Production of nitrogen fixing Azotobacter (SR-4) and phosphorus solubilizing Aspergillus niger and their evaluation on Lagenaria siceraria and Abelmoschus esculentus

Highlights • Drawbacks of chemical fertilizers have attracted the attention of farmers toward biofertilizers.• Nitrogen fixer Azotobacter SR-4 and phosphorus solubilizer Asphergillus Niger were produced and observed to be efficient biofertilzers.• A significant increase in yield parameters were observed when these biofertilizers were evaluated on Bottle gourd and okra.• In conclusion, biofertilizers or microbial inoculants could be used as an alternate source, which are both cost-effective as well as eco-friendly.


Background
Nitrogen (N) and phosphorus (P) are well-known fundamental nutrients needed by plant for their growth and development. To achieve high yield, farming practices require chemical fertilizers that are very costly and may also create environmental problems. Owed to environmental alarm and fear for consumer health, the use of chemical fertilizers in agriculture is presently under debate. Consequently, a specific group of fertilizers were discovered that are known as biofertilizers or bioinoculants and are consisted of microorganisms with plant growth-promoting abilities. Some of these microbial strains are capable of phosphorus solubilizing, nitrogen fixing from air and some produce cellulytic enzymes. Biofertilizers are applied in several ways to soil, to enhance the nutrient availability to the plants. One way is their direct application in soil, other way is seed treatment or application with composite. In either way the biofertilizers are used, they increase the numbers of beneficial microorganisms in the soil to enhance the nutrients availability for the plants.
A number of free-living nitrogen fixing bacteria have been reported as biofertilizers previously [1][2][3]. These bacteria greatly influence the plant growth when used as seed inoculants [4]. Some of them may affect the plants growth directly through synthesis of growth hormones, fixing nitrogen and solubilizing rock phosphates, when used as biofertilizers [5][6][7]. On the other hand, plant growth is also stimulated by phosphate-solubilizing microbes that enhance the available phosphorus and increase the uptake rate of nitrogen, potassium (K), and iron (Fe) [8]. These phosphate solubilizing microbes convert the insoluble phosphates into soluble form using different processes such as exchange reaction, acidification and chelation [9]. It has been reported in an earlier study that plants co-inoculated with biofertilizers have a significance increase in root and shoot biomass, nitrogenase activity and nitrogen fixation [10][11][12][13][14]. Moreover, combined inoculation of P-solubilizing and N-fixing biofertilizers were more effective compared to single inoculation due to the availability of more balanced nutrition for plants [15].
Therefore, in the present study, a bacterial strain Azotobacter (SR-4) and a fungal strain A. niger were used for nitrogen fixation and phosphorus-solubilization, respectively.

Collection of cultures
The bacterial culture Azotobacter (SR-4) and fungal culture A. niger was obtained from the Food and Biotechnology Research Center (FBRC) of PCSIR, Labs. Complex, Lahore, Pakistan. This bacterial strain was previously isolated and identified at the same institute [16].

Inoculum preparation for nitrogen fixer
Stock culture of Azotobacter (SR-4) was maintained on nutrient agar slants and glycerol cultures in nutrient broth and was stored at À80 C. Inoculum was developed in Erlenmeyer flasks using nitrogen free (N-free) medium (2 g sucrose, 0.06 g K 2 HPO 4 , 0.016 g KH 2 PO 4 , 0.02 g NaCl, 0.02 g MgSO 4 , 0.05 g yeast extract, 0.01 g K 2 SO 4 , pH 7) and was incubated on a rotary shaker (360 rpm) at 30 C for 24 h.

Inoculum preparation for phosphorus-solubilizer
A. niger was cultured in potato dextrose agar (PDA) slants for 3-4 days at 30 C and fully-grown slants were stored at 4 C for future use.

Production of Azotobacter in small bioreactor
For fermentation, 1 L N-free media (2 g sucrose, 0.06 g K 2 HPO 4 , 0.016 g KH 2 PO 4 , 0.02 g NaCl, 0.02 g MgSO 4 , 0.05 g yeast extract, 0.01 g K 2 SO 4 , pH 7) was prepared in 1 L bioreactor. 50 ml Azotobacter (SR-4) inoculum, maintained in N-free media, was transferred to bioreactor. Samples were collected from bioreactor daily up to 6 days and were then analyzed for nitrogen.

Solid-State Fermentation for A. niger
For solid-state fermentation, 1 kg wheat bran was moistened with 500 ml water and was sterilized in autoclave at 121 C for 15 min. After cooling to room temperature, the culture of A. niger was inoculated as inoculum and then incubated for 8 days at 30 C in a static inclined position in a steel tray covered with aluminum foil having small pores.

