The application of Klebsiella sp. and Rhizobium radiobacter as biofertilizer and Palm Oil Mills Effluent (POME) as organic fertilizer on growth of Paraserianthes falcataria

The Bio and organic fertilizers are cheap and environmentally friendly source of plant nutrients for agricultural yields and environmental quality improvement. The study aimed to evaluate the application of biofertilizer and Palm oil mills effluent (POME) either singly or in combination on growth of Paraserianthes falcataria under greenhouse condition. The study was laid out in factorial based Completely Randomized Design (CRD) design which was comprised of biofertilizer treatments (Control without bacteria, Klebsiella sp, Rhizobium radiobacter, Klebsiella sp + Rhizobium radiobacter) and five concentrations of POME treatments (0%, 10%, 25%, 50%, 100%) with 3 replicates for each treatment. The results revealed that inoculation with biofertilizer treatments along with POME significantly enhanced plant growth parameters, soil available P, soil phosphatase activity and soil bacterial population. Combination of Rhizobium radiobacter and POME 50% induced the highest increase of shoot length (10.17 ± 0.83cm), root length (28.67±0.88cm), shoot dry weight (0.48± 0.006g), and root dry weight (0.25±0.013g). The highest soil phosphatase activity was obtained in the combination Klebsiella sp. and POME 25% treated soil. The application of Rhizobium radiobacter along with POME at 10% and 25% concentrations reached its highest level of soluble soil P (4.07 ppm) and soil bacterial population (48.33 107 cfu/g of soil) respectively.


Introduction
The increase of continuously intensive land exploitation leads to the decrease of soil organic. Consequently, it causes the decline of soil fertility and land productivity. The addition of organic matter at the time of planting as bio and organic fertilizer is one of the alternative methods to maintain and increase the organic matter level and soil fertility [1,2]. In sustainable agriculture, microbial inoculants and organic fertilizers are affordable and environmentally friendly nutrition sources and are important for the management of soil nutrient [3].
Klebsiella sp. and Rhizobium sp. which are used as biofertilizers, are root-associated bacteria that have an important role as plant growth promoting rhizobacteria (PGPR). These bacteria are able to solubilize both organic and inorganic phosphates, fix nitrogen, produce IAA growth hormone and ACC De aminase activity [4,5,6,7,8,9].
In agricultural sector, waste utilization as nutrients enrichment to improve plant growth is often needed. One of the best agricultural waste that can be converted into organic fertilizer is palm oil  [10]. POME (Palm Oil Mill Effluent), one of the palm oil industry by-products, is oily brownish liquid obtained from the extraction of palm oil. POME can be used as fertilizer on agricultural land due to its macro nutrients content such as N, P and K. The application of POME to the soil is not only able to increase soil fertility but also improve soil structure resulting in better root health and plant growth [11]. Some researchers suggested that the use of organic and sole biofertilizer or a combination of both provided positive response in regard to plant growth, nutrient uptake and availability of plant nutrients [12,13,14].
The objective of this research was to investigate the effectiveness of bacterial inoculants as biofertilizers combined with POME in various concentrations as organic fertilizer on Paraserianthes falcataria in greenhouse-scale.

