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 - 10

6 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, 30

oC, 37

oC, 45

oC and 55

oC 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 reaction-

Orthophosphoric 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 45oC

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 30oC. 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

Legend- LBWS – lower burrow wall soil, LCS- lower control soil, UBWS- upper burrow wall soil, UCS-

Upper 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

LBWS (LM)-30d, 0.12%

LCS(LM)-30d, 9.17%

UBWS(LM)-30d,

33.33%

UCS (LM)-30d, 75%

LBWS(LM)-45d, 0.27%

LCS(LM)-45d, 77.77%

UBWS(LM)-45d, 88.88%

UCS(LM)-45d, 18.60%

LBWS(PC)-30d, 66.60%

LCS(PC)-30d, 50%

UBWS(PC)-30d, 100%

UCS(PC)- 30d, 25%

LBWS(PC)-45d, 50%

LCS(PC)-45d, 33.30% UBWS(PC)-45d,

70%

UCS(PC)-45d, 33.30%

Figure 6. 2: HC value of phosphate solubilising bacteria isolated from burrow wall and control soil of Lampito mauritii and

Pontoscolex corethrurus

0

0.5

1

1.5

2

2.5

3

2

2.25

1.2

2.16

2.5

1.2

2.3

1.85

2.07

1.66

1.26 1.2

2.14

1.33 1.44

1.55

1.2

1.5

2

1.77

1.33

2.66

1.6 1.5

HC

Val

ue

(cm

)

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

0

20

40

60

80

100

120

92

27

39.75

64.25

116.5

59

73.75

79.75

22

41.25

117.25

53

60 58.25

44

64.75

72.5 70.5

43.75

87.55

93.5

64.25

84.9

64.25

IAA

mg/

ml

L. mauritii P. 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 Day 5 Day 7

L.mauritii pH Titrable acidity

pH Titrable acidity

pH Titrable acidity

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) 5.8 2.8 5.7 3.8 5.8 3.6

P.corethrurus

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