solubilization of potassium containing minerals by high
TRANSCRIPT
ORIGINAL ARTICLE
Solubilization of potassium containing minerals by hightemperature resistant Streptomyces sp. isolatedfrom earthworm’s gut
DianFeng Liu1,2,3• Bin Lian1,3
• Bin Wang4
Received: 13 November 2015 / Revised: 15 January 2016 / Accepted: 29 March 2016 / Published online: 9 April 2016
� Science Press, Institute of Geochemistry, CAS and Springer-Verlag Berlin Heidelberg 2016
Abstract A potassium solubilizing bacterial strain des-
ignated EGT, which is tolerant of high temperature, was
isolated from an earthworm’s gut to obtain a bacterium that
can weather potassium-bearing rock effectively through
solid-state fermentation. Molecular phylogeny and 16S
rRNA gene sequence analysis demonstrated the bacterial
strain was a member of the Streptomyces genus. To assess
its potential to release potassium from silicate minerals,
this strain was used to degrade potassium-bearing rock
powder by solid-state fermentation. After fermentation, the
amount of water-soluble Al, Fe and K of the substrate with
active inoculum was higher than those of the control,
which had autoclaved inoculum, and those of the fresh
substrate. The result indicated that the strain had the ability
to weather potassium-bearing rock and could be used as an
inoculum in the production of potassium bio-fertilizer, due
to its potassium release activity from rock and tolerance to
high temperature.
Keywords Weathering � Potassium solubilizing bacteria �Streptomyces
1 Introduction
Potassium (K), an element essential for plant growth, plays
an essential role for enzyme activation, protein synthesis
and photosynthesis (Armengaud et al. 2009; Basak and
Biswas 2009; Lin 2010). It is one of the three main
macronutrients, together with Nitrogen (N) and Phospho-
rus (P), which are needed for crop growth and crop pro-
duction increase (Amtmann and Armengaud 2007;
Sugumaran and Janarthanam 2007; Chen et al. 2008).
Potassium in soil is typically divided into four forms:
water-soluble (solution K), exchangeable, non-exchange-
able and structural or mineral forms (Jalali 2006; Basak
and Biswas 2009). Potassium from the water-soluble pool
is directly available for plant uptake. Water-soluble K is
readily replenished by the soil exchangeable K, which is
held by negatively charged clay minerals and organic
matter in soils. Some non-exchangeable K can become
exchangeable when the water-soluble and exchangeable K
are depleted by plant removal, leaching, or exchange
reactions with other cations. Mineral K, which is the major
proportion of total K in soils, can only become available
very slowly through long-term soil weathering (Peter-
burgsky and Yanishevsky 1961; Basak and Biswas 2009;
Ghiri et al. 2010). Total reserves of K in soil are generally
large, but more than 98 % of them exists in the form of
silicate minerals (microcline, muscovite, orthoclase biotite,
feldspars, etc.) and can’t be directly absorbed by plants
(Sugumaran and Janarthanam 2007). However, crops need
large amounts of potassium during growth. A large amount
of potassium fertilizer must be added into soil to
& Bin Lian
[email protected]; [email protected]
1 Jiangsu Key Laboratory for Microbes and Functional
Genomics, Jiangsu Engineering and Technology Research
Center for Microbiology, College of Life Sciences, Nanjing
Normal University, No. 1, Wenyuan Road, Nanjing 210023,
People’s Republic of China
2 Department of Bioengineering, Puyang Vocational &
Technical Institute, Puyang 457000, People’s Republic of
China
3 State Key Laboratory of Environmental Geochemistry,
Institute of Geochemistry, Chinese Academy of Sciences,
Guiyang 550081, People’s Republic of China
4 Department of Bio-Engineering, Henan Institute of Science
and Technology, Xinxiang 453003, People’s Republic of
China
123
Acta Geochim (2016) 35(3):262–270
DOI 10.1007/s11631-016-0106-6
adequately supply crops with the potassium needed for
sustaining crop production.
