Development and standardization of cyst based liquid

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Development and standardization of cyst based liquid formulation of Azospirillum Bioinoculant Article in Acta Microbiologica et Immunologica Hungarica · July 2007 DOI: 10.1556/AMicr.54.2007.2.7 · Source: PubMed

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Acta Microbiologica et Immunologica Hungarica, 54 (2), pp. 167–177 (2007) DOI: 10.1556/AMicr.51.2007.2.7

DEVELOPMENT AND STANDARDIZATION OF CYST BASED LIQUID FORMULATION OF AZOSPIRILLUM BIOINOCULANT REGUPATHY THAMIZH VENDAN* and MUTHUSAMY THANGARAJU Agricultural College and Research Institute, Tamil Nadu Agricultural University, Tiruchirappalli-620 009 Tamil Nadu, India

(Received: 9 September 2006; revised: 11 November 2006; accepted: 26 November 2006)

Azospirillum bioinoculant is well known for its high nitrogen fixing and plant growth promoting characters. The carrier based bioinoculants generally suffer from shorter shelf life, poor quality, high contamination and low field performance. Therefore, it is necessary to develop alternative new formulation of inoculants where cyst based inoculants can play significant role. The cyst based liquid formulation was developed by inoculating Azospirillum into the cyst inducing minimal salts medium (MSM). One hundred per cent conversion of vegetative cells into cyst cells was noticed in 96 h. The survival of cyst cells in the MSM was observed up to one year and two months and interestingly, the population level of 108 was maintained till the final observation. The cyst cells of Azospirillum accumulated poly-b-hydroxybutyrate (PHB) granules and exhibited desiccation tolerance up to 20 days and temperature tolerance up to 40 ºC. Thus the cyst based liquid formulation has twin advantage of longer shelf life and tolerance to harmful environmental conditions. Regeneration of cyst cells into vegetative cells in different media viz., tap water, sterile water, rice gruel and nutrient broth was studied. The changes started within 3 h and complete return of vegetative cells was observed at 24 h. Although all the media tested favoured regeneration, comparatively quicker regeneration was observed in nutrient broth and followed by rice gruel. Thus, Cyst based liquid formulation of Azospirillum has all the survival advantages and can be used as a potential bioinoculant. Keywords: Azospirillum bioinoculant

* Corresponding author; E-mail : [email protected]

1217-8950/$20.00 © 2007 Akadémiai Kiadó, Budapest

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VENDAN and THANGARAJU

Introduction Azospirillum species are probably the most studied and appeared to have significant potential for commercial applications. These organisms characterized by high nitrogen fixing ability, are found in abundant numbers in the rhizosphere as well as in the intercellular spaces of the roots of certain cereals and other plants [1]. Some of the suggested modes of action for Azospirillum are; secretion of phytohormones, nitrogen fixation, production of undefined signal molecules that can interfere with plant metabolism, nitrite production and the enhancement of mineral uptake by plants [2].Thus they exert beneficial effect on the growth of plants [3] and increase the yield of many crops of agronomic importance. Azospirillum are known to be highly pleomorphic and to change their metabolic activities swiftly in the face of changing environmental conditions [4]. Production of cyst may represent a mechanism by which azospirilla can persists in the rhizosphere during unfavorable conditions [5], such as desiccation, temperature and nutrient limitation and convert into enlarged cyst forms [6]. A frequent observation is that in carrier based inoculants, the number of viable cells decrease from 109 to 107 colony forming units (cfu) per g after 90 days of storage [2]. The most consistent feedback received from the farmers and inoculant producers is the concern about the shelf life of carrier based inoculants, since they have shorter shelf life, which hardly extends beyond three to four months under normal storage conditions. The development of adequate formulations, which would ensure survival, protection of the strain and the application technology, which would allow timely, easy and precise delivery in the field could be a major step towards this goal [7]. Hence techniques to increase the shelf life of inoculants thus become necessary to propagate this technology in large scale. In this study, experiments were conducted to induce the cyst forms in Azospirillum to get a bioinoculant with longer shelf life and their tolerance to adverse conditions.

