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Rock phosphate solubilization by immobilized cells of Enterobacter sp. in fermentation and soil conditions
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PlI:SO960-8524(96)00044-S
ROCK PHOSPHATE SOLUBILIZATION BY IMMOBILIZED CELLS OF ENTEROBACTER SP. IN FERMENTATION AND SOIL CONDITIONS Nikolay Vassilev,* Marcia Toro, Maria Vassileva, Rosario Azcon & Jose Miguel Barea Estacion Experimental de1 Zaidin, CSIC, ProJ Alhareda, I, 18008-Granada, Spairl (Received
19 February
1997; revised version received 17 March 1997; accepted 26 March 1997)
Abstract An Enterobacter sp., a phosphate-solubilizing bacterium, was immobilized in agar: I/arious amounts of the immobilized bioparticles were applied in a repeatedbatch fermentation process in order to solubilize Venezuelan rock phosphate. A total production of 388 mg and 403 mg soluble PI1 was obtained for 2.0 g rock phosphate11 in frasks containing 10 ml and 20 ml agar beads, respectively, that was significantly higher compared to a free-cell single-batch control. whole and destroyed immobilized bioparticles were introduced into a soil enriched with rock phosphate to improve the growth of onion plants. Compared to the control supplemented with a free-cell bacterial suspension, the results showed at least equal plant growth of 80 mglpot and shoot P concentration of 82 uglplant when IO destroyed beads were applied per pot. 0 1997 Elsevier Science Ltd. Key words: Enterobacter
phosphate, growth.
sp., immobilized
solubilization,
fermentation,
isms involving chelation and exchange reactions (Earl et al., 1979; Fox and Comerford, 1992; Gerke, 1992). Filamentous fungi and bacteria have been tested in fermentation systems, or introduced directly into soil, in order to solubilize RP (Taha et al., 1969; Azcon et al., 1976; Khan and Bhatnagar, 1977; Kucey et al., 1989). A large number of phosphatesolubilizing (PS) microorganisms has been found in the rhizosphere of plants but in fact, this constitutes only a small percentage of the total microbial population. It is, therefore, widely accepted that an additional quntity of PS microorganisms has to be introduced into the soil in order to help plants in taking up the phosphorus from insoluble sources. However, no reports of solubilization of rock phosphate in soil conditions by a microorganism in an immobilized state have yet been made. In general, immobilized cells offer some advantages over conventional free-cell systems such as the possibility of continuous operation, a higher cell density in small volumes, and a higher stability (Vassilev and Vassileva, 1992). Recently. we have reported the successful solubilization of inorganic phosphate by immobilized Penicillium variabile and Aspetgillus niger, but only in liquid fermentation systems (Vassilev et al., 1996, 1997). The purpose of this work was to perform an efficient immobilization of Enterobacter sp., and verify its potential application in both fermentation and soil conditions as part of a larger project for microbial solubilization of rock phosphate.
cells, rock plant
INTRODUCTION Phosphorus plays a vital role in plant nutrition, but its concentration in soil solution is in the range of 100-400 g P/ha (Wild, 1988). For this reason, the possibility of practical use of rock phosphate (RP) as a fertilizer has received significant interest in recent years. Unfortunately, rock phosphate is not plantavailable in soils with pH greater than 5.5-6.0, and, even when conditions are optimal, plant yields are as a rule lower than those obtained with soluble phosphate (Khasawneh and Doll, 1978). One very attractive approach for RP solubilization is the application of microorganisms able to excrete organic acids. It has been repeatedly shown that low molecular weight organic acids can strongly increase concentrations of phosphorus in solution by mechan*Author to whom correspondence
METHODS Micoroorganism
and fermentation
media
The microorganism used was Enterobacter sp. (a soil isolate from Granada province, naturally resistant to 150 pg streptomycin/ml), maintained on agar RC medium at 4°C (Ramos and Callao, 1967) by subculturing monthly. The growth medium (RC liquid medium) was used to obtain bacterial mass for
