Phosphate and Molybdenum Content in Liquid Biofertilizer Formulation of N 2 Fixing Endophytic Bacteria and its Characteristic and Growth By : Mieke Rochimi Setiawati, Dedeh Hudaya Arief, Pujawati Suryatmana, and Ridha Hudaya Soil Biology and Biotechnology Laboratory Agriculture Faculty, Padjadjaran University Jl. Raya Bandung Sumedang KM 21, Jatinangor. Bandung 40600 e-mail : miekesetiawati@yahoo.com ABSTRACT Nitrogen-fixing endophytic bacteria which is symbiose with rice plant can be used as biofertilizer because could be give N-fixing as biologically directly to plants. Increasing the role of N 2 fixing endophytic bacteria in broad spectrum needs along with produce its biofertilizer. Liquid media formulation as production media N 2 fixing endophytic bacteria needs ATP energy resources from phosphate and molybdenum as nitrogenase enzyme precursor for high quality of liquid biofertilizer. The objective of the research is to attain the liquid biofertilizer formulation of preeminence N 2 fixing endophytic bacteria with high density of population and nitrogenase activity. The research found that Pseudomonas sp was isolate endophytic bacteria with the most stable nitrogenase activity compared with Burkholderia cepacia and Acinetobacter sp. Pseudomonas sp. population and nitrogenase activity were increase with added phosphate (K 2 HPO 4 ) 0.6 g/l but it was sensitive to high concentration of sodium molybdenum. The molybdenum (Na 2 MoO 4 2H 2 O) necessity for Pseudomonas sp bacteria was 0,01 g/l. The highest biomass productivity of Pseudomonas sp occurred at twelfth hour. Hence, it can be inferred the incubation period for inoculation of endophytic bacteria productivity of Pseudomonas sp. should be done within less than twelve hours. Key words: nitrogen-fixing endophytic bacteria, nitrogenase activity, biofertilizer 1
Introduction The plant kingdom is colonized by a diverse array of endophytic bacteria which form non-pathogenic relationships with their hosts. When beneficial, such associations can stimulate plant growth, increase disease resistance, improve the plant s ability to withstand environmental stresses (e.g. drought), or enhance N 2 fixation (Sturz and Nowak, 2000). Crop sequences can favour the build-up of advantageous associations of bacterial endophyte populations leading to the development and maintenance of beneficial host-endophyte allelopathies. Utilization of endophytic bacteria in sustainable crop production systems will require strategies to create and maintain beneficial bacterial populations and N 2 fixing as biofertilizer for increasing the role of N 2 fixing endophytic bacteria in broad spectrum. The existence of the element phosphorus (P) is very important in the media N 2 fixing endophytic bacteria growth, especially in N 2 transfer of energy in the cell as ATP and ADP. For nitrogen fixing is also required molybdenum (Mo) in sufficient quantities. Molybdenum is required in the fixation nitrogen as a cofactor in the nitrogenase enzyme activity of N 2 fixing endophytic bacteria in growth media. Materials and Methods N 2 fixing endophytic bacteria production media formulation research performed in a batch fermentor type facultative anaerobic equipped with controlled conditions pipe outlet and a mixing tool/agitator at a speed of 120 rpm. Batch fermentor anaerob facultative type Sampling of media formulation culture 2
Preparation liquid media for culture biofertilizer Preparation for Reduction Acetylene Assay analysis Research design used is Complete Random Design Factorial pattern. The first factor is the dose of the element phosphate (KH 2 PO 4 ) consists of 3 levels (0.2 g/l; 0.4 g/l; 0.6 g/l, and 0.8 g/l). The second factor dose of molybdenum (Na 2 MoO 4 2H 2 O) consists of four levels (0.001g/L; 0.01g/L; 0.1g/L; and 1.0 g/l). Combination treatment is repeated two times. The observed parameters are N 2 fixing endophytic bacteria population and its nitrogenase activity. Endophytic bacterial population sampling conducted by turbidimetry method using 620 nm length spectrum of spectrophotometer. Samples were analyzed by calculating increase in bacterial biomass in each treatment given. Analysis conducted by nitrogenase activity method Acetylene Reduction Assay (ARA) were analyzed by using Gas Chromatography (Olivares et al, 1996). Result and Discussion Before determining the needs of phosphate and molybdenum in the liquid biofertilizer formula is carried out ability of N 2 fixing test on endophytic bacteria isolates. Three N 2 fixing endophytic bacteria isolates from previous studies tested nitrogenase activity. The results of the analysis are as follows: 3
