Chapter 2 Isolation, screening, morphological and biochemical characterization of bacterial isolates

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1 Chapter 2 Isolation, screening, morphological and biochemical characterization of bacterial isolates

2 2.1 Introduction Soil is a dynamic, living matrix that is an essential part of the terrestrial ecosystem. It is a crucial resource not only for agricultural production and food security but also towards maintenance of most life processes. Soil is considered as a storehouse of microbial activity, though the space occupied by living microorganisms is estimated to be less than five percent of the total space. The functions of soil microorganisms are central to the decomposition processes and nutrient cycling. They play an important role in soil processes that determine plant productivity. For successful functioning of introduced microbial bioinoculants and their influence on soil health, efforts have been made to explore soil microbial diversity of indigenous community, their distribution and behavior in soil habitats. Soil bacteria are one of the major groups of microbes, which are abundant in rhizosphere soil ranging between 10-6 to 10-8 colony-forming units (cfu) per gram, and some of them have shown great potential for plant health promotion and as biocontrol agents of nematodes (Siddiqui and Mahmood, 1999). These plant growth promoting rhizobacteria (PGPRs) are currently being exploited towards these ends. Antoun and Prevost (2006) gave evidence in the literature indicating that PGPRs can be a true success story in sustainable agriculture. Phosphate solubilizing bacteria (PSBs) are important categories of PGPRs which are used in the form of biofertilizers in various agricultural countries, as significant areas of cultivated soils are deficient in nutrients, for e.g. phosphorus (P) (Xie, 1996). Phosphorous is the major essential macronutrient for plant growth and development and hence is commonly added as fertilizer to optimize yield. PSBs have been used to convert insoluble phosphate into soluble forms available for plant growth (Nahas et al., 1990). It has been shown that PSBs increased P availability in soils and increase mineral content in the plant (Sheng et al., 2002). A combination of application of rock phosphate with other minerals and with co inoculation of bacteria that solubilize them might provide a faster and continuous supply of phosphorous for optimal plant growth. This conversion occurs through acidification, chelation and exchange reactions and results in the production of organic acids, which have become indicators for routine isolation and selection procedures of PSBs (Illmer et al., 1995).

3 The present chapter includes collection of soil samples from different locations, isolation and screening of the phosphate solubilizing bacteria and their morphological and biochemical characterization. 2.2 Materials and Methods Collection of soil sample The soil samples were collected from different locations of the Kukrail forest, Lucknow, Lawns of Integral University, Lucknow and Central Institute of Medicinal, and Aromatic Plants (CIMAP), Lucknow. The soil samples were taken from two zones viz., upper zone, which is cm deep and lower zone, which is cm in depth Isolation and screening of phosphate solubilizing bacteria The soil rhizospheric samples were screened for the presence of phosphate solubilizing bacteria Media preparation for isolation of bacteria The bacteria were grown on nutrient agar media. For this nutrient agar medium comprising of yeast extract 0.3 %; peptone 0.5 %; CuSO g/ml was prepared and ph adjusted to 7.0. This medium was complemented with agar 1.5 % and autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to solidify Serial dilutions of soil samples Soil rhizospheric samples from various locations were taken and serial dilutions were made. For this 1 g sample was taken and added in tube containing distilled water and mixed thoroughly. This represented 10-1 dilution. Under aseptic conditions, 10-2 to 10-9 dilutions of samples were prepared Screening of phosphate solubilizing bacteria The isolates were screened on the basis of plate assay. Two media viz., Pikovskaya s media (Pikovskaya, 1948) and bromophenol blue media were used for screening. The Pikovskaya s

