great interest in soils as genetic reservoirs, the field of
soil microbiology is rapidly evolving (Curtis and Sloan 2005; Rondon et al. 2000).
Although soil microbiological studies of ADE are in their infancy they will benefit
from rapid methodological improvements in the field. Given that ADE sites vary
greatly in their distribution, age and extent (Kern et al. 2003) and soil ecosystems
are highly complex, a suite of microbiological methods is needed to explore these
systems.
Highly evolutionarily-conserved gene sequences, such as the 16S rRNA gene
in Bacteria can be used identify individual organisms or compare microbial population profiles across samples (Marsh et al. 2000; Ranjard et al. 2001; Vinuesa et
al. 1998). Community DNA extracted directly from soil contains a multitude of
diverse 16S rRNA genes which can be isolated and amplified using the polymerase chain reaction (PCR). Collections of 16S rRNA gene fragments can be separated based on numerous criteria. For example, denaturing gradient gel
electrophoresis (DGGE) uses the variable concentration of guanine and cytosine
base pairs in DNA to separate 16S rRNA gene fragments based on distinct melting
properties (Theron and Cloete 2000) and terminal restriction fragment length
polymorphism (T-RFLP) uses variable sites of discreet endo-nuclease activity
along genes to cut fragments into pieces and separate them by molecular weight
(Marsh et al. 2000). Alternatively, individual gene fragments, representing distinct
taxa, can be cloned into genetic libraries and the DNA sequenced directly
(Sambrook and Russell 2001) to reveal both taxonomic groups and functional
genes. Comparing microbial population profiles across samples or as they relate to
experimental variable requires multivariate statistical techniques. Other methods
in soil microbial ecology include culture-based approaches and microscopy.
Cloning and culturing techniques continue to improve methodologically (Leadbetter
2003) and have the advantage of being able to directly test physiology and gene
expression.
15
15.2
15.2.1
301
Current Findings
Isolating and Identifying Organisms
I. For unique soils, such as ADE, culturing techniques offer an excellent opportunity to screen for and reveal novel organisms that can be maintained and studied in
vivo. Most probable number (MPN) estimations only reflect a small portion of soil
organisms, but are a traditional means by which to compare microbial population
levels (Woomer et al. 1990). Bacterial populations have been shown to be high at
four ADE sites, and to remain at high levels with increasing soil depth compared to
adjacent nutrient-poor soils (Fig. 15.1).
II. A comparison of different soil bacterial communities was made based on
growth on different selective media followed by direct screening of 16S rDNA
amplicons subjected to hydrolysis with various restriction enzymes. A phylogeny
of sequenced rDNA (Sambrook and Russell 2001) was used to compare isolates
across soil types, depths and sites.
15.2.2
Microscopy
Log CFU/odw
109
108
107
106
105
104
103
0-30
40-70
Hat
80-120
Buried/50+
DS
0-8
10-40
LG
0-30
50-80
Acu
Fig. 15.1 Most probable number (log10) of bacteria colony forming units (CFU) g1 ODW soil by
depth, soil type (Anthrosol = black bar, adjacent soil = gray bar) at four sites. Mpns were calculated using MPNES software from (Woomer et al. 1990)
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SM Tsai et al.
15.2.3
Molecular Methods
To obtain robust information about the soil microbial community using molecular
approaches, DNA extracted directly from soil must be representative of the whole
community. The presence of BC in ADE may change the surface binding characteristics of soil particles and thus reduce the efficiency of nucleic acid extraction in
ADE soils (Thies and Suzuki 2003). The mechanism behind reduced extraction
efficiency has not been determined exactly, although it is suspected that the BC
itself reduces total nucleic acid yield and thus DNA extracts from ADE may not be
representative of the actual microbial community in either richness or dominance
indices. Efforts from the group were made to develop T-RFLP, DGGE and clone
libraries from ADE and adjacent soils to describe community level differences
across ADE sites. We are also starting identification of microbial metabolites
excreted by bacterial isolates from ADE, to determine the activity and biotechnological potential of natural compounds from these communities.
To study the microbial communities from ADE and adjacent soils, several expeditions to Amazon were made since 2003. The first reported the microbial structures
in three ADE sites, with one showing soil features similar to ADE (Dona Stella site).