Many countries, such as China, India and Brazil, are
important agricultural countries and need large amounts of
fertilizers every year, yet they are deficient in potassium
fertilizer resources (Sun et al. 2009; Basak and Biswas 2010).
However, these countries are fortunate to be rich in low-grade
potassium-bearing rock (PBR) (Chen 2000; Basak and Bis-
was 2009). If these materials can be converted into potassium
fertilizer by some suitable chemical or biological means, the
lack of potassium fertilizer resources would be greatly eased
in those agricultural countries. Some researchers have proved
that composting technology can effectively convert
unavailable K into plant available form because of the acidic
environment prevailing during composting, and the product
of mineral K compost can increase crop yields (Nishanth and
Biswas 2008; Biswas 2010).
The natural weathering rates of minerals are very low.
Some microorganisms must be added into the compost to
degrade potassium-bearing minerals and quickly release
enough K. It has been found that many microorganisms can
enhance the weathering of potassium-bearing minerals,
however most of them cannot survive high temperature. The
temperature inside compost is generally high, oftentimes more
than 50 �C during composting. Therefore, some microbes that
are tolerant to high temperatures are necessary if weathering
potassium-bearing rocks through composting technology.
Earthworms have been described as ecosystem engi-
neers due to their key role in altering the biological activity
and physical structure of soils (Liu et al. 2011; Zangerle
et al. 2011). The role of earthworms in promoting the
weathering of potassium-bearing minerals have been pre-
viously reported, and earthworms’ gut microbes may play
an important role in increasing the rates of mineral
weathering (Basker et al. 1994; Carpenter et al. 2007,
2008; Liu et al. 2011). Therefore, earthworms’ guts are
potential resources from which some potassium solubiliz-
ing microorganisms could be isolated. In this study, a
potassium solubilizing bacterium, which tolerated high
temperature, was isolated from the gut of the earthworms
after they fed on potassium-bearing rock powder for
10 days. Subsequently, this strain was used to degrade
potassium-bearing rock powder by solid-state fermentation
to examine its weathering ability.
2 Materials and methods
2.1 Preparation of K-bearing minerals, earthworms
and other materials
Potassium-bearing rock resource is rich in Luoyang, Henan
Province, China. To develop and utilize the resource, we
collected some PBR from Luoyang. The rock was crushed
and sieved to pass through a 0.074 mm mesh. The mineral
composition and the elemental composition of the rock
were determined using the K-value method of X-ray
diffraction (Rigaku, D/MAx-2200) (Li et al. 2003) and
X-ray Fluorescence (Axios, PW4400), respectively, in the
Institute of Geochemistry, Chinese Academy of Sciences.
The rock’s mineral composition contains feldspar
(70.99 %), quartz (23.45 %), hematite (3.62 %), montmo-
rillonite (1.66 %), illite (0.15 %) and hornblende (0.13 %).
The major element oxides of the PBR are K2O (6.36 %),
Al2O3 (11.52 %), SiO2 (70.36 %), Fe2O3 (0.53 %), CaO
(0.38 %), MgO (0.06 %), Na2O (2.53 %) and Loss on
ignition (LOI, 8.02 %).
The earthworms (Eisenia foetida) were purchased from
Henan Academy of Agricultural Sciences. Oyster mush-
room culture waste was collected from Henan Institute of
Science and Technology. Wheat bran and sugar were
purchased from the market.