4 medium consisted of yeast extract 0.50 (g/l), dextrose (g/l), calcium phosphate 5.00 (g/l), ammonium sulphate 0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l), manganese sulphate (g/l), ferrous sulphate (g/l). Bromophenol media consist of yeast extract 0.50 (g/l), dextrose (g/l), calcium phosphate 5.00 (g/l), ammonium sulphate 0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l), manganese sulphate (g/l), ferrous sulphate (g/l) and 0.5 % of bromophenol blue dye. Both the media were prepared and ph adjusted to 7.0. This medium was complemented with agar 1.5 % and autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to solidify. Bacterial colonies were inoculated on petriplates containing medium for plate assay and the plates were incubated in inverted position in incubator for up to 24 h at 37 C. Positive cultures were screened by observing transparent halo zones in Pikovskaya s medium and yellow halo zone on bromophenol blue medium Pure cultures of phosphate solubilizing bacterial species Positive bacterial colonies were subculture on fresh petriplates containing medium for plate assay. Axenic cultures were isolated by incubating the plates in inverted position in incubator for up to 24 h at 37 C. Positive cultures were screened by observing transparent halo zones in Pikovskaya s medium which is due to the solubilization of insoluble tricalcium phosphate into the soluble form and yellow halo zones appears on bromophenol blue medium which is due to the production of organic acids leading to lowering of ph in the medium Morphological characterization of bacterial isolates by Gram staining procedure The purity and tentative identification of the isolated phosphate solubilizing bacteria was done by Gram s staining procedure. The cell shape and Gram s property of bacteria were examined after staining with standard Gram staining procedure. A thin smear of bacterial isolate was prepared on the glass slide, air-dried and heat-fixed. It was stained in the following sequential order: covered with crystal violet for 30 s, washed with distilled water, covered with Gram s iodine solution for 60 s, washed with 95 % ethyl alcohol, washed with distilled water, counterstained with safranin for 30 s and finally washed with distilled water. The stained and air-dried slides were examined under microscope using oil-immersion objective technique. Gram-positive bacteria retains the color of crystal violet and stain in purple color, while the Gram-negative takes the color of counter stain safranin appear pink in color.

5 2.2.4 Biochemical characterization of phosphate solubilizing bacteria Solubilization index based on colony diameter and halo zone for each PSB indicate the efficiency of solubilization of insoluble phosphate into the soluble one thus forming a transparent halo zone around the colony. Catalase production, oxidase production, urease production and nitrate reduction are valuable criteria for differentiating and identifying various types of bacteria. Hence, the positive bacterial isolates were also analyzed qualitatively for their nitrate reducing, urease, oxidase and catalase producing capabilities Solubilization index (SI) 0.1 ml of each PSB culture preserved in sterile distilled water was placed on Pikovskaya s agar (Pikovskaya, 1948) plates and incubated for seven days. Solubilization Index was measured using following formula (Edi-Premono et al., 1996). Colony diameter + halo zone diameter SI = Colony diameter ph change 1 ml of three days old culture of bacteria in sterile distilled water was added to sterile 100 ml Pikovskaya s broth (PB) medium and kept on shaker for seven days. Sterile uninoculated medium served as control. Initial ph and change in ph was recorded on 3 rd, 5 th and 7 th day by digital ph meter Nitrate reduction test Certain bacteria reduce nitrate to nitrite while others are capable of further reducing nitrite to form nitrogen or ammonia. The isolates were incubated at 37 C overnight followed by addition of 0.5 ml each of sulphanilic acid (0.8 % in 5 N acetic acid) and -naphthylamine (0.5 % in 5 N acetic acid). The appearance of red or pink color indicated the positive test for nitrate reduction Urease test The overnight cultures were inoculated to the test tubes containing sterilized urea broth and incubated for hours at 20ºC ±8ºC. The development of pink color was taken as

6 positive for the test Catalase test Catalase test was performed by growing bacteria in nutrient broth overnight at 37 C followed by addition of a drop of 3 % H 2 O 2. The production of effervescence due to catalasecatalyzed breakdown of H 2 O 2 to molecular oxygen indicated positive reaction Oxidase test To the Trypticase Soy Agar (Himedia) plates, overnight cultures of the test isolates were spotted and plates were incubated for 24 h at 20ºC ± 8º C. After incubation, two to three drops of tetramethyl-phenylenediamine dihydrochloride was added to the surface of the growth of each test organism. The color change to puple or maroon was taken as oxidase positive Analysis of acid phosphatase enzyme activity from various isolates Medium preparation and bacterial culture All the bacterial isolates tested positive in plate assay were subjected to analyses of activity of acid phosphatase enzyme. Bacterial colonies tested positive in plate assay were inoculated in Pikovskaya s broth, poured in test tubes (10 ml/tube) and autoclaved at 15 psi for 15 min. The tubes were incubated in incubator shaker at 120 rpm, 37 C for overnight. 10 ml of above grown bacterial culture was taken and filtered through Whatman no. 1 filter paper. This was considered as enzyme or protein sample. The enzyme acid phosphatase was assayed using para nitrophenyl phosphate (PNP-P) as a substrate. The reaction mixture contained 2.5 ml (0.1 M) sodium acetate buffer (ph 5.8), 1 ml (1 mm) magnesium chloride, 0.5 ml 1 % PNP-P and 0.5 ml of a suitable dilution of enzyme preparation. One ml of the reaction mixture was transferred to 2 ml of 0.2 M sodium hydroxide before and after 15 min incubation at 37 C to stop the reaction. The sodium hydroxide solution added before incubation acts as a control sample for each analysis. The amount of para nitro phenol (PNP) liberated was measured by recording the absorbance at 420 nm using an appropriate calibration curve. Activity is expressed as μmol PNP liberated min -1. The blank was run in a similar manner using distilled water.

