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Isolation and Quality Control of Nitrogen-Fixers and PhosphateSolubilizers for Production and Development of Inoculant

Johnry S. Maloles
University of the Philippines, Los Baos, Laguna, 4031
31 March 2013
Plant growth promoting bacteria (PGPB) could be used for growth promotion and increase in nutrient uptake of agricultural crops. In this study, PGPBs were isolated from soil using selective and differential medium to screen for their capability to fix nitrogen and solubilize phosphate. Four unknown bacterial strains were obtained and showed positive results for nitrogen-fixation and only one for phosphate solubilization, namely R-C1, R-C2, F-C1, F-C2, and PSB, respectively. F-C2 was chosen for further studies since it can rapidly grow on the Dobereiners medium alongside with the PSB. The carrier based PGPB consortium with unknown isolates of F-C2 and PSB was prepared and the shelf life for each inoculant was studied up to four weeks of storage at 28 C. F-C2 initial count was 2.24 x 106 CFU g-1 and finished with 9.33 x 106 CFU g-1 after 4 weeks of storage, indicating that cell viability was retained with significant percent increase in cell count. On the other hand, PSB started with a higher cell count of 2.87 x 108 CFU g-1. With increase in time of storage, viable cell count dramatically decreased reaching a percent cell viability of less than 1% (2.92 x 106 CFU g-1) after 4 weeks, indicating a 100-fold decrease in cell concentration. It can be concluded that the carrier was best for the unknown F-C2 bacterial strain and not for the PSB strain. The present investigation confirms that these chosen bacterial strains are still not ready for commercial production and needs further study. ______________________________________________________________

Production of inoculant for leguminous plants even started in the early 1890s in the UK and the USA (22). The USA is probably still the largest producer of legume inoculants in the world (13). With the growth of the industry for commercial production of inoculants, considerable requirements have been brought up and extensive research was initiated for the vast improvement of the development process of the booming industry (25). And now, inoculant production is widespread worldwide, where commercial production is carried out in all continents including the biggest producing countries Australia, India, Brazil, Uruguay, Argentina, and other European countries (13). The key points on where development of inoculants can be successful depends on two key factors, namely the isolate to be cultured for mass production and the carrying material to be used. The actual steps for production of inoculants involve isolation of microorganism, mass culture production, and preparation of inoculants along with inoculant quality control. Individual organism can be mass multiplied using specific media either as small scale or as large-scale commercial production procedure using fermenters. The desired growth of organisms is then mixed with carrier materials and sealed in culture packets. Quality check of inoculant

is regularly checked prior to distribution of individual biofertilizer culture (11). In considering the microorganism to be used, a significant number of bacterial species are able to exert a beneficial effect upon plant growth. Mostly they are associated with the plant rhizosphere, so they are called as rhizobacteria. This group of bacteria has been termed plant growth promoting rhizobacteria, and among them are strains from genera such as Alcaligenes, Acinetobacter, Arthrobacter, Azospirillum, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Paenibacillus, Pseudomonas, Rhizobium, and Serratia (20, 12). Bacteria associated with plants can be either harmful or beneficial. PGPR may promote growth directly by fixing atmospheric nitrogen, solubilizing minerals such as phosphorus, producing of siderophores that solublize and sequester iron, or by producing plant growth regulators such as phytohormones (15, 23).Indirect growth promotion occurs when PGPB promote plant growth by improving growth restricting conditions (10). This can happen directly by producing antagonistic substances, or indirectly by inducing resistance to pathogens (9). A specific bacterium can affect plant growth by one or more of these mechanisms, and also use different abilities for

