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

bin2368@vip.163.com; hn_ldf@126.com

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).

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

Amtmann A, Armengaud P (2007) The role of calcium sensor-

interacting protein kinases in plant adaptation to potassium-

deficiency: new answers to old questions. Cell Res 17:483–485

Armengaud P, Sulpice R, Miller AJ, Stitt M, Amtmann A, Gibon Y

(2009) Multilevel analysis of primary metabolism provides new

insights into the role of potassium nutrition for glycolysis and

nitrogen assimilation in arabidopsis roots. Plant Physiol

150:772–785. doi:10.1104/pp.108.133629

Banfield JF, Barker WW, Welch SA, Taunton A (1999) Biological

impact on mineral dissolution: application of the lichen model to

understanding mineral weathering in the rhizosphere. Proc Natl

Acad Sci USA 96:3404–3411

Basak BB, Biswas DR (2009) Influence of potassium solubilizing

microorganism (Bacillus mucilaginosus) and waste mica on

potassium uptake dynamics by sudan grass (Sorghum vulgare

Pers.) grown under two Alfisols. Plant Soil 317:235–255

Basak BB, Biswas DR (2010) Co-inoculation of potassium solubi-

lizing and nitrogen fixing bacteria on solubilization of waste

mica and their effect on growth promotion and nutrient

acquisition by a forage crop. Biol Fert Soils 46:641–648.

doi:10.1007/s00374-010-0456-x

Acta Geochim (2016) 35(3):262–270 269

123

Basker A, Kirkman JH, Macgregor AN (1994) Changes in potassium

availability and other soil properties due to soil ingestion by

earthworms. Biol Fert Soils 17:154–158

Biswas DR (2010) Nutrient recycling potential of rock phosphate and

waste mica enriched compost on crop productivity and changes

in soil fertility under potato–soybean cropping sequence in an

Inceptisol of Indo-Gangetic Plains of India. Nutr Cycl Agroe-

cosyst, pp. 1–16

Carpenter D, Hodson ME, Eggleton P, Kirk C (2007) Earthworm

induced mineral weathering: preliminary results. Eur J Soil Biol

43:S176–S183

Carpenter D, Hodson ME, Eggleton P, Kirk C (2008) The role of

earthworm communities in soil mineral weathering: a field

experiment. Mineral Mag 72:33–36

Chen J (2000) Development and utilization of potassium-bearing rock

resource and their prospects. Geol Chem Miner 22:58–64

Chen HC, Wang SY, Chen MJ (2008) Microbiological study of lactic

acid bacteria in kefir grains by culture-dependent and culture-

independent methods. Food Microbiol 25:492–501

Clarridge JE (2004) Impact of 16S rRNA gene sequence analysis for

identification of bacteria on clinical microbiology and infectious

diseases. Clin Microbiol Rev 17(4):840–862

Embley TM, Stackebrandt E (1994) The molecular phylogeny and

systematics of the Actinomycetes. Annu Rev Microbiol

48:257–289

Ghiri MN, Abtahi A, Jaberian F, Owliaie HR (2010) Relationship

between soil potassium forms and mineralogy in highly

calcareous soils of southern Iran. Aust J Basic Appl Sci

4:434–441

Hall K, Arocena JM, Boelhouwers J, Liping Z (2005) The influence

of aspect on the biological weathering of granites: observations

from the Kunlun Mountains, China. Geomorphology

67:171–188. doi:10.1016/j.geomorph.2004.09.027

Jalali M (2006) Kinetics of non-exchangeable potassium release and

availability in some calcareous soils of western Iran. Geoderma

135:63–71. doi:10.1016/j.geoderma.2005.11.006

Janda JM, Abbott SL (2002) Bacterial identification for publication:

when is enough enough? J Clin Microbiol 40:1887–1891. doi:10.

1128/jcm.40.6.1887-1891.2002

Kampfer P, Huber B, Buczolits S, Thummes K, Grun-Wollny I, Busse

HJ (2008) Streptomyces specialis sp. nov. Int J Syst Evol

Microbiol 58(11):2602–2606

Kim J, Dong H, Seabaugh J, Newell SW, Eberl DD (2004) Role of

microbes in the smectite-to-illite reaction. Science 303:830–832.

doi:10.1126/science.1093245

Knight A, Mindell DP (1993) Substitution bias, weighting of DNA

sequence evolution, and the phylogenetic position of Fea’s viper.

Syst Biol 42(1):18–31

Larkin MA et al (2007) Clustal W and Clustal X version 2.0.