should be addressed. 29
30
N. Vassilevet al.
immobilization and contained (g per litre of soil extract-filtrate of l:lO, soikwater): commercial (Panreac) nutrient broth (S), glucose (20), yeast extract (2), pH 7.5. The medium for rock phosphate solubilization (RPS medium), deficient in phosphorus, contained (g per litre of distilled water): glucose (20), NH&l (7.0), MgS04.7H20 (0.5), CaC12.2H20 (O.l), yeast extract (O.l), pH 7.0. Venezuelan rock phosphate (28.9% P205) was added to the RPS medium at a concentration of 2 g/l. Immobilization
and repeated-batch culture
The free-cell cultivation for obtaining cell material for immobilization was performed in 250 ml Erlenmeyer flasks with 100ml growth medium (RC) under agitation at 220 rpm and 30°C for 30 h. The immobilized cells were prepared by mixing a suspension of Enterobucter sp. cells otained after centrifugation (10 000 g, 10 min) of fermentation broth from four flasks (6.10’ CFU/ml) with a 3% solution of agar at 45°C. The mixture was homogenized for 3-4 min at low speed of the homogenizer and then dropped into sterile olive oil to form 2-3 mm biocatalyst beads. Immobilized bacterial cells were separated from the liquid, washed with sterile distilled water and transferred to 250-ml Erlenmeyer flasks (10 ml and 20 ml beads per flask, in triplicate) containing 100 ml of medium RPS for repeated-batch cultivation. The fermentation liquid was centrifuged (1600 g, 10 min) and was further used for analysis after each batch cycle. Free-cell control experiments were carried out without addition of rock phosphate and only to compare pH values registered in both free- and immobilized-cell cultures because of the difficulty of separating rock phosphate particles and free-cell biomass. Experiments were carried out in flasks under agitation at 220 rpm and 30°C. RPS medium was changed every 50 h following the same centrifugation procedure. Soil-plant experiment
The soil used was the top O-20 cm of a Granada (Spain) field soil with pH of 7.5 and containing 15 mg phosphorus/g (Olsen test), total N 0.26% and organic matter 0.8%. Whole and mechanically destroyed immobilized-cell beads were inoculated manually in plastic pots containing 300 g soil-sand
(5:2) steam-sterilized mixture (10 beads per pot, 10’ CFU per bead) in the 5-cm surface layer. An amount of 2 ml liquid (RC medium) bacterial inoculant (10’ CFU per ml), that had been normally applied in previous experiments, was introduced into the control pots and used as a reference treatment. Venezuelan rock-phosphate powder (0.1 mm mesh), at a concentration of 100 ppm, was added to all treatments. One onion seed, sterilized with 10% hypochlorite for 20 min, and then washed three times with sterile water, was placed in each pot. All pots were cultivated in a greenhouse for five weeks under a day/night cycle of 16/8 h, 21/15”C, 50% relative humidity. Throughout the experiment, the pots were weighed every day and water loss from field capacity was replaced by top watering. Analytical methods The number of colony-forming units (CFU) in the
different types of bacterial culture was determined by spreading samples of free-cell culture, mechanically disintegrated immobilized-cell beads, and soil-water extract (1:lO) at low dilutions on to RCagar plates containing 150 pg streptomycin/ml and 150 ,ug cycloheximide/ml. Soluble phosphorus concentration in the fermentation liquid and plants was determined by the molybdo-vanado method described by Lachica et al. (1973). Medium pH was measured with a glass electrode. The five-week-old shoot dry weight was determined after drying at 70°C. RESULTS AND DISCUSSION Fermentation study
Recently, we have reported a successful application of free Enterobucter sp. cells in rock phosphate solubilization in liquid-culture conditions (Nedialkova et al., 1996). In the present study, the immobilized bacterium was also effective in dissolving Venezuelan rock phosphate. The Enterobacter sp., encapsulated in agar beads, was able to acidify the fermentation medium in repeated-batch shake-flask fermentation (Table 1). The level of acidification, with an average difference of about two pH units as
Table 1. pH values and soluble phosphate concentration during repeated-batch immobilized Enterobacter sp. No
1 2 3 5” 6
of batches
cultivation
of 10 and 20 ml of beads with
10 ml beads
20 ml beads
PH*
mg P/l
PH
mgP/l
4.4 * 0.20 4.1* 0.17 4.OkO.12 4.OkO.16 4.3kO.11 4.2 + 0.26
69fl.l 65 f 2.7 65 f 3.0 64 2.8 65+2.1 f 60 f 2.0
4.5 * 0.10 4.1* 0.20 4.lkO.18 4.4f0.16 4.0f0.15 4.5 f 0.10
78+ 1.0 69 f 2.3 65 + 2.8 68 64 k 2.9 2.6 k 59f2.5
Values are f standard deviation for three replicate cultures.* pH of free-cell culture without rock phosphate was 5.8-6.0.