growth promotion at various times during the life cycle of the plant (10). Nitrogen is required for cellular metabolism and is therefore important in plant growth and production of food and feed. Biological nitrogen fixation by capable bacteria is a significant source of available nitrogen for the utilization of agricultural crops (8). For nodulating legumes, nitrogen can be provided through symbiotic fixation of atmospheric N2 by nitrogenase in rhizobial bacteroids. This process of biological nitrogen fixation accounts for 65% of the nitrogen currently utilized in agriculture, and will continue to be important in future sustainable crop production systems (16). Concerning the available phosphorus in soil, plants can only absorb phosphate only in soluble form. The transformation of insoluble phosphate into soluble form is carried out by a number of microbes present in the soil. A large fraction of soil microbes can dissolve insoluble inorganic phosphates present in the soil and make them available to the plants (28). Insoluble phosphate compounds can be solubilized by organic acids and phosphatase enzymes produced by plants and microorganisms For example, PSB have been shown to enhance the solubilization of insoluble P compounds through the release of organic acids and phosphatase enzymes (19). Inoculants normally consist of a mixture of an appropriate bacterial strain or strains and a carrier material. A wide range of carriers has been used as a base for Rhizobium inoculants (4) but peat has remained the preferred material because of the protection it offers to cultures against high storage temperatures and its ability to maintain hydration of the cultures. Peat also usually contains certain essential nutrients which not only help survival of the cultures but also allow further growth and multiplication during storage (18) The objective of this study was to screen for nitrogen-fixing and phosphate solubilizing bacteria from different soil samples, and to further evaluate their capability to retain viability in a certain period of time in carrier mix. Quality control of the developed inoculant was monitored to determine percent cell viability of the plant growth-promoting bacteria.

method. Agar plating method was carried out to select for putative plant growth-promoting bacteria (PGPB) through serial dilution. Ten grams of each soil sample was weighed and diluted in dilution bottle containing 90 mL of saline water. Soil suspension was serially diluted up to 10-6. 0.1 mL of dilutions 10-4 to 10-6 were spread plated in triplicates on selective and differential medium of Dobereiners Medium ( composed of 5 g, Malic Acid; 4 g, KOH; 5 g, Yeast Extract; 1 mL, 1% MnSO4H2O; 1 mL, 10% MgSO47H2O; 2 mL, 10% NaCl; 4 mL, 10% K2HPO4; 0.2 mL, 0.1% NaMoO4; 1 mL, 10% CaCl2; 1 mL, 5% FeSO47H2O; 5 mL, 1M NH4Cl; 3 mL, Bromthymol Blue; 2 g, Agar 1000 mL H2O (pH of 6.5-6.8)) and Pikovskaya Medium (composed of 1 g, Glucose; .05 g, Yeast Extract; .01 g, CaCl2; .025 g, MgSO47H2O; .251 g, CaPO4; 2 g, Agar (g/L)), both containing 100ppm of cycloheximide, and were incubated for 5 weeks at 28 C. Isolates that showed positive results for nitrogen fixation and phosphate solubilization were picked and streaked for isolation twice to ensure purity. Isolates were maintained at Dobereiners and Pikovskaya slants stored at 4 C. Characterization of Nitrogen-Fixers and Phosphate Solubilizer. Cultural and morphological characteristics of the best nitrogen-fixer and phosphate-solubilizer were observed. The unknown bacterial isolates were inoculated in Nutrient Agar (28 g Nutrient Agar in 1 L distilled water) and were streaked for isolation, incubated at 28 C for 24 h. Single colonies were observed and their cultural characteristics were noted including the size, form, margin, consistency, pigmentation, and elevation. Morphological characteristics of the two unknown bacterial isolates were determined by the gram staining technique. Bacterial strains were processed using standard gram staining procedure. A smear of the F-C2 and PSB were prepared on a glass slide, airdried and heat fixed. It was then stained with crystal violet solution for 1 min and washed with tap water. Iodine solution was dropped on the glass slides and let to stain for 1 min. Using 95% ethanol, decolorization was done to remove the excess stain. It was washed again with tap water and safranin was added as a counterstain for 2 min. It was washed again with water and let to air dry. Their gram reaction and appearance under the microscope were noted. Preparation of Starter Culture and Carrier. Selected isolates from Dobereiners Medium and Pikovskaya medium were multiplied in large quantities in appropriate culture broths. A loopful of bacterial growth of nitrogen-fixer and phosphatesolubilizer were transferred to 15 mL of DM and PM,

MATERIALS AND METHODS Isolation, Screening, and Maintenance of Bacterial Isolates. Bacterial strains were isolated from soil from three different sampling sites at the University of the Philippines, Los Baos, Laguna. Soil samples from grassland area, forest soil, and rice paddy soil were aseptically collected prior to the isolation