2.2 Isolation of K-solubilizing bacteria
Seven hundred grams of K-bearing rock powder, mixed
with 200 mL of deionized water, was autoclaved at 121 �C
for 30 min. After cooling, 35 earthworms were placed into
the mineral powder and fed for 10 days. The feeding
containers were covered to avoid light; the laboratory
temperature was about 25 �C during feeding. The earth-
worms were sedated, surface sterilized with ethanol (75 %)
and dissected under sterile conditions after feeding on
mineral powder for 10 days. The whole gut content of each
specimen without gut wall was extracted with a sterile
spatula and transferred into sterile 2 mL tubes. The gut
content was immersed with sterile distilled water, and
shaken at 200 rpm for 10 min at 28 �C to detach the bac-
teria from the mineral particles. The liquid mixture was
allowed to stand for 20 min to obtain the microbe-con-
taining supernatant to isolate the mineral-solubilizing
bacteria. The medium for isolating mineral-solubilizing
bacteria was Aleksandrov’s medium (sucrose 5.0 g,
MgSO4 0.5 g, CaCO3 0.1 g, Na2HPO4�12H2O 2.0 g,
FeCl3�6H2O 0.005 g, potassium-bearing rock powders
1.0 g, agar 18.0 g, H2O 1.0 L, pH 7.0–7.2)(Li et al.
2008), a common medium used to isolate silicate-weath-
ering bacteria. The supernatant was serially diluted, and the
aliquots of each dilution were spread on plates of Alek-
sanderov’s medium and incubated at 28 �C for 2–3 days.
Selected colonies were further purified using the same
medium. The colonies were stored at 4 �C until further use.
The above microbial strains were inoculated to Alek-
sanderov’s medium and incubated at 50 �C for 2 days. The
colonies, which grew well at 50 �C, were selected and stored
on slants for studying the solubility of K-bearing rock powder.
Acta Geochim (2016) 35(3):262–270 263
123
2.3 Morphology and biochemical characterization
of the strain
The mycelia of the isolates, identified as species belonging
to the genus Streptomyces by analyzing their morphologi-
cal characteristics and by biochemical tests, were observed
by using the cover slip method, in which sterile square
cover slips were inserted at an angle of 45� in sterile
Aleksandrov’s agar medium in petri dishes. Individual
cultures would grow to the intersection of the medium and
cover slips. The cover slips were removed after three days
of incubation, air-dried and observed with a microscope.
Morphological characters were photographed (Thakur et al.
2007).
Biochemical tests, including Gram staining, nitrate
reduction, amylohydrolysis, Voges–Proskauer reaction,
and catalase activities, were performed according to the
method we used in a previous study (Liu et al. 2012).
2.4 DNA extraction, amplification and sequencing
Cultures for DNA extraction were prepared by inoculating
100 mL of yeast extract medium (YEM) with 100 lL of
spore suspension. The YEM medium [sucrose 10.0 g, yeast
extract 0.2 g, (NH4)2SO4 0.5 g, CaCO3 1.0 g, MgSO4
0.5122 g, KCl 0.1 g, Na2HPO4�12H2O 2.507 g, H2O
1.0 L, pH 7.0–7.2] was prepared as previously described
(Zhou et al. 2010). After incubating at 37 �C for 48 h on a
rotary shaker at the rate of 180 rpm, the culture was
transferred to a centrifuge tube (2 mL), and centrifuged at
10,0009g for 10 min to concentrate cells into a pellet for
extraction of bacterial genomic DNA. DNA was extracted
using a Bacterial Genomic DNA Extraction Kit (Omega,
US) according to the manufacturer’s instructions.
The 16S rRNA gene was amplified by polymerase chain
reaction (PCR) using bacterial universal primers 16S-fD1
(50-AGAGTTTGATCCTGGCTCAG-30) and 16S-rD1 (50-ACGGTTACCTTGTTACGACTT-30) (Weisburg et al.