Table 1. Nitrogenase activity of N 2 fixing endophytic bacteria isolate culture Isolate code ( isolate species) Nitrogenase activity (nmol C 2 H 4 g -1 DW hour -1 ) on 2007 E16 (Burkholderia cepacia) 225,0 94,0 E26 (Pseudomonas sp.) 254,0 233,0 E36 (Acinetobacter sp.) 263,5 212,0 Nitrogenase activity (nmol C 2 H 4 g -1 DW hour -1 ) on 2008 From the table above E26 isolate (Pseudomonas sp.) has nitrogenase activity which more stable compared to another N 2 fixing endophytic bacteria isolates so that Pseudomonas sp. used in this study. The bacteria Pseudomonas sp growth curve below is the results of analysis conducted on the standard curve and N 2 fixing endophytic bacteria populations during the growth. To make the bacterial growth curve, samples of bacteria cultured were analyzed every three hours for 27 hours. By making bacterial growth curve we can determine the optimum harvest age at the time of producing liquid bio-fertilizers Pseudomonas sp. bacteria. KURVA PERTUMBUHAN B 300 250 Cell (CFU/ml) X 10 6 200 150 100 50 0 0 5 10 15 20 25 30 Waktu (Jam) Figure 1. Bacterial growth curve Pseudomonas sp. N 2 fixing endophytic bacteria Figure 1 shows the growth curve of bacteria Pseudomonas sp consisting of several phases of which phase is the phase lag adaptation of the bacteria Pseudomonas sp, which lasted for 6.5 hours; exponential phase of bacterial growth phase increased with the maximum speed indicated by a sharply rising curve. In the exponential phase, bacteria Pseudomonas sp. propagated by multiple exponential until eventually will produce a maximum population. In the picture above the maximum bacterial population seen after 12 hours of growth that can be achieved is 2.72 x 4
10 8 cfu/ml. Exponential phase is used to determine the optimum harvest age at the time of producing liquid bio-fertilizers bacteria Pseudomonas sp. The next growth phase is marked by stationer phase straight line where the number of bacteria that grow almost equal to the number of dead. Final phase of bacterial growth curve is the phase where the number of deaths of live bacteria is less than the number of dead bacteria. From the growth curve Pseudomonas sp., it appears that the character of the kinetics of the isolates showed that exponential final phase occurs at the 12th hour. This means that the maximum cell production can be harvested at hour 12. This character can be a reference basis for the development of mass production inoculants, for efficiency of time. Because of this long before the characteristic kinetics of these isolates is known, to isolate the biomass production lasted for 24 hours. Thus this growth curve analysis can indicate the maximum production time is appropriate and efficient. Efficiency of isolates of the production time (24 hours - 12 hours) is 12 hours, and at 24 hours to bacterial growth even in death phase (decrease in viability) and this is not recommended for harvesting. Population of N 2 fixing endophytic bacteria From the analysis N 2 fixing endophytic bacteria populations due to treatment phosphate and molybdenum, there was no interaction but each element has independent effect. This is thought to occur due to the phosphate levels are not sufficient for adequate growth of bacteria or a range of phosphate is used not to show the interaction effects for growth. This is evident from the results obtained with the addition of phosphate levels have increased growth trend. Thus the optimum dose to give effect to the increased growth has not appeared. Phosphate elements are given in the media production of N 2 fixing endophytic bacteria at a certain concentration lead to increased bacterial population. Adding the element phosphate (K 2 HPO 4 ) at concentrations of 0.8 g/l causing N 2 fixing endophytic bacteria population increase. Concentrations below 0.6 g/l, K 2 HPO 4 provision does not cause an increase in N 2 fixing endophytic bacteria population (Table 2). According to Leigh et.al. (1995) phosphate functions as a component of ATP formation. ATP is needed to reduce N 2 invitro. ADP produced would inhibit nitrogenase activity, therefore this system should be used phosphorylation of ADP to ATP by the addition of P from the outside. 5
