Phosphate solubilizing bacteria from Earthworm Burrow
Wall soil
“Without the work of this humble creature, who knows nothing of the
benefits he confers upon mankind, agriculture, as we know it, would be very
difficult, if not wholly impossible”
Charles Darwin on Earthworms
Introduction
Phosphorus (P) is an essential element for plant development and growth making up
about 0.2 % of plant dry weight. After nitrogen P the second most limiting element, plays
an important role in plant metabolism by supplying energy required for metabolic
processes (Lal 2002; Vance et al., 2000, 2001). Plants acquire P from soil solution as
phosphate anions. However, phosphate anions are extremely reactive and may be
immobilized through precipitation with cations such as Ca2+, Mg2+, Fe3+ and Al3+,
depending on the properties of a soil. In these forms, P is highly insoluble and
unavailable to plants. Phosphorus has to be converted into soluble forms by phosphatase
enzyme such as acidic and alkaline phosphatases. Among the great variety of enzymes
that are produced by soil microorganisms, during their metabolism (Acosta-Martinez
2000), the enzyme phosphatase is produced to convert P into soluble forms. Several
scientists have reported the ability of different bacterial species to solubilize insoluble
inorganic phosphate compounds, such as tricalcium phosphate, dicalcium phosphate,
hydroxyapatite, and rock phosphate. This group of beneficial bacteria capable of
hydrolyzing organic and inorganic phosphorus from insoluble compounds (Chen et al.,
2006) and making it available for plants are called phosphate solubilizing bacteria (PSB).
They are involved in a range of processes that affect the transformation of soil P and thus
are an integral part of the soil P cycle effectively in releasing inorganic and organic pools
of total soil P through solubilization and mineralization (Hilda and Fraga 1999). These
bacteria convert phosphorus into soluble forms by phosphatase enzyme and produce
amino acids, vitamins and growth promoting substances like indole acetic acid (IAA) and
gibberellic acid (GA3) which help in soil fertility. Many reports suggest that a number of
soil bacteria possess mineral phosphate solubilizing activity (Yahya and Al- Azawi 1989;
Mikanova and Kubat 1994). Strains from the genera Pseudomonas, Bacillus and
Rhizobium are among the most powerful P solubilizers (Rodriguez and Fraga 1999) and
can increase the P uptake by plants by making it available to plants. Phosphatase activity
measurement provides an index of potential availability of phosphatase in soil (Mansell
1981). P-solubilization ability of the microorganisms is considered to be one of the most
important traits associated with plant P nutrition and can improve the effectivity of
mineral P transformation.
Free-living PSB are always present and commonly isolated from soil. Significant
components of the soil are the earthworms which are recognized as important to plant
litter decomposition and fertility of soil playing a complex role which involves the
assistance of microorganisms. Among beneficial soil microbes stimulated by earthworms
are nitrogen-fixing and phosphate solubilizing bacteria, the actinomycetes and
mycorrhizal fungi. Suhane et al., (2007) found that the total bacterial count was more
than 1010/g of vermicompost. It included actinomycetes, Azotobacter, Rhizobium,
Nitrobacter and phosphate solubilizing bacteria, ranging from 102 - 106 per g of
vermicompost. While many studies have examined impacts of earthworm on carbon and
nitrogen fluxes in soils (Bohlen et al., 1997; Bouche and Al- Addan et al., 1997; Lavelle
et al., 1997), less attention has been paid to how and to what extent earthworms influence
the dynamics of soil phosphorus.
Earthworm casts contain large amounts of soluble nitrogen, phosphorus and organic
carbon and burrow linings may be expected to be enriched by these elements. Earthworm
burrow wall hence creates a favorable microhabitat for the soil microflora and plants. The
increased amount of inorganic phosphorus released during cast deposition was related to
and preceded by increased microbial and phosphatase activity (Sharpely and Syers,
1976). High P2O5 content in casts supports phosphatase availability which is required for
growth of root, microbial enhancement and in turn, may help drive biological nitrogen
fixation (Sharpely and Syers 1976). Recently, enhanced phosphate content in the soil and
press mud casts of Lampito mauritti has been reported. Kale and Bano (1986) report as
high as 7.37% of nitrogen and 19.58% of phosphorus as P2O5 in worm’s vermicast.
Satchel and Martin (1984) have found direct correlation between microbial population
and enzyme activity. Microbes like Pseudomonas and Bacillus species are reported to
mineralize phosphate (Dubey and Maheshwari 1999). Reports suggest that enhanced
phosphatase activity in the casts with more microbial population is of microbial origin
rather than by the epithelium of the gut of the earthworm (Vinotha et al., 2000). This
study involves the isolation of PSB from the burrow wall of Lampito mauritii and
Pontoscolex corethrurus.
Methods and Materials
Generation of Soil Samples
The soil sample was generated as described in chapter 2.
Isolation of phosphate solubilizing bacteria