1991). The reaction mixture for PCR amplification was
prepared as follows: 0.4 mM of deoxynucleoside triphos-
phates, 0.4 lM of each primer, 1 9 PCR buffer, 2 mM
magnesium chloride, 1 U of Taq DNA polymerase, and 1 lL
(about 5–15 ng) of template DNA in a final volume of
30 lL. Amplification was made using a touchdown proce-
dure, as follows: the annealing temperature was set at 65 �C
and decreased by 1 �C every cycle until reaching a ‘‘touch-
down’’ at 55 �C. The amplification program consisted of
5 min at 94 �C, and 10 touchdown cycles of denaturation at
94 �C for 1 min, annealing at 65 �C (with the temperature
decreasing 1 �C each cycle) for 1 min, and extension at
72 �C for 2 min, followed by 25 cycles of 94 �C for 1 min,
55 �C for 1 min, and 72 �C for 2 min. During the last cycle,
the length of the extension step was increased to 10 min.
After amplification, the PCR product was checked by elec-
trophoresis in 1.5 % (w/v) agarose gels.
The PCR product was excised from 2 % low melting
point agarose (Sigma, St-Louis, MO) and the DNA was
purified using a Gel Isolation Kit following the manufac-
turer’s instructions (Promega, Madison, WI, USA). The
purified PCR product was sequenced by Shanghai Sangon
Biological Engineering Technology & Services Co., Ltd.
2.5 Phylogenetic analysis
The obtained 16S rRNA gene sequence was compared to
those in the National Center for Biotechnology Information
database using the BLAST procedure (Altschul et al.
1997). The reference sequences with the highest similari-
ties to the query were retrieved from GenBank. The target
16S rRNA gene sequence and the retrieved reference
sequences were aligned with the ClustalX program (Larkin
et al. 2007) with parameters set to its default values.
Alignments were improved by comparison to the secondary
structures and any regions of uncertain alignment were
omitted from the subsequent analyses.
The best-fit model of nucleotide substitution for the data
sets was selected using the program PAUP 4.0b10 (Swof-
ford 2002) and Modeltest 3.06 (Posada and Crandall 1998).
Base composition and sequence variability were examined
using the software package MEGA4.0 (Tamura et al.
2007). A minimum evolution (ME) phylogenetic tree with
1000 bootstrap was then constructed using the Tamura-Nei
model, with the pairwise deletion of gaps by Mega 4.0
package.
The outgroup used for further phylogenetic analysis was
based on the above result of ME. Further phylogenetic
analysis, conducted using PAUP 4.0b10 with ME method,
was performed using heuristic searches with the random
stepwise addition of 10 replicates and tree bisection-re-
connection (TBR) rearrangements in effect. The optimal
model of nucleotide substitution for the ME analyses,
HKY85, was from the result of the best-fit nucleotide
substitution model selection. Bootstrap analyses were
performed for 1000 replicates to evaluate the robustness of
each clade.
2.6 Mineral weathering by the bacterial isolate
The isolate was revitalized on plates of Aleksanderov’s
medium and incubated at 37 �C for 3–4 days to obtain its
spores. The spores and aerial mycelia were scraped off the
agar surface, using a sterilized inoculation loop, when a
mass of gray spores appeared on the plates. The spore
suspension was then prepared in sterile H2O.
The solid-state medium for the assessment of the effect
of bacteria on mineral weathering was prepared as follows:
264 Acta Geochim (2016) 35(3):262–270
123
the waste of the oyster mushroom culture (66 %) was
grounded, dried and mixed with rock powder (16 %),
wheat bran (16 %) and sugar cane dregs (2 %). Water was
added into the mixture; the ratio of water to mixture was
1.1: 1 (v/m). The mixed substrate (about 1.5 kg wet wt)
was poured into heat resistant polypropylene bags and
sterilized (substrate temperature 121 �C) for 30 min.
After the substrate had cooled, it was inoculated with
500 mL of spore suspension (108 spore mL-1). Then, the
substrate was incubated at 37 �C for 14 days. The control,
whose substrate components were the same with the above
treatment, was inoculated with autoclaved inoculum, and
also incubated at 37 �C for 14 days. Each treatment was
carried out on three biological replicates. The substrates,
incubated for 14 days, were sampled to determine their
water-soluble elements’ content.