Materials and methods Developing the cyst based liquid formulation of Azospirillum The Azospirillum culture, Azospirillum lipoferum (Az-204) was grown in N free malic acid (NFb) broth [8] up to OD value of 1.45 and 1.10 to get population of 109 cfu/ml. The cells were harvested by centrifugation at 5000 rpm at 4 °C and washed three times with 100 mM potassium phosphate buffer solution. Twenty

Acta Microbiologica et Immunologica Hungarica 53, 2006

AZOSPIRILLUM BIOINOCULANT

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five milliliter of these cultures were inoculated into one litre cyst inducing medium viz., Minimal Salts Medium (MSM) [9] and another set was maintained with the addition of 10 ml of glycerol (10 mM). They were incubated at room temperature in an environmental shaker at 200 rpm to investigate cyst formation, viable cell population, poly-b-hydroxy butyrate accumulation, desiccation and temperature tolerance of Azospirillum cultures.

Determination of conversion rate of vegetative cells of Azospirillum into cyst cells The conversion rate of vegetative cells into cyst cells of Azospirillum was calculated by taking direct microscopic count using a Haemocytometer by observing morphological difference at 24 h interval. The number vegetative cells and cyst cells per large square were counted and cyst conversion percent was worked out. Cyst conversion percent =

Number of cyst cells in large square

Total number of cells observed in the large square

× 100

Enumerating the viable cell population The population was estimated using NFb medium through the drop plate method [10] at monthly interval.

Estimation of poly-b-hydroxy butyrate content The PHB content was measured [ 11 ] from the Azospirillum culture grown in MSM sampled at 24 h interval of incubation. Bacterial cell suspension was prepared by centrifuging the culture at 8000 rpm for 20 min at 4 °C. One ml of cell suspension was added to one ml of 2 N hydrochloric acid and the mixture was digested at 100 °C for 2 h. After digestion the mixture was cooled and extracted twice with chloroform. The chloroform was then evaporated in a boiling water bath and to the sediment, 5 ml of concentrated sulphuric acid was added. The sample was heated to 100 °C for 10 min in a water bath. After cooling, the absorbance was measured at 235 nm in Systronics-UV-Vis Spectrophotometer-108.


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Desiccation tolerance of Azospirillum cyst cultures One ml of five fold diluted A. lipoferum culture grown in MSM was taken in sterile 1.5 ml eppendorf tubes. The tubes were in turn kept open in a sterile petridish and this set up was incubated at 37 °C in an incubator. The dried cells from the eppendorf tubes were washed with 100 µl of sterile distilled water with vigorous agitation to remove them quantitatively and their viability was determined by plating on NFb medium.

Temperature tolerance of Azospirillum cyst cultures One ml of five fold diluted A. lipoferum culture grown in MSM was taken and kept in the water bath at temperatures 30 °C, 40 °C, 50 °C or 60 °C for 30 min. Then the tubes were removed, rapidly cooled and the population was determined by plating with one ml of culture on NFb medium.

Regeneration of cyst cells of Azospirillum in different media Regeneration of cyst cells of Azospirillum was examined in different media viz., tap water, sterile water, rice gruel and nutrient broth. Cyst inoculum (2.5 × 108 cfu / ml) was added to the above media at 1, 2 and 5 per cent levels individually and incubated on a shaker with 120 rpm at room temperature. The regeneration and multiplication of cells was examined by serial dilution and plating at varied intervals from 0 h to 36 h. All the data obtained from the above experiments were subjected to statistical analysis as per the method detailed by Panse and Sukhatme [12].