Table 2. N 2 fixing endophytic bacteria populations due to differences in concentrations of phosphate and molybdenum Endophytic bacteria K 2 HPO 4 concentration (P) population (cfu x10 9 ) 0,2 g/l (p 1 ) 0.96 a 0,4 g/l (p 2 ) 1.09 a 0,6 g/l (p 3 ) 1.25 ab 0,8 g/l (p 4 ) 1.32 b Na 2 MoO 4 2H 2 O concentration (M) 0,00l g/l (m 1 ) 1.30 b 0,0l g/l (m 2 ) 1.20 b 0,l g/l (m 3 ) 0.96 a 1,0 g/l (m 4 ) 0.86 a Note : Figures marked with the same letter are not significantly different according to Duncan test at 5% level. In contrast to the effects of molybdenum, the results indicate that the increased concentration of high molybdenum gives the likelihood of resistance to the growth of Pseudomonas sp. isolate. This means that the sp Pseudomonas very sensitive to high concentrations of molybdenum. Conclusions can be made that optimum molybdenum concentration for growth of Pseudomonas sp levels are below 0.001 g/l Na 2 MoO 4 2H 2 O. Nitrogenase activity of N 2 fixing endophytic bacteria The influence of the interaction between phosphate concentration and molybdenum not occur in bacterial nitrogenase activity of N 2 fixing endophytic bacteria. Concentrations of phosphate and molybdenum affect nitrogenase activity of N 2 fixing endophytic bacteria. K 2 HPO 4 concentration (P) 0.6 and 0.8 g/l of bio-fertilizers, N 2 fixing endophytic bacteria produces nitrogenase activity which is higher than the concentration of P below. Suspected concentration of phosphate 0.6 g/l or more is sufficient concentration to be able to supply P for phosphorylation of energy for bacterial nitrogenase enzyme production N 2 fixing endophytic bacteria. 6
Molybdenum concentration 0.01 g/l produced the highest nitrogenase activity, below and above the concentration of 0.01 g/l did not increase nitrogenase activity (Table 3). Maximum nitrogenase activity which is the optimum concentration of molybdenum as a precursor in the nitrogenase enzyme activity. The higher nitrogenase activity of endophytic bacteria will produce a large N 2 fixing so that the greater the N 2 is reduced by the bacteria into NH 3. The NH 3 elements will be reduced to NH 4 and then used to form proteins and bacterial biomass. Table 3. Nitrogenase activity due to differences in concentrations of phosphate and molybdenum K 2 HPO 4 concentration (P) Nitrogenase activity (nmol C 2 H 4 g -1 DW hour -1 ) 0,2 g/l (p 1 ) 1.0168 a 0,4 g/l (p 2 ) 1.2823 a 0,6 g/l (p 3 ) 1.5587 b 0,8 g/l (p 4 ) 1.6432 b Na 2 MoO 4 2H 2 O concentration (M) 0,00l g/l (m 1 ) 1.2799 a 0,0l g/l (m 2 ) 1.5093 b 0,l g/l (m 3 ) 1.2377 a 1,0 g/l (m 4 ) 1.0972 a Note: Figures marked with the same letter are not significantly different according to Duncan test at 5% level. In the production process of bio-fertilizers is needed efficiency of input. P applications based on liquid bio-fertilizers, increased bacterial populations and endophytic efficient nitrogenase activity resulting from the provision of K 2 HPO 4 0.6 g/l. With the concentration of those elements able to efficient 0.2 g per liter of liquid bio-fertilizers which are made while the bacterial population endophytic and nitrogenase activity generated as much by giving K 2 HPO 4 0.8g/L. 7
Endophytic bacterial populations as large as a result of granting molybdenum for 0.001 and 0.01 g/l did not cause an increase in nitrogenase activity as much. It is alleged the elements can only increase molybdenum nitrogenase activity that is responsible for N 2 fixation, but did not affect to growth or population N 2 fixing endophytic bacteria. Conclusions 1. Pseudomonas sp was isolate endophytic bacteria with the most stable nitrogenase activity compared with Burkholderia cepacia and Acinetobacter sp. 2. Pseudomonas sp. population and nitrogenase activity were increase with added phosphate (K 2 HPO 4 ) 0.6 g/l, whereas the molybdenum (Na 2 MoO 4 2H 2 O) necessity for Pseudomonas sp bacteria was 0,01 g/l. 3. The highest biomass productivity of Pseudomonas sp occurred at twelfth hour. Hence, it can be inferred the incubation period for endophytic bacteria productivity of Pseudomonas sp. should be done within less than twelve hours. Acknowledgments Financial support came from Andalan Research Padjadjaran University, 2008. We thank to Sri Agustini and Ari Rizki Arviana for technical assistance. References Olivares, F.L., V.L.D. Baldani, V.M. Reis, J.I. Baldani, dan J. Dobereiner. 1996. Occurences of the endophytic diazotrophs Herbaspirillum spp in root, stems and leaves predominantly of Graminae. Biol, Fertil. Soils. 21 : 197-209. Sturz, A.V. and J. Nowak. 2000. Endophytic communities of rhizobacteria and the strategies required to create yield enhancing associations with crops. Appl. Soil Ecology. 15:2, 183-190. 8