31
Rock phosphate solubilization by immobilized cells compared to the free-cell control, was sufficient to overcome the neutralizing effect of rock phosphate. Significant amounts of soluble phosphorus were detected in the medium solution. There was no difference between pH values and rock phosphate solubilization-rate using 10 and 20 ml of immobilized-cell particles. During the studied period of six, 50-h batch fermentation cycles, the soluble phosphorus concentration was maintained at a constant average value in the range of about 66-70 mg/l for one batch period which corresponded to solubilization of 26-27% of the total phosphorus content present in the phosphate rock source. This suggested that the immobilized bacterial cells produced a stable system for rock phosphate solubilization for at least six repeated batches. Similar advantageous behaviour of enhanced stability and catalytic longevity of immobilized microorganisms was recently observed with passively entrapped filamentous fungi applied in a rock phosphate solubilization process (Vassilev et al., 1996; Vassilev et al., 1997). The magnitude of the results reported here could be appreciated by the fact that using free Enterobacter sp. cells the maximum amount of 60 mg soluble phosphorus/l was achieved only after 21 day fermentation, all conditions being equal (Nedialkova et al., 1996). Despite some decrease in the soluble P concentration at the end of the process, the immobilized bacterium maintained its activity for nine repeated-batch cycles, although the amount of soluble phosphorus fell to half of the maximum (data are not shown). Soil-plant
experiment
In order to evaluate the feasibility of applying immobilized bacterial cells in rock phosphate solubilization in soil conditions, the activity of immobilized Enterobucter sp. was measured using as criteria the plant growth and plant phosphorus concentration. Overall, the plant dry matter production was markedly improved by the introduction of Enterobucter sp. to the experimental soil as compared with the non-inoculated control (Table 2). Taken individually, the effect of the immobilized bacterium on plant growth was at least comparable to that of free-cell inoculated soil in the case of destroyedbead inoculation, when in both treatments an increase of 66% was registered over the control
grown in soil without Enterobucter sp. Similarly, the phosphorus content of plants was almost equal (82-86 &plant) in the presence of these two forms of the bacterial inoculant. The beneficial effect of the whole beads on providing plants with soluble phosphorus was less spectacular in comparison to that of the destroyed-bead treatment. However, expressing the results as phosphorus in dry tissue (%), the whole-bead immobilized bacterium appears to be more effective (0.13%) than destroyed-bead (0.10%) and free-cell (0.10%) inoculants. The reason for this dissimilarity is probably the slow rate of cell-release and the low mobility of cells escaped from the whole beads. Such behaviour of immobilized bacterial cells was reported by Dommergues et al. (1979) when Rhizobium juponicum entrapped in polyacrylamide gel blocks was introduced into soil. Bearing in mind the above considerations, it was not surprising that the total number of bacterial cells measured at the end of the experiment