Data from this study are shown on Figs. 15.1 and 15.2. Another expedition collected
ADE soils from two regions: Balbina Lake (Figs. 15.3 and 15.4), at a site named
Green
fluorescence
indicates live
organisms
Fig. 15.2 Confocal microscopy of black carbon from ADE using fluorescent microscopy images
demonstrated the presence of live (in green) microorganisms on BC surfaces, indicating that
BC can serve as a habitat for microbes, despite its chemical recalcitrance, despite its chemical
recalcitrance (See Color Plates)
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Fig. 15.3 Global positioning system (GPS) for the ADE soil from Balbina Lake (Central
Amazon) - 130'26,4';S and 6005'34';W and for the adjacent soil - 130'27';S and 6005'33';W.
There is a typical high vegetation richness above the terra preta soils, as shown from Fig. 15.1.
On the left, it is shown how the soil cores were collected at different depths (See Color Plates)
Fig. 15.4 Global positioning system (GPS) for the ADE soil from the National Forest of Caxiuan
(Eastern Amazon), with global positioning system (GPS) data for the ADE (Mina I) of 140'45,5';S
and 5120'71';W (See Color Plates)
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SM Tsai et al.
were Acidobacteria 27.2%, Proteobacteria 14.2%, Firmicutes 3.8%, Verrucomicrobia 3.8%, Nitrospira 1.3%, Planctomycetes 1.3%, Actinobacteria 0.4% e
Gemmatimonadetes 0.4%. In this survey, the low soil pH may be one major aspect
which may have directly influenced the bacterial diversity in those soils, as
Acidobacteria were present in higher frequency when compared to other phylla
(Fig. 15.7). In addition, the above-ground vegetation from the adjacent pristine forest
in Caxiuan-Par may have also influenced the frequency of the prevalent phylla,
which was not so clearly observed at the Balbina Lake. Four 16S rRNA clone libraries were obtained (Fig. 15.5), using genomic DNA extracted from the soil samples as
templates in the PCR reactions. The PCR-products were cloned into the pGEM-T
vector and 980 clones were selected and searched using the GenBank (NCBI-USA)
and the RDP II program. Data analyses indicated predominance of unknown microorganisms, representing 41.6% among the sequences from ADE-Balbina, 68.3%
from Adjacent-Balbina, 84.8% from ADE-Mina (Terra Preta) and 47.7% from
Adjacent-Mina. In ADE-Balbina (Fig. 15.6), the predominant phylum was Firmicutes,
representing 37.1% of the total sequences from that site, followed by Proteobacteria
(9.6%), Verrucomicrobia (5.6%) Acidobacteria (2.5%), Gemmatimonadetes (2.5%),
Actinobacteria (0.5%) and Nitrospira (0.5%). On the other hand, in the adjacent soil
ADJ-Balbina, the predominant phylla were Proteobacteria (15.1%), Acidobacteria
(12.5%), Firmicutes (2.3%), Nitrospira (1.1%) and Verrucomicrobia (0.8%).
The estimates of the Operational Taxonomic Units (OTUs) richness using
Bootstrap directly corroborated the diversity values obtained from the Simpson and
Extraction
Plate inoculation
and screening
Insert + vector
Insert
Vector
DNA sequencing
Quantification of
plasmid DNA
Amplification of
the gene 16S rRNA
Sequencing analysis
Fig. 15.5 Main steps for the bacterial 16S rRNA clone library construction (See Color Plates)
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1.1%
0.5%
0.5%
2.5%
9.6%
15.1%
0.8%
5.6%
2.5%
Balbina
Terra Preta
41.6%
37.1%
12.5%
Balbina
Adjacent
Unclassified
Firmicutes
Acidobacteria
Verrucomic robia
Gemmatimonadetes
Proteobacteria
Actinobacteria
Nitrospira
Unclassified
Firmicutes
Acidobacteria
Verrucomicrobia
Proteobacteria
Nitrospira
2.3%
68.3%
Fig. 15.6 Clone libraries from ADE (Terra Preta) soil and its adjacent soil from Balbina Lake
(Central Amazon) (See Color Plates)
X
1.1%
1.1%
1.4%
0.4%
6.5%
1.3%
1.3%
0.4%
0.4%
3.8%
3.8%
4.7%
Unclassified
Acidobacteria
Proteobacteria
Verrucomicrobia
Firmicutes
Nitrospira
Planctomycetes
Mina
Terra Preta
14.2%
47.7%
Mina
Adjacent
Unclassified
Acidobacteria
Proteobacteria
Verrucomicrobia
Firmicutes
Actinobacteria
Gemmatimonadetes
Nitrospira
Planctomycetes
27.2%
84.8%
Fig. 15.7 Clone libraries from ADE (Terra Preta) soil and its adjacent soils from the Caxiuan
National Forest (Eastern Amazon) (See Color Plates)
Table 15.1 Estimates of OTUs richness and diversity index calculated from the four 16S rrna