7 Reaction showing acid phosphatase enzyme activity with para nitrophenyl phosphate substrate Determination of protein content by Lowry s method 500 l of bacterial culture was taken in microfuge tube and protein was precipitated with equal volume of ice-cold 20 % trichloroacetic acid (TCA) and kept at 4 C overnight. The pellet was recovered by centrifuging at 12,000 rpm for 5 min at room temperature and decanting the supernatant. The pellet was washed with 0.1 ml ice-cold 10 % TCA and ice-cold acetone. Depending on the pellet size, it was dissolved in ml of 0.1 N NaOH. The solution was subjected to heating for 5 min in boiling water bath and vortexed vigorously. The protein content was determined by Lowry s method (Lowry et al., 1951). For protein content determination, 0.5 ml of protein solution was taken in test tube and 2.5 ml of alkaline solution [prepared by mixing 2 % Na 2 CO 3 solution (in NaOH), 2 % sodium potassium tartrate and 1 % CuSO 4.5H 2 O in 100:1:1] was added. The contents were mixed well and the tubes were incubated at room temperature for 10 min. This was followed by addition of 0.25 ml of 1.0 N Folin s reagent. The contents in the tube were mixed thoroughly and after 10 min, absorbance at 660 nm against reagent blank was determined spectrophotometrically using bovine serum albumin fraction V as standard.

8 2.3 Results and Discussion Isolation and screening of phosphate solubilizing bacteria Different types of soil samples, viz., Normal soil, Usar soil and Sandy soil were collected from various locations (Fig. 2.1). (1) Normal soil (2) Usar soil (3) Sandy soil Figure 2.1 Different types of soils Based on serial dilution forty-five bacterial isolates from different soil samples were isolated, from which 21 bacterial isolates having potential phosphate solubilizing ability were observed by plate assay. Various bacteria, which were isolated from soil, were preserved on the nutrient agar media (Fig.2.2). The shape, color and type of colony were estimated on the nutrient media and further the Gram s staining was performed

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10 Figure 2.2 Bacterial isolates having phosphate solubilizing potential growing on nutrient agar media The tricalcium phosphate present in Pikovskaya s medium in insoluble form was converted into the soluble form by the phosphate solubilizing bacteria (phosphate solubilizers), thereby giving a clear zone around a positive colony (Fig.2.3).

11 Figure 2.3 Bacteria showing phosphate solubilization leading to formation of clear zone in Pikovskaya's medium In bromophenol blue medium (Fig.2.4) all the positive bacterial isolates were capable of producing organic acids, which led to change in ph and thereby color change from blue to yellow. Figure 2.4 Bacteria showing yellowing of bromophenol blue medium Bacterial isolates were characterized by analyzing their shape, presence of catalase, oxidase, urease and nitrate reduction abilities. The isolates were examined and the results are given in Table 2.3. Variations were observed among the isolates in their colony morphology Morphological characterization of PSBs by microscopic examination

12 All the twenty-one isolates were studied for their Gram reaction property, cell morphology using standard Gram s staining procedure, and data are presented in Table 2.3. The isolates, as shown (Fig.2.5) were found to be purple and pink colored, Gram-positive and Gram-negative respectively, of varying size under microscope using oil immersion technique

13 21 Figure 2.5 Gram s staining slides of bacterial isolates Biochemical characterization of isolates Solubilization index (SI) All the 21 isolates were able to solubilize tricalcium phosphate (TCP) in Pikovskaya s agar medium and the diameter of the zones of solubilization indicated wide variations among the isolates (2 to 3.2) (Table 2.1). Table 2.1 Comparative analysis of solubilization indices of 21 isolates Bacterial isolates Solubilization index (SI) PKN 1 2±0.17 PKU 5 2.9±0.12 PKS 6 2.5±0.1 PKU 4 2.4±0.15 PKS 5 2.4±0.12 PKN 2 2.8±0.13 PBS 4 3.4±0.14