was higher in the free-cell-inoculated than in the immobilized soil (9 x lo3 cells/pot) whole-bead (2.1 x lo3 cells/pot) and destroyed-bead (5.1 x lo3 cells/pot) treatments. Nevertheless, even at concentration, the immobilized this reduced bacterial culture was able to ensure a sufficient rate of solubilization of rock phosphate present in the experimental soil. On the other hand, the high amount of bacterial mass encapsulated in the gel matrix should be noted, and this might guarantee a continuous flow of cells to the environment for a long period of time. It is important to note that as the aim of this experiment was to assess the effect only of the immobilized bacterium, inoculation with AM fungi and other rhizosphere microorganisms to the soil-plant system was consciously avoided. For the same reason, only wet-gel inoculum preparations were applied to the soil in order to prevent the agar beads from possible contaminations during drying. It is now widely accepted that immobilized microbial cells overcome some of the problems associated with survival, stability, efficacy, storage, transportation and ease of application. Although this approach is well known, particularly in applying nitrogen-fixing and biocontrol microorganisms (Van Elsas and Heijnen, 1990), to our knowledge this study is the first attempt to use immobilized microbial cells for solubilization of rock phosphate in soil. However,
Table 2. Shoot dry weights and P contents of onion plants not inoculated or inoculated with immobilized Enterobacter sp. P concentration
Dry weight of shoot (mg)
Treatment
In dry tissue (5%)
&plant 48f 1.9 79f 1.4 56k2.3 80 + 2.9
Non-inoculated Free cells Whole beads Destr. beads Values are f standard
deviation
for five replicate
cultures.
49 &-2.3 86+3.1 74 * 2.2 82k2.9 Results are significant
at
P = 0.001.
0.10 0.1 I 0.13 0.10
32
N. Vassilevet al.
further research is needed to optimize the immobilization procedure applying various gel carriers and additives, as well as to enhance the effectivity of introduced PS microorganisms in soil conditions.
REFERENCES Azcon, R., Barea, J. M. & Hayman, D. S. (1976). Utilization of rock phosphate in alkaline soil by plants inoculated with mycorrhizal fungi and phosphate-solubilizing bacteria Soil Biol. Biochem., 8, 135-138. Dommergues, Y. R., Diem, H. G. & Divies, C. (1979). Polyacrylamide-entrapped Rhizobium as an inoculant for legumes, Appl. Environ. Microbial., 37, 779-781. Earl, K. D., Syers, J. K. & McLaughlin, J. R. (1979). Origin of the effects of citrate, tartrate, and acetate on phosphate sorption by soils and synthetic gels Soil Sci. Sot. Am. J., 43,674-678. Fox, T. R. & Comerford, N. B. (1992). Influence of oxalate loading on phosphorus and aluminum solubility in spodosols Soil Sci. Sot. Am. J., 56, 290-294. Gerke, J. (1992). Phosphate, aluminum, and iron in the soil solution of three different soils in relation to varying concentration of citric acid Z. Pflanzenemahr: Bodenk., 55,339-343. Khan, J. A. & Bhatnagar, R. M. (1977). Studies on solubilization of insoluble phosphates by microorganisms. I. Solubilization of Indian phosphate rock by Aspergillus niger and Penicillium sp.. Fert. Technol., 14, 329-333. Khasawneh, F. E. & Doll, E. C. (1978). The use of phosphate rock for direct application to soils. Adv. Agron., 30, 159-206.