Green house experiment
Rhizobium radiobacter (InaCC B834) and Klebsiella sp. (InaCC B833), used as biofertilizers, were obtained from the InaCC Collection, Microbiology Division of the Biology Research Center, LIPI. The abilities of these bacteria in fixing N, ACC deaminase activity, producing IAA, solubilizing inorganic and organic phosphates have been tested in vitro [15] . For the inoculants preparation, 1 ml of each Rhizobium radiobacter and Klebsiella sp. was inoculated on 250 ml Erlenmeyer containing Yeast mannitol (YM) broth and liquid Pikovskaya respectively [16]. The erlenmeyer was incubated on a shaker at the speed of 120 rpm for 3 days at room temperature. Bacterial culture was centrifuged at the speed of 12000 rpm for 10 minutes and the pellets obtained were diluted into sterile aquadest until the cell density was 10 9 cfu/ml. The bacterial solution was used as seed inoculant.
Palm Oil Mill Effluent (POME), used as organic fertilizer, was taken from ponds in PTP VIII Cikasungka, West Java. Paraserianthes falcataria seeds were obtained from the collection of Bogor Botanical Garden. The seeds were sterilized in sodium hypochlorite then rinsed with sterile distilled water three times. The seeds were germinated in sterile Petri dish which had been coated with sterile filter paper beforehand. The sprouts of P. falcataria were inoculated by being immersed in liquid culture of Rhizobium radiobacter and Klebsiella sp. for 60 minutes. Inoculated sprouts then were planted in pots filled with 300 grams of soil. Non-inoculated plants were used as controls.
Planting was carried out in a greenhouse in Microbiology -Research Center for Biology -LIPI Cibinong. As much as 300 grams of soil was loaded into the pot. Then each pot was given POME solution based on the treatment and incubated for 2 weeks. Three Paraserianthes falcataria sprouts were transferred into each pot. Harvesting was carried out when the plants was 60 days old. The IS BIOREV 2018 IOP Conf. Series: Earth and Environmental Science 308 (2019) 012057 IOP Publishing doi:10.1088/1755-1315/308/1/012057 3 observed variables included root and shoot length (cm), root and shoot dry weight (mg), soil available P (ppm), phosphatase activity (unit) and total bacterial population (cfu/g of soil).

Analysis of available P content, Phosphatase and Total bacterial population in soil
Samples of soil rhizosphere during the harvesting (2 months after planting) from each treatment were analyzed to measure the level of available P, phosphatase activity and total bacterial population.
2.2.1. The measurement of available P. Available P content were determined using the Olsen method, soil samples were extracted with the Olsen's extracting solution (sodium bicarbonate, pH 8.5). Place on a mechanical shaker for 30 minutes at room temperature and centrifuge the suspension for 10 minutes, 12000 rpm. Measured the supernatant colorimetrically [17].

The measurement of phosphatase activity.
A mix of 1 gram of soil sample, 4 ml of substrate p-NPP 0.115 M and 5 ml sterile aquadest was put on a shaker for 30 minutes at room temperature. The mixture was centrifuged at the speed of 12,000 rpm for 15 minutes. As much as 0.5 ml of acetate buffer was added into 0.1 ml of supernatant and incubated for 15 minutes. After incubation, 0.5 ml of 0.5M CaCl2 and 2 ml of 0.5M NaOH were added. The absorbance of the sample was measured at the wavelength of 880 nm. The standard and blank were treated in the similar way as sample. A pnitrophenol solution with a concentration of 1-6 ppm was used as standard, while distilled water was used as blank [18].

Total bacterial population in soil.
The pour plate method was used to count the bacterial population [19]. As much as 1 gram of soil was diluted into 9 ml of sterile aquadest to obtain a 10 -1 dilutions. From the dilution a dilution series up to 10 -7 was prepared. As much as 0.1 ml from 10 -3 ,10 -5 and 10 -7 dilution was taken into a sterile Petri dish, then poured over with nutrient agar on top. Plates were incubated for 7 days at room temperature. The number of viable cells was measured by CFU (colony forming unit/ g of soil).

Statistical analysis
The acquired data were analyzed using SPSS and values were given as means ± SD for triplicate samples. Duncan's Multiple Range Test (DMRT) was applied to test the significance of treatment means at P ≤ 0.05.