Figure 1. Bacterial isolates from rice paddy soil and forest soil labelled as R-C1, R-C2, F-C1, and F-C2 streaked for isolation in Dobereiners medium, incubated for 48 h at 28 C. respectively, and were incubated for 48 h at 28 C without shaking. Five milliliters of the broth was transferred to 100 mL seed culture of DM and PM, and was incubated for 4 d at ambient temperature in the rotary shaker. After the cells were acclimatized on seed broths, 10 mL of the cultures were transferred to 200 mL of sterile Nutrient Broth (NB) and incubated for 6 d at 28 C with shaking. Number of the bacteria was estimated by performing simple windows test technique to check for the required turbidity of the mother culture. Change in the medium were also consistently monitored in every step to ensure that the bacterium being investigated still possesses the capability to fix nitrogen or solubilize phosphate. Carrier used for this study was provided by the Agricultural Laboratory of the Institute of Molecular Biology and Biotechnology, Los Baos (UP-BIOTECH). Carrier weighs 80 g each composed of 50% charcoal and 50% clay placed in a low density polythene bag. The carriers were sterilized at 121 C for 1 h for three consecutive days prior to mixing of the inoculant. The quality of the mother culture and inoculant were determined by performing spread plating in Nutrient Agar plates by serial dilution technique. Plates were incubated for 24 h at 28 C and total viable cell count was computed. Preparation of Final Inoculant and Quality Control. Fifteen millilitres of the mother culture of the nitrogen-fixer and phosphate-solubilizer were aseptically transferred to the prepared sterile carriers by pipetting. The bags were thoroughly kneaded to ensure the absorption of the liquid culture into the carrier. Inoculants were let to cure and incubate at 28 C. Initial count of the viable cell concentration was determined by performing spread plating technique. The populations of individual plant growth promoting bacteria in the inoculant carriers were assessed at weekly intervals up to 1 month with three bags as representative for each. Percent cell viability was also computed for comparison. RESULTS Isolation and Screening. After 5 w of incubation of the spread plated agars in the Dobereiners and Pikovskaya Medium, a total of five isolates were selected based on their cultural growths and reactions in the selective and differential medium. Four isolates were found to exhibit nitrogen fixation capability based on the color change of the Dobereiners medium from light green to blue-green (Figure 1). Two of the isolates were from the rice paddy soil and the other two were from the forest soil, namely R-C1, R-C2, F-C1, and F-C2, respectively. Only one isolate showed a zone of clearing in the Pikovskaya medium which indicated a positive result for phosphate solubilization. The phosphate solubilizer was isolated from the forest soil and was labelled as the PSB (Figure 2).

Figure 2. Phosphate-solubilizing bacteria in Pikovskaya Agar Plate after five weeks of incubation at 28 C.

Figure 3. Gram staining reaction of unknown bacterial isolates F-C2 and PSB. Cultural and Morphological Characterization The unknown F-C2 isolate was considered for further study since it can rapidly fix nitrogen and it can propagate very rapidly alongside with the phosphate solubilizer unknown bacterial strain. The two cultures were streaked for isolation and their cultural characteristics were observed for characterization. Nitrogen fixer F-C2 had a diameter of about 1 mm after 24 h of incubation, an entire and umbonate colony, with yellowish pigmentation, and creamy consistency. The phosphate solubilizer had a growth of about 1.5 mm in diameter after 24 h of incubation, an entire and umbonate colony, and creamy in color and consistency. Morphological characteristics of the two unknown strains were also investigated to have an idea of the possible identification of the isolates. Fresh cultures were obtained and were subjected to standard gram staining procedure. The nitrogen fixer F-C2 was a gram-negative, cocci bacterium, while the phosphate solubilizer was a gram-positive, short rods bacterium that usually come in pairs (Figure 3). Starter Culture and Carrier Quality Check. The quality of the prepared mother culture in nutrient broth and carrier were monitored by simple serial dilution and spread plating technique. Both of the mother cultures of the nitrogen-fixer and phosphate solubilizer strains reach the required minimum number of cells per mL of 108 (Table 1). Higher cell density was recorded for the phosphate solubilizer (8.7 x 109 CFU mL-1) mother culture than the nitrogen-fixer (1.5 x 108 CFU mL-1), making a more than ten-fold higher than the other. Carrier had a total count of 1.5 x 103 CFU g-1 of bacteria, which is a relatively low count and makes the carrier a good material for the inoculant production to proceed. Table 1. Initial cell count of the mother cultures and total number of contaminants in the carrier after sterilization. Seed Inoculant and Carrier Quality 10-6 PSB 879 863 810 F-C2 27 14 5 10 carrier