Results and discussion A gradual increase in the conversion per cent of cyst formation from vegetative cells was observed with laps of time. In the present study, at 96 h, 100 per cent conversion of vegetative cells in to cyst cells was recorded (Fig. 1), where fructose and KNO3 were used as the carbon and nitrogen sources respectively. It was proved that media containing fructose and KNO3 favoured flocculation and formation of cyst cells by providing nutrient stress to Azospirillum cells [6, 13,



171

35 30

Cells (%)

25 20 15 10 5 0 0

24

48

72

96

Hours

Vegetative cells

Cyst cells

Figure 1. Assessment of conversion of vegetative cells of Azospirillum into cyst cells in the minimal salts medium (MSM)

14]. The reason behind the flocculation is believed to be connected with the production of exocellular polymers, particularly b-linked polysaccharides [6]. Berg et al. [4] reported that the capsular forms (C forms) of azospirilla were obtained on a limiting carbon and nitrogen medium and these forms are responsible for cell aggregation and synthesis of polysaccharides necessary for encystation. The survival of cyst cells of Azospirillum was assessed in the minimal salts medium (MSM) and MSM amended with glycerol (10 mM) up to the period of 420 days (Table I). The results revealed that the population level of 1010 was maintained up to 150 days of observation in both media. The population at 109 was noticed from 180 days up to 330 days in both MSM and MSM + glycerol treatments. Interestingly, the 420 days observation showed population level of 108 in both treatments. This work is in line with Inamdar et al. [15] where they reported the long life span of cyst based inoculant of Azotobacter and observed the survival for over two years. MSM amended with glycerol (10 mM) supported higher number of cells as compared to the MSM alone. It might be due to the fact that glycerol has high water binding capacity and may protect cells from the effects of desiccation by slowing the drying rate. Production of reserve material of PHB granules in Azospirillum cultures grown in MSM and MSM amended with glycerol was studied in this experiment. The PHB accumulation increased constantly up to 108 h and thereafter a decline was noticed (Table II). Suresh Babu et al. [16] found an increase in accumulation


172


Table I Survival of cyst cells of Azospirillum in the cyst based liquid formulation Days

Population of Azospirillum (log cfu/ml) MSM* MSM + glycerol

30 60 90 120 150 180 210 240 270 300 330 360 390 420

11.01 10.99 10.95 10.93 10.92 10.01 10.00 10.00 9.98 9.96 9.94 8.98 8.96 8.90 Sed

11.02 11.00 10.98 10.95 10.94 10.85 10.04 10.04 10.02 10.02 9.97 9.02 9.00 8.99 CD (0.05)

Days Media Interaction

0.007 0.002 0.010

0.014 0.005 0.020

* Minimal salts medium. Table II Poly-b-hydroxybutyrate (PHB) content of Azospirillum grown in MSM Time (h) MSM

PHB (mg/g dry wt. of cells) MSM + glycerol

0 12 24 36 48 60 72 84 96 108 120 132

0.000 0.315 0.658 0.921 0.988 1.114 1.791 2.216 2.921 3.847 3.152 2.852

0.000 0.318 0.664 0.947 0.991 1.120 1.804 2.219 2.933 3.852 3.157 2.861

Sed CD (0.05)

0.002 0.004

0.003 0.006

of PHB up to 96 h and a decline afterwards which is in conformity with the present results. Under carbon and nitrogen limiting conditions, the Azospirillum cells tend to form abundant PHB granules [6]. PHB production and its role in survival of var-



173

ious stress and deleterious conditions in the A. brasilense were studied [17]. Kadouri et al. [18] reported the synthesis and utilization of PHB as a carbon and energy source under stress conditions. The cyst formation and its tolerance against stress conditions by Azotobacter was reported earlier [19, 20]. The cyst cells of Azospirillum exhibited tolerance to desiccation and survived for 20 days when subjected to desiccation tolerance tests (Fig. 2). It was observed that the cyst cells of Azospirillum are resistant to desiccation from a few hours to one month [5]. It is also apparent that the PHB rich cyst cells confer more resistance to desiccation than the PHB deficient vegetative cells. MSM+ glycerol treatment recorded higher number of viable cells as compared to MSM alone. The inference of the present study is in agreement with desiccation tolerance experiment conducted by Sadasivan and Neyra [21] with cyst form of Azospirillum cells. Temperature tolerance study showed that above 50 ºC, there were no viable cells of cyst based Azospirillum (Fig. 3), whereas Berleman and Bauer [22] reported that 81.8 per cent survival of cyst cells of Rhodospirillum centenum when incubated at 52 ºC for as long as 30 min. However in the present study, the Azospirillum cyst cells were tolerant up to 40 ºC. The cyst formation process is generally accompanied by the production of a thick coat or capsule [21] and Bleakley et al. [23] attributed the acquisition of resistance of cysts to cyst coat against ultraviolet treatment, temperature and desiccation. The capacity to form cyst endows Azospirillum with the ability to survive under adverse environmental conditions. However, the success of this survival strategy depends on the presence of an efficient mechanism for returning to the vegetative state under favourable conditions. In this study, the regeneration of cyst 11