Kucey, R. M. N., Jansen, H. H. & Leggett, M. E. (1989). Microbially mediated increases in plant-available phosphorus Adv. Agron., 42,199-228. Lachica, M., Aguilar, A. & Yanez, J. (1973). Analisis foliar. Metodos analiticos en la Estacion Experimental de1 Zaidin Anal. Edaf Agrobiol., 32, 1033-1047. Nedialkova, K., Toro, M. and Barea, J. M. (1996) Ability of three phosphate-solubilizing rhizobacteria to release phosphorus from several rock phosphate sources. In Mycorrhizas in Integrated Systems, ed. C. Azcon-Aguilar and J. M. Barea, pp. 649-652. European Commision, Brussels. Ramos, A. & Callao, V. (1967). El empleo de la solubilizacion de fosfatos en placa coma tecnica diferencial bacteriana Microbial, Espan., 20, l-2. Taha, S. M., Mahmoud, S. A. Z., Halim-Al-Damaty, A. & Abd-El-Hafez, A. M. (1969). Activity of phosphate-dissolving bacteria in Egyptian soils Plant Soil, 31, 149-160. Van Elsas, J. D. & Heijnen, C. E. (1990). Methods of introduction of bacteria into soil. A review. Biol. Fert. Soils, 10,127-133. Vassilev, N. & Vassileva, M. (1992). Production of organic acids by immobilized filamentous fungi. Mycol. Res., 96, 563-570. Vassilev, N., Fenice, M. & Federici, F. (1996). Rock phosphate solubilization with gluconic acid produced by immobilized Penicillium variabile P16. Biotechnol. Tech.,
10,584-588. Vassilev, N., Vassileva, M. & Azcon, R. (1997). Solubilization of rock phosphate by immobilized Aspergillus niger: Bioresource Technology, 59, l-4. Wild, A. (1988) Plant nutrients in soil: Phosphate. In Soil Conditions and Plant Growth, ed. A. Wild, pp. 695-742. Longman Scientific and Technical, Essex.
PlI:SO960-8524(96)00044-S
ROCK PHOSPHATE SOLUBILIZATION BY IMMOBILIZED CELLS OF ENTEROBACTER SP. IN FERMENTATION AND SOIL CONDITIONS Nikolay Vassilev,* Marcia Toro, Maria Vassileva, Rosario Azcon & Jose Miguel Barea Estacion Experimental de1 Zaidin, CSIC, ProJ Alhareda, I, 18008-Granada, Spairl (Received
19 February
1997; revised version received 17 March 1997; accepted 26 March 1997)
Abstract An Enterobacter sp., a phosphate-solubilizing bacterium, was immobilized in agar: I/arious amounts of the immobilized bioparticles were applied in a repeatedbatch fermentation process in order to solubilize Venezuelan rock phosphate. A total production of 388 mg and 403 mg soluble PI1 was obtained for 2.0 g rock phosphate11 in frasks containing 10 ml and 20 ml agar beads, respectively, that was significantly higher compared to a free-cell single-batch control. whole and destroyed immobilized bioparticles were introduced into a soil enriched with rock phosphate to improve the growth of onion plants. Compared to the control supplemented with a free-cell bacterial suspension, the results showed at least equal plant growth of 80 mglpot and shoot P concentration of 82 uglplant when IO destroyed beads were applied per pot. 0 1997 Elsevier Science Ltd. Key words: Enterobacter
phosphate, growth.
sp., immobilized
solubilization,
fermentation,
isms involving chelation and exchange reactions (Earl et al., 1979; Fox and Comerford, 1992; Gerke, 1992). Filamentous fungi and bacteria have been tested in fermentation systems, or introduced directly into soil, in order to solubilize RP (Taha et al., 1969; Azcon et al., 1976; Khan and Bhatnagar, 1977; Kucey et al., 1989). A large number of phosphatesolubilizing (PS) microorganisms has been found in the rhizosphere of plants but in fact, this constitutes only a small percentage of the total microbial population. It is, therefore, widely accepted that an additional quntity of PS microorganisms has to be introduced into the soil in order to help plants in taking up the phosphorus from insoluble sources. However, no reports of solubilization of rock phosphate in soil conditions by a microorganism in an immobilized state have yet been made. In general, immobilized cells offer some advantages over conventional free-cell systems such as the possibility of continuous operation, a higher cell density in small volumes, and a higher stability (Vassilev and Vassileva, 1992). Recently. we have reported the successful solubilization of inorganic phosphate by immobilized Penicillium variabile and Aspetgillus niger, but only in liquid fermentation systems (Vassilev et al., 1996, 1997). The purpose of this work was to perform an efficient immobilization of Enterobacter sp., and verify its potential application in both fermentation and soil conditions as part of a larger project for microbial solubilization of rock phosphate.