2.7 Determination of water-soluble K, Al, Fe and Ca
The samples for determining elemental concentration were
prepared by drying them in an oven at 70 �C. To assess the
isolate’s potential for enhancing mineral weathering, con-
centrations of water-soluble K, Al, Fe and Ca of the three
samples, including the fresh solid-state medium, the sub-
strate inoculated with autoclaved inoculum and the one
inoculated with living bacterium, were determined by
inductively coupled plasma atomic emission spectrometry
(ICP-AES, Optima 2100 DV, Perkin Elmer, USA).
Determination of the concentration of water-soluble K,
Al, Fe and Ca was performed using the methods as pre-
viously described (Liu et al. 2011) with the following
modifications: five grams of a dried sample was mixed with
50 mL of ddH2O in a 250 mL flask followed by 30 min of
vigorous shaking. After shaking, the samples were cen-
trifuged at 8000 rpm for 10 min. The supernatants were
filtered through a 0.45 lm filter paper to obtain a purified
extract. The concentrations of water-soluble K, Al, Fe and
Ca in the purified extract were determined by ICP-AES.
3 Results
3.1 Isolation of mineral potassium-solubilizing
bacteria tolerant to high temperature
After isolation and repeated purification, seven fast-growing
microbial strains were obtained from the Alexander silicate
medium at 28 �C. These strains were inoculated into the
Alexander silicate medium and cultivated in an incubator at
50 �C. One strain was found growing in the medium and
forming a strain colony; this strain was labeled ‘EGT’.
Strain EGT grew well in the solid Alexander silicate med-
ium at 28 and 37 �C. At 50 �C, however, the rate of growth
was reduced and the strain colonies were small. EGT’s
colonies appeared to be white, dry and intensely woolly.
After a while, gray spores appeared on the surface of the
colonies (see Fig. 1). Furthermore, white mycelium pellets
formed when EGT was put into a liquid medium at a con-
stant temperature of 28/37 �C for 28 h with agitation at 180
r/min. The Strain EGT was Gram-positive.
The results of the measurements, including nitrate
reduction, amylohydrolysis and catalase activities, were all
positive; The Voges-Proskauer reaction, however, pro-
duced a negative result. The results of morphological
observations were given in Fig. 1. After the strain grew on
for 3 days, a mass of gray spores appeared on the aerial
hyphae. Straight spore chains could be clearly observed
with an optical microscope.
3.2 Identification and phylogeny of strain EGT
The 16S rRNA gene sequence of Strain EGT derived from
clones has been submitted to GenBank (accession number
JF701918). The gene sequence length of 16rRNA is 1381 bp,
Fig. 1 Colonies of the strain EGT and optical micrograph (9 400) of
its mycelium. The top figure showed the colonies of the strain EGT
grown on solid Aleksanderov’s medium at 28 �C for three days. Gray
spore had been formed. The bottom figure showed the morphological
character of the strain EGT’s mycelium observed with an optical
microscope
Acta Geochim (2016) 35(3):262–270 265
123
among which A occupies 22.3 % (308 bp), T 18.0 %
(249 bp), G 33.8 % (467 bp), C 25.9 % (357 bp), A ? T
40.3 % (557 bp) and C ? G 59.7 % (824 bp). The four base
group percentages are similar to those in the 16S rRNA gene
sequences of other Streptomyces in the GenBank.