Bioinformatics 23:2947–2948. doi:10.1093/bioinformatics/

btm404

Li YH, Xu FG, Wang J, Song ZK (2003) Quantitative analysis is of

ettringite in cement hydration products by ‘‘value K’’ method of

XRD. Chin J Spectrosc Lab 20:334–337

Li ZG, Luo YM, Teng Y (2008) Research method of soil and

environmental microbiology. Science Press, Beijing

Lian B, Fu PQ, Mo DM, Liu CQ (2002) A comprehensive review of

the mechanism of potassium releasing by silicate bacteria. Acta

Mineral Sin 22:179–183

Lin YH (2010) Effects of potassium behaviour in soils on crop

absorption. Afr J Biotechnol 9:4638–4643

Liu DF, Jiang GF (2005) Molecular phylogenetic analysis of

Acridoidea based on 18S rDNA with a discussion on its

taxonomic system. Acta Entomol Sin 48(2):232–241

Liu D, Lian B, Wang B, Jiang G (2011) Degradation of potassium

rock by earthworms and responses of bacterial communities in

its gut and surrounding substrates after being fed with mineral.

PLoS One 6:e28803. doi:10.1371/journal.pone.0028803

Liu D, Lian B, Dong H (2012) Isolation of Paenibacillus sp. and

assessment of its potential for enhancing mineral weathering.

Geomicrobiol J 29:413–421. doi:10.1080/01490451.2011.

576602

Nishanth D, Biswas DR (2008) Kinetics of phosphorus and potassium

release from rock phosphate and waste mica enriched compost

and their effect on yield and nutrient uptake by wheat (Triticum

aestivum). Bioresour Technol 99:3342–3353

Oskay M (2009) Comparison of Streptomyces diversity between

agricultural and non-agricultural soils by using various culture

media. Sci Res Essay 4:997–1005

Peterburgsky AV, Yanishevsky FV (1961) Transformation of forms

of potassium in soil during long-term potassium fertilization.

Plant Soil 15:199–210. doi:10.1007/bf01400454

Posada D, Crandall KA (1998) MODELTEST: testing the model of

DNA substitution. Bioinformatics 14:817–818

Sheng XF (2005) Growth promotion and increased potassium uptake

of cotton and rape by a potassium releasing strain of Bacillus

edaphicus. Soil Biol Biochem 37:1918–1922

Srivibool R, Jaidee K, Sukchotiratana M, Tokuyama S, Pathom-aree

W (2010) Taxonomic characterization of Streptomyces strain

CH54-4 isolated from mangrove sediment. Ann Microbiol

60:299–305. doi:10.1007/s13213-010-0041-4

Sugumaran P, Janarthanam B (2007) Solubilization of potassium

containing minerals by bacteria and their effect on plant growth.

World J Agric Sci 3:350–355

Sun AW, Zhang WF, Du F, Gao LW, Zhang FS, Chen XP (2009)

China’s development strategy on potash resources and fertilizer.

Mod Chem Ind 29(9):10–16

Swofford DL (2002) PAUP* phylogenetic analysis using parsimony

(* and other methods), Version 410b10. Sinauer Associates,

Sunderland

Syed DG, Agasar D, Kim CJ, Li WJ, Lee JC, Park DJ, Xu LH, Tian

XP, Jiang CL (2007) Streptomyces tritolerans sp. nov., a novel

actinomycete isolated from soil in Karnataka, India. Antonie van

Leeuwenhoek 92(4):391–397

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular

evolutionary genetics analysis (MEGA) software version 4.0.

Mol Biol Evol 24:1596–1599

Thakur D, Yadav A, Gogoi BK, Bora TC (2007) Isolation and

screening of Streptomyces in soil of protected forest areas from

the states of Assam and Tripura, India, for antimicrobial

metabolites. J Mycol Med 17:242–249

Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S

Ribosomal DNA amplification for phylogenetic study. J Bacteriol

173:697–703

Xu P, Zhang LP, Yu LY (2001) Classification of Streptomyces with

the V-2 variable region in 16S rDNA. Biodivers Sci

9(2):129–137

Youssef GH, Seddik WMA, Osman MA (2010) Efficiency of natural

minerals in presence of different nitrogen forms and potassium

dissolving bacteria on peanut and sesame yields. J Am Sci

6:647–660

Zangerle A, Pando A, Lavelle P (2011) Do earthworms and roots

cooperate to build soil macroaggregates? A microcosm exper-

iment. Geoderma 167–168:303–309. doi:10.1016/j.geoderma.

2011.09.004

Zhao F, Sheng XF, Huang Z, He LY (2008) Isolation of mineral

potassium-solubilizing bacterial strains from agricultural soils in

Shandong Province. Biodiver Sci 16:593–600

Zhou XY, Du Y, Lian B (2010) Effect of different culture conditions

on carbonic anhydrase from Bacillus mucilaginosus inducing

calcium carbonate crystal formation. Acta Microbiol Sin

50:956–962

270 Acta Geochim (2016) 35(3):262–270

123