The soil samples were air dried and used for isolation of PSB.

Aliquots of serial diluted soil samples (10-5) were aseptically pour plated on the
Pikovskaya’s medium containing suspended insoluble phosphate compound
(tricalcium phosphate) and bromothymol blue indicator.

The plates were incubated for 24-48 hours at 37oC.

Bacterial colonies causing clear phosphate solubilizing yellow halos following pH
drop through the release of organic acids were selected and isolated.

The total number of bacteria in the plate was counted, so also the colonies with a
clear halo.

The percentage of phosphate solubilising bacteria were calculated as follows

The diameter of clear zone was measured in addition to the colony diameter.

The pure cultures were maintained in Pikovskaya’s medium
Calculation of the hydrolyzing capacity (HC) value
HC value on the Pikovskaya’s agar was indexed as the diameter of the colony plus the
clear zone around it divided by the diameter of the colony (Hankin and Anagnostakis
1977; Hendricks et al., 1995).
Selected isolates from L.mauritti and P.corethrurus burrow wall and control soil showing
high HC value and phosphatase activity were used for further study. Phosphatase activity
and titrable acidity at different pH and temperature was estimated for 3, 5 and 7 days. The
Pikovskaya’s broth with pH range of 4, 5, 6, 7, 8 and 9 and incubation temperature of
20oC, 30oC, 37oC, 45oC and 55oC were used for this study.
Estimation of Phosphatase activity
Phosphatase is an enzyme which is used for non-specific phosphomonoesterases.
Phosphatases liberate inorganic phosphate from organic phosphate ester liberating
phosphorus (phosphate)(pi). These enzymes catalyse the following reactionOrthophosphoric monoester + H2O → alcohol + pi
Depending on their pH optims, phosphatases have been classified into two group’s
namely acid and alkaline phosphatase. Acid phosphatases function optimally at acidic pH
(4.0-5.5) and it hydrolyses a number of phosphomonoester and phosphoproteins. Alkaline
phosphatases give maximum activity at alkaline pH (8-10) and catalyses the hydrolysis of
numerous phosphate esters, such as esters of primary and secondary alcohol, sugar
alcohol, phenols and amines. Phosphatase activity or phosphorous solubilization potential
of PSB strains isolated was estimated in the supernatant at different pH and temperature.
Procedure

Pikovskaya’s broth was added with known amount of tricalcium phosphate as a
substrate.

The flasks were inoculated with PSB strains isolated (OD 0.2(A600)).

Uninoculated flasks were used as control.

The flasks were incubated at 30oC for 3, 5 and 7 days.

5ml of the culture was centrifuged and the phosphatase activity estimated in the
supernatant (Tatabai and Bremner 1969).

The phosphatase activity was calculated by referring to a standard graph prepared
with p-nitro phenol (100µg/ml).

Enzyme activity was expressed as µg of p-nitrophenol released/mg cell
Quantitative analysis of IAA production PSBs

Modified Pikovskaya’s broth containing 1% tryptophan as a substrate was
aseptically inoculated with pure cultures of the isolates.

This was incubated at 30oC overnight in a rotary shaker (120 rpm).

1.5 ml bacterial culture was centrifuged at 2,000 rpm for 5 minutes.

To 1 ml of the supernatant 2ml of FeCl3- HClO4 reagent was added.

After
25
minutes
of
incubation
the
absorbance
was
read
in
UV-
spectrophotometer at 530 nm.