Evaluation of biofertilizers
The effect of these biofertilizers on plant's height, length and width of leaf and the number of fruits per plant was tested in L. siceraria and A. esculentus. For this purpose, three treatments were applied; In 1 st treatment, 500 g plant seeds were inoculated with 250 g phosphorus solubilizing biofertilizer, in the 2 nd treatment seeds were treated with 250 mL nitrogen fixing biofertilizer and in the 3 rd treatment seeds were co-inoculated with both phosphorus (250 g) and nitrogen biofertilizers (250 mL) ( Table 1). The biofertilizers were used to make slurry with water (phosphorus solubilizing) and soil (nitrogen fixing), and the slurry coated seeds were then air dried and were sown in soil. However, same quantity of untreated seeds was used as control.

Efficiency of Azotobacter as nitrogen fixer
The nitrogen fixing efficiency of Azotobacter (SR-4) is equal to the mg of nitrogen produced per gram of carbon utilized. The nitrogen efficiency of Azotobacter (SR-4) was determined by the Kjeldahl method [17]. The samples were centrifuged at 4000 rpm and 4 C for 10 min. Then 2 ml of supernatant was mixed with 10 ml K 2 Cr 2 O 7 solution and 20 ml of H 2 SO 4 and was heated for 1 min. In the following step, 200 ml of H 2 O was added again with 4-5 drops of ferroin indicator. Titration of the above solution was conducted against 0.5 N FeSO 4 solution and total carbon was measured from the total volume of FeSO 4 solution used.

Efficiency of A. niger as phosphorus solubilizer
Similarly, in determination of phosphorus solubilizing activity, the samples were centrifuged at 4000 rpm and 4 C for 10 min and supernatant was collected. Vanadomolybdate reagent was prepared by mixing ammonium molybdate 5% (W/V), ammonium vanadate 0.25% (W/V) and diluted nitric acid with water in 1:3 ratios and was used to measure the soluble phosphorus [18]. However, Heinonen method was used to analyze phytase and phosphatase [19]. 1 mL of supernatant was incubated with same quantity of phytic acid and tricalcium phosphate (substrate for phytase and phosphatase) in 200 mM glycine buffer (pH 5) at 35 C for 1 h. After incubation, 1 mL citric acid (1 M) was added to stop enzyme activity. In the last step, 4 mL of reagent mixture containing 2.5% (W/V) solution of ammonium molybdate, 5 N H 2 SO 4 and acetone in 1:1:2 ratio was added, vortexed and the optical density was observed at 400 nm. 1 IU for phosphatase and phytase was equal to 1 mM of phosphorus released/mL/min.

Statistical analysis
The findings were statistically compared through LSD test using SPSS V16. The significant difference was represented with different letters and non-significant difference was represented with similar letters with yield values.

Nitrogen fixing efficiency of Azotobacter
Carbon utilization and nitrogen fixationwas determined at different intervals of fermentation. For this purpose, samples (n = 6) were collected after every 12 h for 3 days of fermentation. After 12 h of incubation, carbon utilization was 0.34 g/100 mL and was increased to 0.61 g/100 mL by 72 h. Similarly, N fixation capacity was increased from 8.0 mg/100 mL to 21.40 mg/100 mL by 72 h of incubation. Consequently, with increase in cell mass the carbon contents of medium decreases lead to increased utilization of carbon. The efficiency of Azotobacter (SR-4) (mg of nitrogen produced per gram of carbon utilized) was 23.52 N mg/gC after 12 h and increased to 35.08 N mg/gC with increase in fermentation time to 72 h ( Table 2).

Phosphorus solubilizing efficiency of A. niger
Phosphorus solubilizing efficiency was measured in solid state fermentation at different intervals of fermentation. Enzymatic activity was increased from 0-170IU for phosphatase and 0-133IU for phytase during 0-48 h of incubation. However, decline in concertation of both phosphatase and phytase was observed after 48 h. Similarly, maximum soluble phosphorus of 835 ppm was observed after 48 h of incubation which support the increased production of phosphate degrading enzymes by A. niger (Table 3)

Biofertilizers effect on Lagenaria siceraria and Abelmoschus esculentus
The field trials of biofertilizers on selected plants (L. siceraria and A. esculentus) showed significant increase in plant height, leaf length/width, fruit size and number of fruits per plant when compared with controls/untreated plants. Furthermore, plants coinoculated with both the N fixing Azotobacter and phosphorus solubilizing A. niger have enhanced performance than those treated with each biofertilizer alone (Tables 4 and 5).