14 PIN 4 2.2±0.13 PKU 3 2.7±0.1 PIN 3 2.1±0.12 PKN 3 3.3±0.1 PCN 3 2.1±0.15 PKS 4 3.2±0.12 PUS 4 2.5±0.13 PIN 2 2.1±0.14 PKU 1 2.3±0.13 PKN 4 2.3±0.1 PKS 3 3±0.12 PIU 1 2.1±0.15 PKU 2 2.3±0.09 PCN 6 3.1±0.13

15 PKN 1 PKU 5 PKS 6 PKU 4 PKS 5 PKN 2 PBS 4 PIN 4 PKU 3 PIN 3 PKN 3 PCN 3 PKS 4 PUS 4 PIN 2 PKU 1 PKN 4 PKS 3 PIU 1 PKU 2 PCN 6 SI (cm) Bacterial isolates Figure 2.6 Histogram showing solubilization indices of bacterial isolates ph change All the 21 isolates were allowed to grow on Pikovskaya s broth (PB) supplemented with TCP (0.5 % w/v). These bacterial isolates showed decrease in ph with time. Minimum ph was observed after 7 th day (Table 2.2). The ph lowered down due to the liberation of the organic acids in liquid media (Nahas, et al., 1996).

16 Table 2.2 Comparative analysis of ph change on 3 rd, 5 th and 7 th days Bacterial isolates ph 3 rd day ph 5th day ph 7th day PKN PKU PKS PKU PKS PKN PBS PIN PKU PIN PKN PCN PKS PUS PIN PKU PKN PKS PIU PKU PCN The minimum ph of 3.2 was shown by isolate PKN 3 after 7 th day. The bacterial isolates, PKS 4, PBS 4, PKS 3, PCN 6 and PKU 5 significantly decreased the ph of media up to 3.3, 3.6, 3.8, 3.6

17 and 3.4 respectively after 7 th day of growth (Fig.2.7). Therefore, these bacterial isolates were considered of exhibiting high phosphate solubilizing efficiency. 7 ph 3rd day ph 5th day ph 7th day PKN 1 PKU 5 PKS 6 PKU 4 PKS 5 PKN 2 PBS 4 PIN 4 PKU 3 PIN PKN 3 PCN 3 PKS 4 PUS 4 PIN 2 PKU 1 PKN 4 PKS 3 PIU 1 PKU 2 PCN 6 Figure 2.7 Histogram showing comparative analysis of bacterial isolates on the ph on different days of growth Nitrate reductase test Nitrate, present in the broth, is reduced to nitrite, which may then be reduced to nitric oxide, nitrous oxide, or nitrogen. The nitrate reduction test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-

18 sulfanilic acid) which then reacts with α-naphthylamine to give a red precipitate (prontosil). The isolates, which reduce nitrate into nitrite and further reduce nitrite into ammonia, indicates the presence of pink color, showing positive results (Fig.2.8). Figure 2.8 Positive nitrate reductase test Urease test Microorganism, which can produce a urease enzyme, attacks the carbon and nitrogen bond amide compound with the liberation of ammonia gas. Phenol red acts as an indicator dye. Due to the production of ammonia, the yellow media turns deep pink in color indicating hydrolysis of urea (Fig. 2.9). Figure 2.9 Positive urease test

19 Catalase and oxidase test The production of catalase enzyme breaks the hydrogen peroxide into water and oxygen, which comes in the form of effervescence, indicating positive catalase reaction (Fig.2.10). The oxidase test is a test which helps in determining if bacteria can produce certain cytochrome c oxidases which reduces N,N,N,N -tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-pphenylenediamine (DMPD) results in dark-blue to maroon color when oxidized, and colorless when reduced. Oxidase-positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron-containing hemoprotein) (Fig. 2.11). Figure 2.10 Positive catalase test Figure 2.11 Positive oxidase test Table 2.3 Tentative identification of bacterial isolates Bacterial isolates Gram positive or negative Shape Nitrate reduction test Urease test Catalase production test Oxidase test PKN 1 Positive Rod-shaped Positive Negative Positive Positive PKU 5 Negative Rod-shaped Positive Positive Positive Positive PKS 6 Positive Spherical Negative Positive Positive Positive PKU 4 Negative Rod-shaped Positive Negative Positive Negative PKS 5 Negative Rod-shaped Positive Positive Positive Negative