cells, rock plant
INTRODUCTION Phosphorus plays a vital role in plant nutrition, but its concentration in soil solution is in the range of 100-400 g P/ha (Wild, 1988). For this reason, the possibility of practical use of rock phosphate (RP) as a fertilizer has received significant interest in recent years. Unfortunately, rock phosphate is not plantavailable in soils with pH greater than 5.5-6.0, and, even when conditions are optimal, plant yields are as a rule lower than those obtained with soluble phosphate (Khasawneh and Doll, 1978). One very attractive approach for RP solubilization is the application of microorganisms able to excrete organic acids. It has been repeatedly shown that low molecular weight organic acids can strongly increase concentrations of phosphorus in solution by mechan*Author to whom correspondence
METHODS Micoroorganism
and fermentation
media
The microorganism used was Enterobacter sp. (a soil isolate from Granada province, naturally resistant to 150 pg streptomycin/ml), maintained on agar RC medium at 4°C (Ramos and Callao, 1967) by subculturing monthly. The growth medium (RC liquid medium) was used to obtain bacterial mass for
should be addressed. 29
30
N. Vassilevet al.
immobilization and contained (g per litre of soil extract-filtrate of l:lO, soikwater): commercial (Panreac) nutrient broth (S), glucose (20), yeast extract (2), pH 7.5. The medium for rock phosphate solubilization (RPS medium), deficient in phosphorus, contained (g per litre of distilled water): glucose (20), NH&l (7.0), MgS04.7H20 (0.5), CaC12.2H20 (O.l), yeast extract (O.l), pH 7.0. Venezuelan rock phosphate (28.9% P205) was added to the RPS medium at a concentration of 2 g/l. Immobilization
and repeated-batch culture
The free-cell cultivation for obtaining cell material for immobilization was performed in 250 ml Erlenmeyer flasks with 100ml growth medium (RC) under agitation at 220 rpm and 30°C for 30 h. The immobilized cells were prepared by mixing a suspension of Enterobucter sp. cells otained after centrifugation (10 000 g, 10 min) of fermentation broth from four flasks (6.10’ CFU/ml) with a 3% solution of agar at 45°C. The mixture was homogenized for 3-4 min at low speed of the homogenizer and then dropped into sterile olive oil to form 2-3 mm biocatalyst beads. Immobilized bacterial cells were separated from the liquid, washed with sterile distilled water and transferred to 250-ml Erlenmeyer flasks (10 ml and 20 ml beads per flask, in triplicate) containing 100 ml of medium RPS for repeated-batch cultivation. The fermentation liquid was centrifuged (1600 g, 10 min) and was further used for analysis after each batch cycle. Free-cell control experiments were carried out without addition of rock phosphate and only to compare pH values registered in both free- and immobilized-cell cultures because of the difficulty of separating rock phosphate particles and free-cell biomass. Experiments were carried out in flasks under agitation at 220 rpm and 30°C. RPS medium was changed every 50 h following the same centrifugation procedure. Soil-plant experiment
The soil used was the top O-20 cm of a Granada (Spain) field soil with pH of 7.5 and containing 15 mg phosphorus/g (Olsen test), total N 0.26% and organic matter 0.8%. Whole and mechanically destroyed immobilized-cell beads were inoculated manually in plastic pots containing 300 g soil-sand
(5:2) steam-sterilized mixture (10 beads per pot, 10’ CFU per bead) in the 5-cm surface layer. An amount of 2 ml liquid (RC medium) bacterial inoculant (10’ CFU per ml), that had been normally applied in previous experiments, was introduced into the control pots and used as a reference treatment. Venezuelan rock-phosphate powder (0.1 mm mesh), at a concentration of 100 ppm, was added to all treatments. One onion seed, sterilized with 10% hypochlorite for 20 min, and then washed three times with sterile water, was placed in each pot. All pots were cultivated in a greenhouse for five weeks under a day/night cycle of 16/8 h, 21/15”C, 50% relative humidity. Throughout the experiment, the pots were weighed every day and water loss from field capacity was replaced by top watering. Analytical methods The number of colony-forming units (CFU) in the