The amount of IAA produced per milliliter culture was estimated using a standard
curve.
Results
The percentage of PSB isolated on Pikovskaya’s agar is depicted in Figure 6.1. The
percentage of PSB in both the 30 days upper and lower burrow wall and 45 days lower
burrow wall soil of L. mauritii was lesser than in their respective control soil. It was
significant that the upper burrow wall soil of L. mauritii at 45 day trials showed highest
percentage of PSB (88.88%) among all samples studied. Whereas in P. corethrurus
worked soils both trails showed higher percentage of PSB compared to their respective
control. All colonies on Pikovskaya’s agar showed the ability to break down phosphate in
the 30 days upper burrow wall soil sample. Fourteen isolates from L.mauritti and 10
isolates from P. corethrurus were identified by Gram staining. Most of the isolates were
Gram +ve, sporulating and non sporulating rods.
In L.mauritii the highest HC value was seen among isolate from control soil (2.5 cm).
Among the isolates from burrow wall soil of L. mauritii, highest activity was seen in
UBWS-P6 (2.14 cm) isolated from 45 days sample followed by UBWS-P2 (2.07 cm)
isolated from 30 day sample (Figure 6.2). Among isolates from P. corethrurus higher HC
value was seen in isolates from burrow wall soil sample. Isolate UBWS-P10 from 30
days showed the highest HC value of 2.66 and isolates from 30 days burrow wall soil
LBWS-P1 showed HC value of 2 cm. UBWS-P10 though showed high HC value, did not
show high phosphatase activity and hence was not used for further study.
The phosphate utilizing bacteria isolated from earthworm burrow wall soil was assayed
for IAA activity. Isolates LBWS-P4 from 45 days burrow wall soil of L. mauritii
produced the highest IAA (117.25mg/ml) among both control and sample, followed by
UCS-P5 (116.5mg/ml) which was isolated from 45 days control soil (Figure 6.3). Most
other isolates from burrow wall soil produced lesser IAA compared to isolates from
control soil. IAA was estimated in 6 isolates from burrow wall soil of P.corethrurus and
4 isolates from control soil. Highest IAA production was seen in isolates UBWS-P9 (93.5
mg/ml) followed by LBWS-P8 (87.55 mg/ml) and LBWS-P2 (84.9mg/ml), all of which
were burrow wall isolates. The results show that most isolates from burrow wall soil from
P.corethrurus produce more IAA than the isolates from burrow wall soil of L.mauritti
Among the 14 isolates from L.mauritii and P.corethrurus, 4 each from burrow wall soil
and 1each from control soil were used to study the effect of pH and temperature on
phosphatase activity. A reduction in the pH of the medium and increase in the titrable
acidity was noticed in all isolates in Pikovskaya’s broth (Table 6. 1). In L. mauritii the
highest phosphatase activity was produced by the isolate from 30 days upper burrow
wall soil; UBWS-2 (25.44 IU) at pH 5 on day 7 followed by pH 6 on day 5 (20.8 IU)
(Table 6.2). An activity of 12.16 IU and 10.02 IU was seen to be produced by isolates
from 45 days upper burrow wall soil UBWS - 5 and UBWS -7 respectively. All other
isolates showed activity less than 10 IU. The isolates from the control soil showed very
less phosphatase activity at all pH. Isolates from P. corethrurus showed lesser
phosphatase activity compared to isolates from L. mauritii. The highest activity was
observed in isolate from 45 days lower burrow wall LBWS-2 (9.87 IU).
All isolates showed high phosphatase activity in day 5 and day 7 trials at 20, 30 and 45 oC
in L. mauritii and P. corethrurus (Table 6.3). In L. mauritii the highest activity was seen
to be produced by 30 days upper burrow wall isolate UBWS-2 (27.3 IU) at 45oC day 5.
Isolate UBWS-3 isolated from 30 days burrow wall soil showed highest activity at 45oC
both at day 5 (21.5 IU) and day 7 (22.7 IU). Isolate UBWS- 7 from 45 days burrow wall