20 PKN 2 Negative Rod-shaped Positive Positive Positive Negative PBS 4 Negative Rod-shaped Positive Negative Positive Negative PIN 4 Positive Rod-shaped Positive Negative Negative Negative PKU 3 Negative Rod-shaped Positive Positive Positive Negative PIN 3 Positive Rod-shaped Positive Positive Positive Negative PKN 3 Positive Rod-shaped Positive Positive Positive Negative PCN 3 Positive Rod-shaped Positive Negative Positive Positive PKS 4 Positive Rod-shaped Positive Positive Positive Positive PUS 4 Positive Rod-shaped Positive Negative Negative Negative PIN 2 Positive Spherical Positive Positive Positive Negative PKU 1 Negative Rod-shaped Positive Positive Positive Positive PKN 4 Positive Rod-shaped Positive Positive Positive Negative PKS 3 Positive Spherical Positive Positive Positive Negative PIU 1 Positive Rod-shaped Positive Positive Positive Positive PKU 2 Negative Rod-shaped Positive Positive Positive Positive PCN 6 Negative Rod-shaped Positive Positive Positive Positive Quantitative analysis of acid phosphatase enzyme activities The tentative PSBs were grown on Pikovskaya s broth containing 0.5 % TCP and their phosphatase activities were measured. The phosphatase activity was estimated in the supernatant of broth taken after centrifugation at 10,000 X g at 4 C. The main mechanism for the solubilization of insoluble organic and inorganic phosphate was due to production of an enzyme acid phosphatase, which catalyzes hydrolysis of phosphate to liberate inorganic phosphorus (Pi). Thus, the isolates were evaluated for their acid phosphatase producing ability by measuring Pi ability. Among all the positive isolates, six bacteria exhibited significantly higher amount of acid phosphatase enzyme activity, including bacteria PKN 3, PKU 5, PKS 4, PBS 4, PKS 3 and PCN 6 (Fig. 2.12). Moreover, these isolates also showed relatively higher solubilization index. Thus, the solubilization index and the acid phosphatase enzyme activity are directly proportional to each other indicating that high enzymatic activity results in the formation of large halo zone. The acid phosphates activities of all the isolates are presented in Table 2.4.

21 Table 2.4: Acid phosphatase enzyme activity of bacterial isolates Bacterial isolates Acid phosphatase activity (nmole/ml) Sp. Ac. (Ac./mg protein) PKN ± ±0.025 PKU ± ±0.019 PKS ± ±0.013 PKU ± ±0.016 PKS ± ±0.018 PKN ± ±0.013 PBS ± ±0.016 PIN ± ±0.019 PKU ± ±0.012 PIN ± ±0.013 PKN ± ±0.016 PCN ± ±0.017 PKS ± ±0.012 PUS ± ±0.012 PIN ± ±0.021 PKU ± ±0.021 PKN ± ±0.021 PKS ± ±0.016 PIU ± ±0.018 PKU ± ±0.014 PCN ± ±0.015

22 PKN 1 PKU 5 PKS 6 PKU 4 PKS 5 PKN 2 PBS 4 PIN 4 PKU 3 PIN 3 PKN 3 PCN 3 PKS 4 PUS 4 PIN 2 PKU 1 PKN 4 PKS 3 PIU 1 PKU 2 PCN 6 nmoles (Pi/ml) Bacterial isolates Figure 2.12 Histogram showing acid phosphatase activity of bacterial isolates 2.4 Conclusion The results indicated existence of variation among the isolated PSBs at morphological and biochemical levels. Some of the isolates showed white colored pigmented colonies while others formed light green and light orange colonies. The variation in colony color could be due to the production of different types of pigments. Several bacterial species are known to produce various kinds of pigments, the allocation of these pigments in the genus is uncertain (Palleroni et al., 1970). In addition to this, these isolates also showed diversity in shape from round to irregular shaped colonies. Furthermore, the round shaped colonies were non-spreading where as the irregular shaped colonies were spreading type. PSBs also exhibited diversification in cell shape, Gram property, nitrate reductase, urease oxidase, and catalase activities. Prominent halo zones were found in case of positive PSB isolates on Pikovskaya s agar. Based on transparent halo zone the isolates that exhibited higher SI also exhibited higher acid phosphatase activity. However, morphological and biochemical characterization of isolates did not give complete information about the isolates. Hence, polyphasic approach involving molecular marker analyses along with phenotypic evaluation is essential for identification of bacterial isolates and determination of genetic variation. Thus, all the 21 isolates were further subjected to diversity analysis with respect to their molecular analysis but the isolates, which

23 show highest solubilization index, high values of ph reduction and high values of acid phosphatase, will be taken into consideration.