different types of bacterial culture was determined by spreading samples of free-cell culture, mechanically disintegrated immobilized-cell beads, and soil-water extract (1:lO) at low dilutions on to RCagar plates containing 150 pg streptomycin/ml and 150 ,ug cycloheximide/ml. Soluble phosphorus concentration in the fermentation liquid and plants was determined by the molybdo-vanado method described by Lachica et al. (1973). Medium pH was measured with a glass electrode. The five-week-old shoot dry weight was determined after drying at 70°C. RESULTS AND DISCUSSION Fermentation study
Recently, we have reported a successful application of free Enterobucter sp. cells in rock phosphate solubilization in liquid-culture conditions (Nedialkova et al., 1996). In the present study, the immobilized bacterium was also effective in dissolving Venezuelan rock phosphate. The Enterobacter sp., encapsulated in agar beads, was able to acidify the fermentation medium in repeated-batch shake-flask fermentation (Table 1). The level of acidification, with an average difference of about two pH units as
Table 1. pH values and soluble phosphate concentration during repeated-batch immobilized Enterobacter sp. No
1 2 3 5” 6
of batches
cultivation
of 10 and 20 ml of beads with
10 ml beads
20 ml beads
PH*
mg P/l
PH
mgP/l
4.4 * 0.20 4.1* 0.17 4.OkO.12 4.OkO.16 4.3kO.11 4.2 + 0.26
69fl.l 65 f 2.7 65 f 3.0 64 2.8 65+2.1 f 60 f 2.0
4.5 * 0.10 4.1* 0.20 4.lkO.18 4.4f0.16 4.0f0.15 4.5 f 0.10
78+ 1.0 69 f 2.3 65 + 2.8 68 64 k 2.9 2.6 k 59f2.5
Values are f standard deviation for three replicate cultures.* pH of free-cell culture without rock phosphate was 5.8-6.0.
31
Rock phosphate solubilization by immobilized cells compared to the free-cell control, was sufficient to overcome the neutralizing effect of rock phosphate. Significant amounts of soluble phosphorus were detected in the medium solution. There was no difference between pH values and rock phosphate solubilization-rate using 10 and 20 ml of immobilized-cell particles. During the studied period of six, 50-h batch fermentation cycles, the soluble phosphorus concentration was maintained at a constant average value in the range of about 66-70 mg/l for one batch period which corresponded to solubilization of 26-27% of the total phosphorus content present in the phosphate rock source. This suggested that the immobilized bacterial cells produced a stable system for rock phosphate solubilization for at least six repeated batches. Similar advantageous behaviour of enhanced stability and catalytic longevity of immobilized microorganisms was recently observed with passively entrapped filamentous fungi applied in a rock phosphate solubilization process (Vassilev et al., 1996; Vassilev et al., 1997). The magnitude of the results reported here could be appreciated by the fact that using free Enterobacter sp. cells the maximum amount of 60 mg soluble phosphorus/l was achieved only after 21 day fermentation, all conditions being equal (Nedialkova et al., 1996). Despite some decrease in the soluble P concentration at the end of the process, the immobilized bacterium maintained its activity for nine repeated-batch cycles, although the amount of soluble phosphorus fell to half of the maximum (data are not shown). Soil-plant
experiment
In order to evaluate the feasibility of applying immobilized bacterial cells in rock phosphate solubilization in soil conditions, the activity of immobilized Enterobucter sp. was measured using as criteria the plant growth and plant phosphorus concentration. Overall, the plant dry matter production was markedly improved by the introduction of Enterobucter sp. to the experimental soil as compared with the non-inoculated control (Table 2). Taken individually, the effect of the immobilized bacterium on plant growth was at least comparable to that of free-cell inoculated soil in the case of destroyedbead inoculation, when in both treatments an increase of 66% was registered over the control