Vegetative cells

10

Cyst cells (MSM)

log cfu /ml

Cyst cells (MSM + glycerol)

9 8 7 6 0

2

4

6

8

10

12

14

16

18

20

Days

Figure 2. Desiccation tolerance of cyst cells of Azospirillum in the cyst based liquid formulation


174


Vegetative cell

12

Cyst cells (MSM) Cyst cells (MSM + glycerol)

log cfu / ml

10

8

6

4 30

40

50

60

Temperature o C

Figure 3. Temperature tolerance of cyst cells of Azospirillum in the cyst based liquid formulation

cells was studied in different media and found that regeneration occurs much more rapidly than the formation of cyst cells. Within 3 h of plating on nutrient medium, cyst cells under phase contrast microscopy showed a decrease in the width and brightness of the highly refractile outer coat (Table III). Fast regeneration was noticed in nutrient broth followed by rice gruel and it might be due to its supply of suitable carbon, nitrogen, vitamin and trace elements for the growth of Azospirillum. After 24 h, complete return of the vegetative cell was observed with a recurrence of vibriod and spiral shapes and active cell motility. Similar results were obtained by Berleman and Bauer [22] from germination of Rhodospirillum cyst cells. Interestingly, sterile water and tap water also promoted regeneration of cysts which clearly indicated that neither a carbon nor nitrogen source is specifically required for regeneration. Among the cyst inoculum levels, 5 per cent level was found to be optimum and induced higher population and quicker regeneration as compared to other inoculum levels. The cyst based liquid formulation of Azospirillum has twin advantage of longer shelf life and tolerance to adverse conditions such as temperature and desiccation. Hence, it can be concluded that the accumulation of PHB and formation of cyst cells are critically important for improving the shelf life, efficiency and reliability of commercial inoculants. Thus cyst based liquid inoculant of Azospirillum therefore can be more reliable, potential bioinoculant which could safely be distributed to the remote agricultural areas.


3.11 3.26 3.37

Nutrient broth 1% 2% 5%

Media Concentration Time Interaction

3.09 3.24 3.32

3.03 3.15 3.28

Sterile water 1% 2% 5%

Rice gruel 1% 2% 5%

3.01 3.12 3.24

0h

Tap water 1% 2% 5%

Inoculum level

5.24 5.34 5.42

5.19 5.30 5.36

5.13 5.29 5.31

5.06 5.15 5.30

3h

Sed 0.001 0.001 0.001 0.005

7.32 7.39 7.45

7.28 7.36 7.32

5.21 7.32 7.38

5.12 7.25 7.39

6h

8.33 8.44 8.45

8.25 8.38 8.30

8.20 7.28 8.32

7.15 8.28 8.36

9h

9.34 9.36 9.46

8.30 9.33 9.30

8.19 8.29 9.26

8.00 8.26 8.33

12 h

Population of Azospirillum (log cfu /ml)

Regeneration of cyst cells of Azospirillum in different media

Table III

CD(0.05) 0.003 0.002 0.003 0.010

9.34 9.37 9.48

9.26 9.34 9.34

8.23 9.21 9.25

8.12 8.29 9.23

24 h

8.28 8.29 8.33

8.26 8.29 8.29

7.28 8.24 8.25

7.10 7.21 8.17

36 h

AZOSPIRILLUM BIOINOCULANT 175


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Development and standardization of cyst based liquid

Development and standardization of cyst based liquid

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