Greenhouse experiment
3.1.1. The Effect of biofertilizer and POME on shoot and root length. The application of the biofertilizer significantly increased the length of shoots. Plants that were inoculated with Rhizobium radiobacter (R), Klebsiella sp. (B) and mixture of both (M) showed an increase in shoot length ranging from 39.3-48.2% compared to control without biofertilizer. Similar result was reported by [20] that there was an increase in the length of chickpeas shoots and roots inoculated with phosphate solubilizing rhizobia which had the ability to solubilize P and produce IAA (Figure 1).  Figure 1. Effect of biofertilizer and POME on the length of shoot and root Note: K=Control without Inoculation, R=R. radiobacter, B=Klebsiella sp. M= R. radiobacter + Klebsiella sp, P0=Control without POME, P1=POME 10%, P2=25%,P3=50%, P4=100% 3.1.2. The effect of biofertilizer and POME on shoot and root dry weight. In figure 2, there was an escalation in shoot and root dry weight in plants treated with biofertilizer. The application of Klebsiella sp. and Rhizobium radiobacter as biofertilizers improved shoot and root dry weight compared to plant control without inoculants and POME. Similar result was reported by [21] found that inoculation with phosphate solubilizing Rhizobium leguminosarum biovar phaseoli increased the dry weight of lettuce and corn plants. Correspondingly, [9] study provided that the inoculation of Vigna. radiate, V. tetragonoloba and V. unguiculata with phosphate solubilizing and IAA producer Klebsiella pneumonia showed significant differences in the wet and dry weight of plants compared to controls.
According to [22,23,24], the application of PGPR which has various abilities in solubilizing phosphate, producing phosphatase, IAA and ACC deaminase activity, was more effective in increasing shoot and root length, as well as shoot and root dry weight. This is in accordance with the research results that Klebsiella sp and Rhizobium radiobacter which belong to Plant Growth Promoting Rhizobacteria (PGPR), have the ability to nitrogen fixation, ACC deaminase activity, produce IAA, solubilize inorganic P and phosphatase activity (Table 1). [25] reported that several strains of Rhizobium and Klebsiella, Azospirillum, Azotobacter, aside from possessing the ability to solubilize inorganic phosphate from P source, namely Ca3(PO4)2 also had phosphatase activity. Some Klebsiella species such as K. oxytoca Rs-5 showed the ability to produce IAA and ACC deaminase activity [26], K. variicola AY13 produced IAA [27], K. pneumoniae solubilized P, while producing IAA [9]. Similarly, Rhizobium isolated from plant legumes nodules produced IAA, ACC deaminase and solubilized both inorganic and organic P [28,29].
The usage of POME as organic fertilizer demonstrated positive plant growth responses. Plants treated with POME concentrations of 10%, 20%, 50% and 100% showed significant increase in shoot length, dry weight of roots and shoots compared to plant control without POME. This result is in accordance with the research of [30], that reported an increase in the growth of corn plants treated with fermented POME compared to controls.
The combination of biofertilizer and POME stimulated and improved the growth of P. falcataria. In addition to the length of shoots and roots, dry weight of shoots and roots showed the highest increase in plants inoculated with Rhizobium radiobacter at 50% POME concentration. The  [31] reported that the combination of bio-organic fertilizer would increase plant height and dry weight of ginger plants and also increased the population of bacteria and actinomycetes. [14] suggested that the application of organic fertilizers and biofertilizers can increase plant height and leaf number. It is predicted that the biofertilizer used in conjunction with organic fertilizer help the plants to develop better and absorb more nutrients.

Analysis of available P content, phosphatase and total bacterial population in soil
3.2.1. The effect of biofertilizer and POME on soil available P. The effects of biofertilizer and organic POME fertilizer application on available P in soil at harvest (2 months after planting) are presented in Figure 3. All biofertilizer treatments resulted in positive responses of the increase in available P, compared to controls. The highest level of available P (1.84 ppm) was obtained on the soil treated with biofertilizer of mixed Klebsiella sp. and Rhizobium radiobacter inoculant. This result corresponds the observations of [32], reported that several bacteria such as Rhizobium, Klebsiella and Pseudomonas were able to solubilize insoluble phosphates thus become available to plants. The application of biofertilizers on selected strains increased available P in the soil, it is probably a result of the presence of larger microbial activity in solubilizing P bound or mineralizing organic material [33].
The application of POME of 10, 25, 50 and 100% concentrations as organic fertilizer significantly higher available P than control without POME.
[34] found that available phosphorus, soil pH, organic matter, total nitrogen, exchangeable acidity and bulk density of the soil is affected by the use of POME The combination of biofertilizer and organic fertilizer treatment was more effective in increasing the amount of available P than control and treatment of sole biofertilizers or organic fertilizers only. The maximum available P (4.07 ppm) occurred in soil treated with Rhizobium radiobacter inoculant along with POME 10%.