grown in soil without Enterobucter sp. Similarly, the phosphorus content of plants was almost equal (82-86 &plant) in the presence of these two forms of the bacterial inoculant. The beneficial effect of the whole beads on providing plants with soluble phosphorus was less spectacular in comparison to that of the destroyed-bead treatment. However, expressing the results as phosphorus in dry tissue (%), the whole-bead immobilized bacterium appears to be more effective (0.13%) than destroyed-bead (0.10%) and free-cell (0.10%) inoculants. The reason for this dissimilarity is probably the slow rate of cell-release and the low mobility of cells escaped from the whole beads. Such behaviour of immobilized bacterial cells was reported by Dommergues et al. (1979) when Rhizobium juponicum entrapped in polyacrylamide gel blocks was introduced into soil. Bearing in mind the above considerations, it was not surprising that the total number of bacterial cells measured at the end of the experiment was higher in the free-cell-inoculated than in the immobilized soil (9 x lo3 cells/pot) whole-bead (2.1 x lo3 cells/pot) and destroyed-bead (5.1 x lo3 cells/pot) treatments. Nevertheless, even at concentration, the immobilized this reduced bacterial culture was able to ensure a sufficient rate of solubilization of rock phosphate present in the experimental soil. On the other hand, the high amount of bacterial mass encapsulated in the gel matrix should be noted, and this might guarantee a continuous flow of cells to the environment for a long period of time. It is important to note that as the aim of this experiment was to assess the effect only of the immobilized bacterium, inoculation with AM fungi and other rhizosphere microorganisms to the soil-plant system was consciously avoided. For the same reason, only wet-gel inoculum preparations were applied to the soil in order to prevent the agar beads from possible contaminations during drying. It is now widely accepted that immobilized microbial cells overcome some of the problems associated with survival, stability, efficacy, storage, transportation and ease of application. Although this approach is well known, particularly in applying nitrogen-fixing and biocontrol microorganisms (Van Elsas and Heijnen, 1990), to our knowledge this study is the first attempt to use immobilized microbial cells for solubilization of rock phosphate in soil. However,
Table 2. Shoot dry weights and P contents of onion plants not inoculated or inoculated with immobilized Enterobacter sp. P concentration
Dry weight of shoot (mg)
Treatment
In dry tissue (5%)
&plant 48f 1.9 79f 1.4 56k2.3 80 + 2.9
Non-inoculated Free cells Whole beads Destr. beads Values are f standard
deviation
for five replicate
cultures.
49 &-2.3 86+3.1 74 * 2.2 82k2.9 Results are significant
at
P = 0.001.
0.10 0.1 I 0.13 0.10
32
N. Vassilevet al.
further research is needed to optimize the immobilization procedure applying various gel carriers and additives, as well as to enhance the effectivity of introduced PS microorganisms in soil conditions.
REFERENCES Azcon, R., Barea, J. M. & Hayman, D. S. (1976). Utilization of rock phosphate in alkaline soil by plants inoculated with mycorrhizal fungi and phosphate-solubilizing bacteria Soil Biol. Biochem., 8, 135-138. Dommergues, Y. R., Diem, H. G. & Divies, C. (1979). Polyacrylamide-entrapped Rhizobium as an inoculant for legumes, Appl. Environ. Microbial., 37, 779-781. Earl, K. D., Syers, J. K. & McLaughlin, J. R. (1979). Origin of the effects of citrate, tartrate, and acetate on phosphate sorption by soils and synthetic gels Soil Sci. Sot. Am. J., 43,674-678. Fox, T. R. & Comerford, N. B. (1992). Influence of oxalate loading on phosphorus and aluminum solubility in spodosols Soil Sci. Sot. Am. J., 56, 290-294. Gerke, J. (1992). Phosphate, aluminum, and iron in the soil solution of three different soils in relation to varying concentration of citric acid Z. Pflanzenemahr: Bodenk., 55,339-343. Khan, J. A. & Bhatnagar, R. M. (1977). Studies on solubilization of insoluble phosphates by microorganisms. I. Solubilization of Indian phosphate rock by Aspergillus niger and Penicillium sp.. Fert. Technol., 14, 329-333. Khasawneh, F. E. & Doll, E. C. (1978). The use of phosphate rock for direct application to soils. Adv. Agron., 30, 159-206.
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