10-7 96 80 86 1 4 1 10-3 1 0 0

CFU/mL 8.7 x 109

1.5 x 108 CFU/g 1.5 x 103

2 0 1

Quality Check of Mixed Inoculant and Carrier. Nitrogen-Fixer. Three representative bags containing the mixed mother culture and carrier were monitored for a weekly interval up to four weeks with three replicates each. The average of the three bags was taken to compute for the percent cell viability at each sampling point as can be seen on Figure 4. A starting 2.24 x 106 CFU g-1 was obtained in the initial cell count after the mother culture and carrier were mixed. This initial count of bacterial cells did not reached the required minimum CFU g -1 of 108, however, the experiment was still carried out. After one week of storage at 28 C, cell count increased by three-fold from 2.24 x 106 to 7.91 x 106 CFu g-1,

Sample A Viable Cell Count (CFU/g) 2.62E+08 3.28E+07 4.10E+06 5.12E+05

Sample B

Sample C

Expon. (Average)

8.00E+03 0 1

2 3 4 Time (week) Figure 4. Growth profile of the nitrogen fixing F-C2 unknown bacterial strain inoculated in the carrier showing exponential average with weekly sampling interval up to 1 mo, stored at 28 C. Sample 1 Viable Cell Count (CFU/g) 2.62E+09 3.28E+08 4.10E+07 5.12E+06 6.40E+05 8.00E+04 2 3 4 Time (week) Figure 5. Growth profile of the phosphate solubilizing PSB unknown bacterial strain inoculated in the carrier showing exponential average with weekly sampling interval up to 1 mo, stored at 28 C. corresponding to a 352.98% cell viability. On the second and third week of sampling, viable cell count reached up to 3.01 x 107 (1342%) and 2.29 x 107(1022%) CFU g-1, respectively. And on the last sampling, forth week, computed cell concentration of 9.33 x 106 CFU g-1 was obtained, corresponding to a 416% cell viability. This erratic observation on the trend of the cell concentration per gram of the carrier might be caused by the uneven distribution of the inoculant to the carrier during the mixing process. Sampling procedure might also have affected the total number of cell count in each sampling time. The nitrogen-fixing bacteria might be compatible to the carrier which best suited for the acclimitazation of the inoculant. Phosphate Solubilizer. A starting 2.97 x 108 CFU g-1 was determined for the F-C2 inoculant mixed in the carrier (Figure 5). Three bags with three replicates each for sampling was monitored weekly for one month and all were averaged for easy monitoring. A decreasing trend of cell count was observed for all the sampling bags containing the carrier and inoculant. From week zero to week one, a notable decrease was already recorded with only 70% of computed cell viability (2.97 x 108 to 2.10 x 108 CFU g-1) remaining. Week two almost have a ten-fold decrease in viable cell count with only 3.78 x 107 CFU g-1, corresponding to a 12.72% cell viability. This trend of decreasing cell count continued up to week 4, as supported by the exponential average, reaching up to only .98% cell viability with 2.92 x 106 CFU g-1. Most of the bacterial cells probably did not survive or acclimatized with the carrier during the curing process and resulted to less than 1% of viable cells at the end of the four weeks monitoring period. 0 1 Sample 2 Sample 3 Expon. (Average)