The 16S rRNA gene sequence was analyzed by BLAST in
the sequence base of GenBank. The results show that the most
similar 16S rRNA gene sequences all belong to Streptomyces
variants, indicating that EGT is one of the Streptomyces. The
differences between the EGT and the thirteen 16S rRNA
Fig. 2 The divergences of 16S rRNA gene sequences between the strain EGT and some Streptomyces species
Streptomyces aureofaciens (HQ132791) Streptomyces variabilis (EU841659) Streptomyces erythrogriseus (AJ781328) Streptomyces pseudogriseolus (AB184516) Streptomyces griseorubens (AB184139) Streptomyces labedae (AB184704)
EGT Streptomyces almquistii (AY999782)|
Streptomyces althioticus (HQ132792) Streptomyces althioticus (AB184112)
Streptomyces matensis (AB184221) Streptomyces xylophagus (AB184526)
Streptomyces viridochromogenes (AB184075) Streptomyces viridochromogenes (EU812168)
Streptomyces tendae (NR_025871) Streptomyces tritolerans (DQ345779)
Streptomyces tendae (EU741192) 49100
99
5551
70
75
54
98
70
19
0.001
Group I
Group II
Group III
Out group
Fig. 3 The minimum evolution tree for Tamura–Nei distance matrix of the nearly complete 16S rRNA gene sequences of the strain EGT and
some Streptomyces species using the software package MEGA4.0. Numbers on nodes correspond to percentage bootstrap values for 1000
replicates
266 Acta Geochim (2016) 35(3):262–270
123
gene sequences of Streptomyces are shown in Fig. 2. It can be
seen that the sequence difference is negligible. In particular,
the divergence from S. griseorubens, S. labedae, S. erythro-
griseus, S. variabilis and S. pseudogriseolus is only 0.1 %,
which is within the criterion 16S rRNA B3 % proposed for
agreement (Embley and Stackebrandt 1994).
Multiple alignments using Clustal X, suggest that the
strain’s 16S rRNA gene sequence is 1384 bp (including
gap), among which A occupies 22.3 %, T 18.2 %, C 25.8 %,
G 33.7 %, A ? T 40.5 % and C ? G 56.5 %. We also
observe that the concentration of C ? G is higher than that of
A ? T, the average sequence divergence is 0.5 % (p-dis-
tance), and the average value of the transforma-
tion/transversion ratio is 1.273. We note that the disparity
between the base groups greatly affects the reconstruction of
the phylogeny (Lockhart et al. 1994). The results reveal that
there are differences in the base groups; these differences
must be considered when the phylogeny is analyzed. In
addition, when the value of transformation/transversion is
less than 2.0, the mutation of the gene sequence is in a sat-
uration state and is easily affected by evolutionary noise.
Consequently, errors may occur if extra weights are not
added when reconstructing phylogeny (Knight and Mindell
1993; Liu and Jiang 2005). The average value of transfor-
mation/transversion in the experiment is 1.273 and this has to
be accounted for during the phylogeny analysis. The result of
a hierarchical likelihood test performed using Model test
shows that the best model in the phylogeny analysis is the
Hasegawa–Kishino–Yano (HKY) model. This model con-
siders both the differences in the base groups and the trans-
formation/transversion ratio, and is thus suitable for
phylogeny analysis of the current data group.
Figure 3 shows the ME phylogeny based on the
Tamura-Nei model derived using MEGA 4.0 software.
Four branches are given: the first branch includes S.
aureofaciens, S. variabilis, S. erythrogriseus, S. pseudo-
griseolus, S. griseorubens, S. labedae and the strain EGT;
the second branch includes S. xylophagus, S. althioticus, S.
matensis and S. almquistii; the third includes S. viri-
dochromogenes; and the fourth S. tendae and S. tritolerans.
Figure 3 reveals that these latter, fourth-branch strains are
the sister group of the other bacteria. Thus, these two
strains have been used as outgroups when the phylogeny
was reconstructed using PAUP 4.0 software.