soil showed an activity of 22.9 IU on day 5 at 30 oC. In P. corethrurus isolate UBWS- 5
from 45 days upper burrow wall soil showed the highest activity of 48.9 IU at 55oC.
Discussion
The earthworm-microbe interactions in terrestrial ecosystems are known to influence soil
fertility and plant growth by changing soil nutrient cycling and the physical environment.
The increase of phosphorus in soil that passes through the intestinal tract of earthworm is
probably due to several factors (i) a significantly great pH of gut contents along the
earthworm intestinal tract (Barois and Lavelle 1986); (ii) large amount of mucus secreted
in earthworm gut, which releases carboxyl groups from carbohydrates that can block and
compete for phosphorus sorbing places, and in turn, increases soluble phosphorus; (iii) an
increase in the microbial activity during digestion processes (Lopez-Hernandez et al.,
1993). Reports suggest that the plant phosphorus uptake was upto three times higher in
the presence of P. corethrurus. The higher concentration of phosphorus found in
earthworm casts in the available form, especially H2PO4- and HPO4-, are usually
beneficial for plant growth (Mackay et al., 1983).
This study showed a high percent of bacteria with the ability to produce the enzyme
phosphatase in the burrow wall soil compared to control soil with the upper burrow wall
of P. corethrurus showing 100% PSB. A study of PSB from seawater and sediment
samples from various sites around the Indian Peninsula showed that 14% of the isolates
had the ability to solubilise phosphate (De Souza et al., 2000). The present results
showed contrasting percentages of PSB in L.mauritii and P.corethrurus. In the burrow
wall soil of L.mauritii the percentage of PSB isolated was less compared to the burrow
wall soil of P. corethrurus. Other reports of PSB include less than 102 cfu g-1 of soil in
Northern Spain (Peix et al., 2001); from 26- 46% of the total soil microflora (Chabot et
al., 1993). Reports from Wan et al., (2004) show that inoculation of both earthworms and
PSB in to soil had significant effects on microbial growth and enzymatic activity, thus
enhancing the release of available P and further accelerating P transformation.
There is increasing evidence that PSB improve plant growth due to biosynthesis of plant
growth substances rather than their action to release available phosphorus (Ponmurugan
and Gopi 2006). The present study on the production of growth promoting substances
indicated that all isolates were able to produce phytohormones such as IAA. All the
strains of phosphobacteria were able to solubilize inorganic phosphate. Phosphate
solubilizing bacteria are capable of producing physiologically active auxins that may
have pronounced effects on plant growth. The cultures release greater quantities of IAA
in the presence of a physiological precursor, tryptophan, in a culture medium. Production
of IAA varies greatly among different species and is also influenced by culture
conditions, growth stage and availability of substrate (Brown 1972; Vijila 2000). It was
found that isolates from earthworm burrow wall of P.corethrurus produce more IAA
compared to the isolates from L.mauritii.
PSB strains isolated from burrow wall soil of L.mauritti and P.corethrurus were able to
grow and solubilize phosphates from Pikovskaya’s broth. The increased bacterial growth
with decrease in pH and production of organic acids resulted in considerable amount of
phosphorus solubilized. There was clear relationship established between bacterial
growth and phosphorus solubilization. These results are consistent with the report of
Rodriguez and Fraga (1999), Whitelaw (2000), Jeon et al., (2003), Maliha et al. (2004)
and Chen et al. (2006), which showed that solubilization of Ca-P complexes were
mediated specially by the decreasing pH of the medium. Joseph and Jisha (2008)