clone libraries, from ADE and adjacent soils of Balbina Lake Central Amazon and the National
Forest of Caxiuan Mina I, Eastern Amazon
Estimates of OTUs richness
D index
Libraries
SN
ON
ACE
Chao1
JK
ADE Balbina
198
119
629.4 429.4
436.2
Adjacent Balbina
265
134
284.1 237.6
284.5
ADE Mina I
278
147
252.8 224.0
233.1
Adjacent Mina I
239
141
373.7 291.7
324.5
OTUs = Operational Taxonomic Units D Index = Diversity Index
SN = Sequence Number ON = OTU Number
Sp = Simpson Index Sh = Shannon Index
Boot
Sp
Sh
156.5
167.3
182.1
179.8
0.0259
0.0102
0.0070
0.0082
4.286
4.608
4.783
4.705
Shannon indexes (Table 15.1), Unique UTOs using Jackknife estimator were correlated with a higher percentage of the low frequencies of phylla in all the four
clone libraries. The non-parametric ACE and Chao1 methods to estimate the OTUs
richness also corroborated the Jackknife values.
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15.3
SM Tsai et al.
Black C clearly has an impact on soil nutrient dynamics and ecological processes
(Zackrisson et al. 1996), but it is unclear how such a variety of carbon species and
their chemical characteristics impact microbial populations and to what extent soil
microbes interact with BC. The degree to which BC alters the flow of organic matter in ADE, either through its effect on microbial biomass or on the chemistry of
degrading C also needs to be explored. Pools of SOM can be subdivided based on
decomposition rates as labile, slow and passive pools. Measuring these different C
pools and relating them to microbial communities contributes information to models of organic matter decomposition, which can then be adapted for use in different
ecosystems (Sohi et al. 2001).
Understanding the functional diversity associated with organic matter degradation in ADE, and soil in general, may pose as much of a challenge to understanding
soil processes as trying to describe the actual taxonomic diversity. In an experiment
varying C substrate quality, Degens et al. (2000) described both the richness and
evenness of soil microbial populations in similar soils under different management
practices, and responded to how these specific substrate additions in different combinations. By comparing respiratory response to added amino acids, simple sugars
and carboxylic acids, significant differences were observed in catabolic activity
between pasture and cropped soils.
The biochemical diversity and complexity of organic residues in the soil ecosystem is matched by species and metabolic diversity. Measuring the microbial communities associated with distinct C pools will illuminate how ADE has retained
stable fertility for hundreds to thousands of years.
In many tropical forests, turnover of organic material occurs rapidly and near to
the soil surface, leading to a rapid loss of soil organic matter when forests are
burned and land is used for agriculture (Sanchez and Logan 1992). In ADE, the
higher fertility at greater soil depth appears to be stabilized by the presence of BC
and leads to large and diverse microbial populations. Understanding the biogeochemical processes involved in maintaining of fertility of ADE soils may lead to
new technologies for soil management in the tropics (Glaser et al. 2002), and provide a novel strategy for mitigating atmospheric CO2 by sequestering BC in soils,
which may also serve as a nucleus for improved soil fertility.
From the first surveys, there is a clear idea that low soil pH may play an important
role on microbial diversity in tropical soils, as Acidobacteria were present in higher
frequency when compared to other phylla. In addition, the above-ground vegetation
from the adjacent pristine forest in Caxiuan-Par may have also influenced the
frequency of the prevalent phylla, which was not clearly found at the Balbina Lake.
Acknowledgements The authors wish to acknowledge and thank the valuable support
from Dr. Wenceslau Geraldes Teixeira (EMBRAPA-CPAA, AM), Sandoval do Nascimento
Morais (INPA/AM) and Jean Charles Peixoto (UFAM/AM) for the soil collections from
Hidroeltrica da Lagoa Balbina AM. This study was supported by CNPq Proc. 485516/20063
and FAPESP Proc. 2006/067000.
15
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