The ME phylogeny based on the HKY85 model was
established using PAUP 4.0 software (seen in Fig. 4). The
35
10
Streptomyces aureofaciens (HQ132791)
Streptomyces labedae (AB184704)
Streptomyces variabilis (EU841659)
Streptomyces erythrogriseus (AJ781328)
Streptomyces pseudogriseolus (AB184516)
Streptomyces griseorubens (AB184139)
EGT
Streptomyces althioticus (AB184112)
Streptomyces matensis (AB184221)
Streptomyces xylophagus (AB184526)
Streptomyces althioticus (HQ132792)
Streptomyces almquistii (AY999782)|
Streptomyces viridochromogenes (AB184075)
Streptomyces viridochromogenes (EU812168)
Streptomyces tendae (NR_025871)
Streptomyces tritolerans (DQ345779)
Streptomyces tendae (EU741192)
86
99
100 56
22
27
48
66
93
62
27
21
24
Group I
Group II
Group III
Out group
Fig. 4 The minimum evolution
tree for HKY85 distance matrix
based on nearly complete 16S
rRNA gene sequences of the
strain EGT and some
Streptomyces species using the
program PAUP 4.0b10.
Numbers on nodes correspond
to percentage bootstrap values
for 1000 replicates
Acta Geochim (2016) 35(3):262–270 267
123
topological features of the phylogeny in this figure are
similar to those in Fig. 3. In addition to the outgroups, the
ingroups of the phylogenetic tree also have three branches
that are similar to those in Fig. 3. Furthermore, the strain
EGT also belongs to the first branch here. These two
phylogenetic trees verify that EGT is one of the
Streptomyces.
3.3 Effects of strain EGT on weathering of PBR
After the PBR powders were degraded by EGT through
solid-state fermentation, the changes in the aqueous con-
centrations of major elements were tested to assess the
isolate’s potential for enhancing mineral weathering. The
results are shown in Fig. 5 and indicate that the proportions
of water-soluble Al, Fe and K in the substrate incubated with
live inoculum for 14 days were higher than those obtained
from the control with autoclaved inoculum and the fresh
substrate. The amounts of aqueous Al, Fe and K in the active
bacterial culture were 23.08 %, 123.19 % and 30.99 %,
respectively, higher than that of the fresh substrate and were
45.45 %, 52.48 % and 9.13 %, respectively, higher than that
of the abiotic control incubated with autoclaved inoculum.
The results reveal that EGT has a significant effect on the
weathering of potassium-bearing mineral powders.
4 Discussion
4.1 Phylogeny and molecular identification of strain
EGT
Streptomyces is the main genus of the actinomycete
microorganism. It is the most prevalent among the acti-
nomycetes in soil, and contributes to the soil ecology and
circulation of materials (Oskay 2009; Srivibool et al.
2010). Streptomyces have a diverse range of species and
metabolism, and are important microbial resource for
humans. At present, the number of Streptomyces granted
legal names exceeds 600 (Kampfer et al. 2008). Since the
1940s, some 55 % of the physiologically active substances
from 12,000 microorganisms have been produced by
Streptomyces (Xu et al. 2001).
Considering its 16S rRNA gene sequence and molecular
phylogeny, the strain EGT obtained from the earthworm
intestinal tract can be identified as a species of Streptomyces.
Fresh substrate Control Bacterium treating
Wat
er-s
olub
le A
l (m
g/kg
)
Fresh substrate Control Bacterium treating
Wat
er-s
olub
le C
a (m
g/kg
)
Fresh substrate Control Bacterium treating
Wat
er-s
olub
le F
e (m
g/kg
)
Fresh substrate Control Bacterium treating
Wat
er-s
olub
le K
(mg/
kg)
Fig. 5 Amount of water-soluble elements released by the strain EGT after incubating for 14 days through solid-state fermentation (Each value
represented the average of three biological replicates). Fresh substrates were the solid-state medium before composting. Controls were incubated
for fourteen days by autoclaved inoculum. Error bars are ± SD (n = 3)
268 Acta Geochim (2016) 35(3):262–270
123
The identification and classification of the microbial strain
underlies its application. However, all of the systems used to
identify bacteria, regardless of whether they are phenotypic
or genotypic, have their limitations, because no single test
methodology provides results that are 100 % accurate (Janda
and Abbott 2002). The main approach used for the identifi-
cation and classification of prokaryotic species is based on its
16S rRNA gene sequence and phylogenetic analysis. Ber-
gey’s classification system, widely accepted by microbiol-
ogists and currently considered as the best approximation to
an official classification, is based on the homologous and
phylogenetic analysis of 16S rRNA genes and the classical
microscopic and biochemical observation of the relatedness
of the organisms (Clarridge 2004). Strains whose similarity
to the 16S rRNA gene sequence are more than 95 % can be
classified into a genus (Clarridge 2004), and the strains
whose similarity are more than 97 % can be classified into a
species (Embley and Stackebrandt 1994).