indicated that phosphate solubilizing organisms are capable of reducing pH of culture
medium. Reports suggest that the increased amount of inorganic P released during cast
deposition was related to and preceded by increased microbial and phosphatase activity
(Vinotha et al., 2000). A similar activity is also possible in the burrow wall since not all
earthworms cast at the soil surface; most species that deposit casts do so in their own
burrows. In this study isolates from the burrow wall showed increased phophatase
activity than the isolates from the control soil.
Extensive use of chemicals as fertilizers to improve plant health and productivity and for
control of pathogens has disturbed the ecological balance of soil and has led to the
depletion of nutrients. Hence there is a need to search for alternative strategies to improve
soil health without causing damage to environment as well as soil. Currently, the main
purpose in managing soil phosphorus is to optimize crop production and minimize P loss
from soils. PSB have attracted the attention of agriculturists as soil inocula to improve
plant growth and yield. When PSB is used with rock phosphate, it can save about 50% of
the crop requirement of phosphatic fertilizer. Given the negative environmental impacts
of chemical fertilizers and their increasing costs, the use of PSB is advantageous in the
sustainable agricultural practices. Phosphate solubilizing bacteria especially are slowly
emerging as important organisms used to improve soil health and earthworm burrow wall
can be a tremendous source of these organisms. This study concludes that bacteria in the
burrow wall soil have better ability to produce plant growth promoters and phosphatase
activity thereby increasing soil fertility and plant growth
UCS(PC)-45d, 33.30%
LBWS (LM)-30d,
0.12%
LCS(PC)-45d, 33.30%
UBWS(PC)-45d,
70%
LCS(LM)-30d, 9.17%
UBWS(LM)30d,
33.33%
UCS (LM)-30d,
75%
LBWS(LM)-45d,
0.27%
LCS(LM)-45d, 77.77%
LBWS(PC)-45d, 50%
UCS(PC)- 30d, 25%
UBWS(LM)-45d,
88.88%
UBWS(PC)-30d,
100%
LCS(PC)30d,
50%
LBWS(PC)-30d,
66.60%
UCS(LM)-45d,
18.60%
Legend- LBWS – lower burrow wall soil, LCS- lower control soil, UBWS- upper burrow wall soil, UCSUpper control soil, PC- P. corethrurus, LM- L. mauritii.
Figure 6.1: Percentage of phosphate solubilising bacteria isolated from burrow wall
and control soil of Lampito mauritii and Pontoscolex corethrurus
3
2.66
2.5
2.5
2.3
2.25
2.16
2
1.85
2
HC Value (cm)
2.14
2.07
2
1.77
1.66
1.5
1.2
1.2
1.26 1.2
1.33
1.44
1.55
1.6
1.5
1.2
1.5
1.33
1
0.5
0
L. mauritii
P. corethrurus
Figure 6. 2: HC value of phosphate solubilising bacteria isolated from burrow wall and control soil of Lampito mauritii and
Pontoscolex corethrurus
100
117.25
116.5
120
93.5
92
87.55
84.9
79.75
IAA mg/ml
80
73.75
72.5 70.5
64.75
64.25
60
59
60
64.25
64.25
58.25
53
41.25
39.75
44
43.75
40
27
22
20
0
L. mauritii
P. corethrurus
Figure 6.3: IAA value of phosphate solubilising bacteria isolated from burrow wall and control soil Lampito mauritii and
Pontoscolex corethrurus
Table 6.1: pH and titrable acidity of isolates from burrow wall and control soil of
Lampito mauritii and Pontoscolex corethrurus
Initial pH 6.8
Day 3
Titrable
acidity
Day 5
pH
Day 7
Titrable
acidity
pH
Titrable
acidity
L.mauritii
pH
UBWS-2 (30d)
4.7
3.9
4.7
4.2
4.9
4.2
UBWS-3 (30d)
4.9
3.2
5.1
4.5
5.4
4.2
UBWS-5 (45 d)
5
3.6
5.2
3.82
5
4.6
UBWS-7 (45d)
4.7
2.6
3.5
2.2
4.8
3.2
LCS-2 (30 d)
P.corethrurus
5.8
2.8
5.7
3.8
5.8
3.6
LBWS-8 (30d)
5.8
2.9
6.2
4.4
6.8
4.3
UBWS-9 (30 d)
5.8
3
6
4.2
6.8
4.3
LBWS-2 (45 d)
5.8
3.1
6
4.2
6.1
3.6
UBWS-5 (45 d)
5.8
3
6.2
4.5
6.6
3.4
LCS-4 (45d)
5.5
2.7
5.8
4.1
5.9
5.3
Table 6.2: Effect of pH on phosphatase activity of isolates from burrow wall and control soil of Lampito mauritii and Pontoscolex
corethrurus
Table 6.3: effect of temperature on phosphatase activity of isolates from burrow wall and control soil of Lampito mauritii and
Pontoscolex corethrurus