Maria Vassileva

Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes

Rock phosphate (RP) is an important natural material traditionally used for the production of phosphorus (P) fertilizers. Compared with chemical treatment, microbial solubilization of RP is an alternative environmentally mild approach. An overview of biotechnological techniques, mainly based on solubilization processes involving agro-industrial residues, is presented. Potential advantages of composting, solid-state fermentation, and liquid submerged fermentation employing free and immobilized microorganisms that produce organic acids and simultaneously solubilize RP are discussed. Subsequent introduction of the final fermented products into soil-plant systems promotes plant growth and P acquisition.

Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition

This paper reviews current knowledge of the production of organic acids by immobilized microorganisms with a simultaneous solubilization of rock phosphate in fermentation and soil conditions. The most widely applied methods are based on the passive immobilization in preformed porous carriers and entrapment of the microbial cells in natural gels. In general, immobilized systems show higher acid producing and rock phosphate solubilizing activity than freely suspended cells. The potential of gel-entrapped P-solubilizers and mycorrhizal fungi as microbial soil inoculants is also pointed out. Some advantages and constraints of using immobilized cells are discussed and a special emphasis on further research is given.

Rock phosphate solubilization by Aspergillus niger grown on sugar-beet waste medium

Solubilization of rock phosphate by Aspergillus niger was studied in solid-state fermentation on sugar-beet waste. This combination was selected after testing three agroindustrial waste materials, namely rice hulls, sugar-beet waste and alperujo. Sugar-beet waste was the best substrate for fungal growth with 69% mineralization, followed by rice hulls and alperujo. The fungus was successfully cultivated on sugar-beet waste supplemented with 3.0 g/l rock phosphate, acidifying the medium and thus decreasing the pH to 3–3.5. Solubilization of insoluble phosphate increased during the first half of the process, reaching a maximum of 292 μg phosphate/ml, although a part of it was probably consumed by the mycelium.

Rock phosphate solubilization by immobilized cells of Enterobacter sp. in fermentation and soil conditions

An Enterobacter sp., a phosphate-solubilizing bacterium, was immobilized in agar. Various amounts of the immobilized bioparticles were applied in a repeated-batch fermentation process in order to solubilize Venezuelan rock phosphate. A total production of 388 mg and 403 mg soluble P/l was obtained for 2.0 g rock phosphate/l in flasks 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 mg/pot and shoot P concentration of 82 μg/plant when 10 destroyed beads were applied per pot.

SOLUBILIZATION OF ROCK PHOSPHATE BY IMMOBILIZED ASPERGILLUS NIGER

Aspergillus nigel; an acid-producing filamentous fungus, was immobilized on polyurethane foam. Various amounts of foam cubes and spore suspension were tested in order to obtain an efficient immobilization process. The best combination selected for further experiments was 0.2 g polyurethane foam and 3 ml spore suspension. Immobilized cells were reused, with higher levels of acid formation being maintained for longer periods (at least 240 h) than for free cells. The highest titratable acidity, of about 315 mmole/l, was reached with 0.5 cm foam cubes after 3 × 48 h batch cultures in a laboratory shake-flask experiment. Rock phosphate (2.5g/l and 5.0g/l) solubilization was carried out in repeated 48 h batch fermentation using A. niger immobilized on polyurethane foam cubes. The greatest accumulation of soluble P,, approximately 360 #g/ml, was obtained after the second batch in flasks with 5 g/l rock phosphate, but process efficiency was higher at lower concentration of the rock phosphate. After 10 days of culture a total production of 1.2-1.6 mg/ml soluble P was obtained depending on the concentration of rock phosphate in the medium.

Application of an encapsulated filamentous fungus in solubilization of inorganic phosphate

Spores of Aspergillus niger were encapsulated in agar, calcium alginate and k-carrageenan and further applied in citric acid production during six repeated batch cultivations. Rock phosphate (RP) at concentrations of 3 g l-1 and 7 g l-1 was supplemented to the culture medium to test encapsulated-fungus solubilizing capability. The highest average citric acid productivity of 0.15 g l-1 h-1 was reached with alginate-bead-encapsulated A. niger on RP-free culture medium while agar seemed to be the most suitable carrier on RP-supplemented medium. Accordingly, the highest average soluble P concentration of 0.20 g l-1 batch-1 was obtained with agar-cell beads as compared with other encapsulated systems.

Effect of encapsulated cells of Enterobacter sp on plant growth and phosphate uptake

Cells of Enterobacter sp., a phosphate-solubilizing bacterium, were encapsulated in calcium-alginate beads supplemented with skim milk. Free-cell and encapsulated-cell formulations were introduced into a soil, amended with rock phosphate, to compare their effect on non-mycorrhizal and mycorrhizal Lactuca sativa plants. Bacterial inoculation affected plant growth and P uptake but encapsulated-cell formulation caused the better plant response. Combined inoculation of encapsulated cells and Glomus deserticola provided the best microbial inoculum and enhanced plant growth by 96% as compared to the uninoculated control. The higher number of bacterial cells (log 4.4 CFU/g soil) detected in soil inoculated with encapsulated cells stimulated the plant mycorrhization. The continuous flow of bacterial cells from the skim-milk-enriched carrier beads appeared to be the main reason for the enhanced plant growth and P uptake.

Preparation of gel-entrapped mycorrhizal inoculum in the presence or absence of Yarowia lipolytica

Two arbuscular mycorrhizal fungi (Glomus deserticola and Glomus fasciculatum) were entrapped in calcium alginate, alone or in combination with a phosphate-solubilizing yeast (Yarowia lipolytica) and, after storage for 60 days, were inoculated into soil microcosms with tomato as the test plant. The average extent of root colonization by gel-entrapped G. deserticola and G. fasciculatum were 32 ± 5.6 and 24 ± 12.1%, respectively. Improved infective potential and colonization efficiency were observed when Y. lipolytica was co-entrapped with the mycorrhizal fungi. The best value, 49%, of mycorrhizal colonization was in roots of plants inoculated with G. deserticola co-entrapped with Y. lipolytica.

Animal bones char solubilization by gel-entrapped Yarrowia lipolytica on glycerol-based media.

Citric acid was produced with free and k-carrageenan-entrapped cells of the yeast Yarrowia lipolytica in single and repeated batch-shake-flask fermentations on glycerol-based media. Simultaneous solubilization of hydroxyapatite of animal bone origin (HABO) was tested in all experiments. The highest citric acid production by free yeast cells of 20.4 g/L and 18.7 g/L was reached after 96 h of fermentation in the absence and presence of 3 g/L HABO, respectively. The maximum values for the same parameter achieved by gel-entrapped cells in conditions of single batch and repeated-batch fermentation processes were 18.7 g/L and 28.1 g/L registered after 96 h and the 3d batch cycle, respectively. The highest citric acid productivity of 0.58 g L−1 h−1 was obtained with immobilized cells in repeated batch mode of fermentation when the added hydroxyapatite of 3 g/L was solubilized to 399 mg/L whereas the maximum efficiency of 89.0% was obtained with 1 g/L of HABO.