Discussion
The bio-fertilizers due their environment friendly nature as compared to the chemical fertilizers is a method of choice for the modern agriculture. Therefore, the present research work was aimed to produce and evaluate Azotobacter (SR-4) as a nitrogen fixer and A. niger as phosphorus solubilizer, that are important constituents needed by the plants. These strains were obtained from the Food and Biotechnology Research Center (FBRC) of PCSIR, Labs. Complex, Lahore, Pakistan. Their inoculums were developed and maintained and were grown in large scale. Then their efficiency as biofertilizers were evaluated and tested in field trails on selected plants in three different treatment schemes.
Azotobacter species have been known to releases variety of growth-promoting substances in addition to nitrogen like indole acetic acid, vitamins B and gibberellins [20,21]. Similarly, Azotobacter species excrete ammonia in the rhizosphere hence helps in plant improvement [22]. In the present study, the Nfixation capacity of Azotobacter (SR-4) was measured by analyzing the concertation of nitrogen in the medium using Kjildhal method [17]. It has been reported that the excess of carbon compound and   0  0  3  8  275  18  0  4  12  360  42  19  5  16  400  84  58  6  20  475  117  112  7  24  710  145  125  8  36  785  154  131  9  48  835  170  133  10  60  775  152  125  11  72  690  136  120 shortage of combined nitrogen in the media greatly affect the activity of nitrogen fixing microorganisms [23]. Increase in Azotobacter (SR-4) activity was observed with increase incubation time. This increase in activity is due to increase in cell mass and hence the increased nitrogenase enzymes production by Azotobacter that fix nitrogen (Table 2). Our findings of increase in efficiency of Azotobacte with increase in incubation time is consistent with other studies, where similar trend in behavior has been reported [24]. Furthermore, we observed higher nitrogen fixing efficiency as compared to other published reports [25,26]. Variation in efficiency of Azotobacter may be due to difference in strains being used in different studies. Sometimes, the efficiency might be different due to amount of dissolved oxygen that affect the carbon consumption rate as well as nitrogen fixation. Although, it has been shown that Azotobacter sp. is usually considered as nitrogen fixer, however, addition of small quantity of nitrogen in the medium reduce the fermentation time due to short lag phase and generation time [25]. This assumption was tested by increasing the incubation time and hence maximum nitrogen fixation was observed in the current study. Due to this Nfaxing capability of Azotobacter, extracellular proteins and ammonia are secreted in nitrogen free medium that are accessible to the plants. Similarly, Azotobacter (SR-4) was found to be efficient nitrogen fixer able to fix 23.52-35.08 mg N/g of carbon oxidized which correspondent to 8-21.40 mg N/g sucrose consumed within 72 h (Table 2). A similar trend of increase in cell count with increase in incubation time has been previously observed which reaches to its peak after two weeks [26]. Although, an aerobic condition is required for growth of Azotobacter, while low oxygen tension or anaerobic conditions are optimum for enzymatic activity of a system, such as nitrogenase responsible for nitrogen fixation. Similarly, compare to liquid media, soil provides more suitable condition for Azotobacter growth and subsequently nitrogen fixation by providing superior equilibrium between anaerobiosis and aerobiosis.
We observed an efficient phosphorus solubilizing activity in A. niger which agrees to previous reports [27][28][29]. A significant increase in the concertation of phytase, phosphatase and soluble phosphorus was also found after 48 h of fermentation along with decrease in soluble phosphorus concentration. This decrease may be due to utilization of phosphorous by fungus mycelia. Our findings are consistent to an earlier study where Aspergillus has shown maximum growth after incubation of 50 h under favorable conditions [30].
The increase in concentration of enzymes observed in our study is higher than that reported in literature [31]. Although, fungi produce these enzymes to solubilize phosphate and phytic acid for its own growth, consequently, a considerable amount of phosphorus become available to plants as well. These N fixing and phosphorus solubilizing biofertilizer were tested in field trails on selected plants in 3 different treatments (Tables 4 and 5). Previous studies have reported that co-inoculation of strains has enhanced root growth, shoot biomass, N% as well as total plant nitrogen in many crops [10][11][12]. Similarly, co-inoculations have considerably increased the yields as compared to single inoculation in soybean, pea, chickpea, groundnut, mungbean and in other crops [31][32][33][34][35][36][37]. There are several reports available on the inevitability and applicability of bacterial fertilizers and phosphate dissolving and N 2 -fixing bacteria [24,[38][39][40][41][42]. Azotobactor when applied to green gram and rice have shown a significant improvement in seed germination and root nodules [42]. Furthermore, Aspergillus sp. has been considered as plant growth promoting fungi by solubilizing phosphorus and can travel long distance as compared to bacteria [39,41]. Interestingly, the co-inoculation of phosphorus solubilizing fungi and nitrogen fixing bacteria were found effective in overcoming drought stress in legume plants as well [40].
Considering these fact and findings it can be said that the effects can be highly variable in practical agriculture. However, it further signifies that combined inoculation of seeds with A. niger and Azotobacter may replace costly and environment toxic fertilizers with environment friendly biofertilizers [43].

Conclusion
It is concluded that biofertilizers, including the nitrogen fixing and phosphorus solubilizing biofertilizer, can be produced cost effectively and can be used for the sustainable and environmental friendly yield enhancement of vegetables. Higher yield than the control showed that the produced biofertilizers have no adverse effect on plant growth of bottle guard and okra. Therefore, the used microbial strains have been proved potential for the routine agriculture practices with additional benefits either separately or in combination or may be supplemented to the chemical fertilizers to reduce their impact on environment.

Conflict of interest
There is no conflict of interest in the research work.  The values with different letters are significantly different. The values with different letters are significantly different.