Despite the EGT being confirmed as one of the Strepto-
myces by its phylogenetic position and similarity to the 16S
rRNA gene sequence with other Streptomyces, the species
name of this strain cannot currently be ascertained. The
maximum sequence divergence of the 16S rRNA gene
between EGT and the thirteen Streptomyces downloaded
from GenBank is just 1.2 %, among which the sequence
divergence between EGT and the five bacteria, including S.
griseorubens, S. pseudogriseolus. S. erythrogriseus, S. vari-
abilis and S. labedae, is only 0.1 %. This suggests a general
lack of suitability for Streptomyces. The strains can however
be classified into a species when their 16S rRNA gene has a
similarity greater than 97 %. This is generally used as a cri-
terion for most prokaryotic species. It seems the 16S rRNA
gene does not have sufficient resolution to differentiate the
genus and the species (Kampfer et al. 2008). Due to the
complexity of the Streptomyces classification and a lack of
acknowledged standard, the conception of species for this
genus in the systematics has always been a difficult problem
(Syed et al. 2007). To unequivocally identify the EGT strain,
extensive phenotypic and genotypic testing is required.
4.2 Effect of EGT on the weathering of PBR
Microorganisms play an important role in the process of
mineral weathering. They promote the formation of soil
and they offer plants nutrients such as phosphorus, potas-
sium and silicon (Banfield et al. 1999; Kim et al. 2004; Hall
et al. 2005). For instance, potassium-solubilizing bacteria
can release potassium from minerals, promote the growth
of plants, improve harvests, strengthen the anti-virus ability
of plants and improve soil quality (Sheng 2005a; Basak and
Biswas 2009, 2010; Youssef et al. 2010). It has a wide
application in agriculture.
Many microorganisms have been found to possess this
potassium-releasing ability, e.g. Pseudomonas, Burkholde-
ria, Acidithiobacillus ferrooxidans, Bacillus mucilaginosus,
B. edaphicus and B. circulans, etc. (Lian et al. 2002; Sheng
2005b; Zhao et al. 2008). However, there are still limitations
to the use of these bacteria as microbial fertilizers. First,
microbial fertilizers are usually manufactured using com-
posting at a high temperature, often above 50 �C. As these
bacteria’s normal living temperature is far lower than 50 �C,
they may die at the high temperatures used. In addition to
this, although some bacteria, such as Bacillus mucilaginosus,
have good potassium-releasing capability, and they also
release excessive amounts of polysaccharide during the
fermentation, which makes the fermentation broth very thick
and viscid. This makes it difficult to produce the required
liquid inoculum for microbiological fertilizers that use the
liquid state deep-seated fermentation method.
In contrast, the strain EGT has many advantages. Firstly,
it can successfully weather potassium-bearing mineral
powders and it can resist high temperatures and survive at
50 �C. Secondly, procedures for seed preparation are
simple due to the large number of spores yielded by the
EGT after the desired incubating time. Therefore, EGT is a
good choice for use in microorganism fertilizers.
Acknowledgments This work was supported by the National Natural
Science Foundation of China (41173091, U1204405), Aid Project for
the Leading Young Teachers in Henan Provincial Institutions of Higher
Education of China (2012GGJS-284), and Natural Science Foundation
of Henan Educational Committee, China (12B180027, 14B180010).
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