DISCUSSION Nitrogen-fixing and phosphate solubilizing bacteria were isolated from the rice paddy and forest soils, and none of the isolates from the grassland area was positive for both determinative tests. This was not expected because nitrogen-fixers can be isolated from tropical grasses that have associative symbioses with the microorganisms (6). Soils from different environments were processed for the isolation of nitrogen-fixing and phosphate solubilizing bacteria because they contain different populations of bacteria, which are generally influenced by the soil properties like pH, salinity, chemical composition, and coarseness of the soil particles (11). Mode of action of the bacteria to be positive on the Dobereiners medium was to fix nitrogen from the atmosphere since the media was lacking of a nitrogen source. Accumulation of ammonia (NH3) on the medium due to the fixing capability of the bacteria results to the increase in pH making the media turn from light green to blue-green due to the indicator dye bromthymol blue. On the other hand, Pikovskaya medium was used for the detection of phosphate solubilizing PGPB. Phosphate dissolving soil microorganisms play a part in correcting the phosphorus balance of crop plants. A positive phosphate solubilizer is indicated by a clear zone surrounding the isolated colony, where the tricalcium phosphate in the media is solubilized by the microorganism to get access of the phosphate ion (17). Based on the observed cultural and morphological characteristics of the F-C2 and PSB isolates, probable identification of the unknown isolates cannot be deduced. More morphological tests should be done to identify the unknown isolates of bacteria like oxidative-fermentative test, catalase test, oxidase test, methyl red test, voges-prokauer test, and motility test (27). Molecular techniques for identification of the bacterial strains would be a much better option where DNA can be extracted and 16S DNA could be amplified using PCR and be sequenced and would give the most probable identities of the unknown isolates. The carriers were autoclaved for three consecutive days at 121 C for 1 h to eradicate all the vegetative cells and possible spores that were present in the inoculant. It is very important to sterilize the carrier to make sure that the longevity of the inoculant be achieved. The use of non-sterile carriers is much cheaper, however, the quality and shelf-life of the inoculant would be very much affected (2). A method of autoclaving peat for 4 h at 121 C was also found to be a satisfactory method (3). There are other ways on how to sterilize carriers which depends mainly on

the type of the container in which the carrier is packed but also on the facilities available. Other methods that can be employed are flash-drying, gamma irradiation, and chemical sterilants (5). Cell concentration of F-C2 and PSB had a different exponential trend which may be due to some factors associated with the proper selection of bacterial strain and carrier. For the nitrogen-fixer F-C2, cell concentration was maintained higher than the initial cell count of 2.24 x 106 CFU g-1. However, the starting initial count already did not pass the required number of viable cell count of 1 x 108 CFU g-1. In cases when the seed broth or mother culture do not reach the required 1 x 108 CFU g-1, the prepared inoculum should be discarded and a new build-up culture should be made. On the other hand, the initial cell concentration from the carrier with the PSB inoculant reached the minimum viable cell count required of 2.97 x 108 CFU g-1. On the monitoring of the cell viability from week zero to week four, cell concentration was observed to decrease dramatically reaching up to only 2.92 x 106 CFU g-1. Percent cell viability on the forth week was less than one percent. These trends in the cell concentration of bacterial cells can be linked to two major factors when considering development of inoculants, the selection of the bacterium and the proper carrier to be used (2). Inoculants have to be designed to provide a dependable source of beneficial bacteria that survive in their corresponding carriers. Choosing the right carrier is really important because it takes the major portion of the inoculant. A good carrier should have one essential characteristic which is the capacity to deliver the right number of viable cells in good physiological condition at the right time (7). To date, no known universal carrier or formulation is presently available for all types of microorganisms (24). Other carriers that can be used can be of plant waste material origin, powders, slurries, granular, and liquids, which all depend on the market availability, cost, and the needs of a particular crop under specific environmental conditions (21). One of the major problems encountered in this experiment was the loss of viability of the bacterial cells in the inoculant after sometime. Developed inoculant lacking the capability to retain its viability for a long period of time is generally unacceptable in the agricultural market (14). The most commonly used carrier since 1950s up to date is finely ground peat although soils high in organic matter and mixtures of soil plus peat or compost or lucerne meal have been used successfully (26). In summary, the selection of an isolate with superior characteristics compatible with a specific carrier should be properly chosen and be optimized. It is better to have the identity of the nitrogen-fixer or

phosphate solubilizer prior to the production of the seed inoculant to have a better knowledge of the optimum conditions needed for maximum biomass production from seed inoculum. Isolates should also be screened for other important metabolites that might be produced or excreted that may be beneficial for agricultural crops. Different strategies might also be developed for the production of inoculants with mixed culture to make the inoculants more effective in providing nutrients to the plant crops. Recombinant DNA technology would also be a great tool to manipulate bacterial strains, creating superior bacterial strains that are able to produce a broad spectrum of enzymes needed to increase free nutrients in soils. For the inoculants to be commercially produced and be available to the working farmers, extensive research should be done first with actual field testing to determine the safety of use of such PGPBs as biological agents.






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