The effect of biofertilizer and POME on the activity of phosphatase. The application of
Klebsiella, sp. displayed significant effect on phosphatase activity with an increase of 28%, followed by rhizobium radiobacter (11%) compared to controls without inoculants. [22] that reported an increase in phosphatase and organic P mineralization due to the treatment of phosphate solubilizing bacteria inoculant, and phosphatase activity was considered as the major contributor to the increase in phosphorus in the soil.   The application of low concentration POME without inoculants enhanced phosphatase activity and increased while the usage of high concentration POME caused a decrease on observed parameters. The soil applied with POME concentration of 10% showed a rise in phosphatase activity of around 5.4% compared to controls without POME. On the contrary, the application of POME of 25, 50 and 100% concentrations resulted in lower level of soil phosphatase than controls. This was also observed by [35] who reported that phosphatase and dehydrogenase activity in POMEcontaminated soils decreased significantly compared to control soil samples (Figure 4).
The application of Klebsiella sp. inoculant along with 25% POME concentration showed the highest soil phosphatase activity (64.72 units).

3.2.
3. The effect of biofertilizer and POME on soil bacterial population. The population of soil bacteria treated with biofertilizer showed significant difference when compared to the control without inoculants. Plants inoculated with Rhizobium radiobacter produced the highest number of soil bacterial population (38.33 x 10 7 cfu /g soil).
The soil treated with POME 10% and 25% demonstrated an increase in bacterial population. Although in the higher concentration of POME treatment, 50 and 100%, the total count of bacterial population tended to be lower than the control. Likewise, [36,37,38] reported that heterotrophic bacterial population, phosphate solubilizing bacteria, nitrifying and lipolytic decreased significantly in soil contaminated with high concentrations of POME; contrariwise with low concentration. The combination of biofertilizer and organic fertilizer treatment was more effective in increasing the bacterial population than control and treatment of sole biofertilizers or organic fertilizers only. Rhizobium radiobacter combined with 25% POME concentration resulted in the highest bacterial population (48.33 x 10 7 cfu/g of soil). [39] stated that the application of biofertilizers in combination with organic fertilizers increased soil bacterial populations more effectively than using sole biofertilizers or organic fertilizers only. Similar observations have been reported by [40], which discovered a significant increase in soil microflora such as bacteria, fungi and actinomycetes and soil enzyme activities such as phosphatase and dehydrogenase in soil treated with phosphate solubilizing IS BIOREV 2018 IOP Conf. Series: Earth and Environmental Science 308 (2019) 012057 IOP Publishing doi:10.1088/1755-1315/308/1/012057 8 bacterial biofertilizer combined with organic fertilizer in the form of compost. The activity of phosphatase, soil microbial populations and organic matter content can be increased by the application of organic fertilizers [41,42,43].

Conclusion
Plants inoculated with sole Klebsiella sp. and Rhizobium sp. or POME or a combination of both showed an increase of growth in Paraserianthes falcataria. All biofertilizer treatments and combinations with organic fertilizers presented positive response in regard to the increase of available P, phosphatase and total count of bacterial population in soil. The application of low concentration POME without inoculants increased phosphatase activity and soil bacterial population, whereas the use of high concentration POME resulted in a decrease of those parameters.