Optimization of oxalic acid production by fungi for biotechnological solubilization of rock phosphate

Amaro, Jeniffer Kelly Cortes; Xavier, Laura Vieira; Ribeiro, Michelle Miranda Araújo de Carvalho; Vieira, Bruno Sérgio; Mendes, Gilberto de Oliveira

doi:10.1590/1678-992X-2021-0076

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Agricultural Microbiology • Sci. agric. (Piracicaba, Braz.) 80 • 2023 • https://doi.org/10.1590/1678-992X-2021-0076 copy

Optimization of oxalic acid production by fungi for biotechnological solubilization of rock phosphate

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

This study describes a biotechnological strategy for producing and applying oxalic acid to solubilize phosphorus (P) from rock phosphate (RP). We evaluated six fungal species (Aspergillus niger FS1, Penicillium islandicum FS41, Pleurotus ostreatus PO1, Rhizoctonia solani Rhiz555, Sclerotium rolfsii Sr25, and Sclerotinia sclerotiorum Scl134) and three culture media (potato dextrose broth, Tsao and Strasser media) to maximize oxalic acid production. Among the fungal isolates tested and culture media, S. rolfsii Sr25 and Tsao medium showed efficient oxalic acid production. Tsao medium was optimized following a response surface methodology after initial screening of factors affecting RP solubilization. The optimized concentrations were 1 g L^–1 NaNO₃, 100 g L^–1 glucose, 2 g L^–1 KH₂PO₄, 4.5 g L^–1 yeast extract, and 25 mg L^–1 MgSO₄·7H₂O used for 20 days of incubation. Under these conditions, 71 mmol L^–1 oxalic acid was obtained, representing a three-fold increase over production under non-optimized conditions (20 mmol L^–1). Under optimized conditions, oxalic acid produced by S. rolfsii Sr25 reacted with low-solubility RP and solubilized 100 % of the P contained in ore. Thus, using S. rolfsii Sr25 to produce oxalic acid seems a promising biotechnological alternative for P solubilization from RP.

Keywords:
phosphorus; microorganism; oxalate; fertilizer

Factor	Lower level (–1)	Upper level (+1)	Effectᵃ
Glucose (g L^–1)	10	100	3.893^* * Significant by the t test (p < 0.05). ᵃEffect = m+ – m-– (m+ and m--– represent the average oxalic acid produced for the upper and lower level of each factor, respectively).
NaNO₃ (g L^–1)	1	3	–2.021
KH₂PO₄ (g L^–1)	0.5	2	5.291^* * Significant by the t test (p < 0.05). ᵃEffect = m+ – m-– (m+ and m--– represent the average oxalic acid produced for the upper and lower level of each factor, respectively).
Yeast extract (g L^–1)	1	3	6.208^* * Significant by the t test (p < 0.05). ᵃEffect = m+ – m-– (m+ and m--– represent the average oxalic acid produced for the upper and lower level of each factor, respectively).
MgSO₄.7H₂O (g L^–1)	0.025	1	–3.754^* * Significant by the t test (p < 0.05). ᵃEffect = m+ – m-– (m+ and m--– represent the average oxalic acid produced for the upper and lower level of each factor, respectively).
MnCl₂.4H₂O (g L^–1)	0	0.2	0.986
pH (7.0)	Unbuffered	Buffered	1.815
Incubation time (d)	10	20	10.268^* * Significant by the t test (p < 0.05). ᵃEffect = m+ – m-– (m+ and m--– represent the average oxalic acid produced for the upper and lower level of each factor, respectively).

	Glucose	KH₂PO₄	Yeast extract	MgSO₄.7H₂O	Time
	------------------------------------ g L^–1 ------------------------------------				d
Base level (0)^a a Related to the experiment shown in Table 1, representing the average value between the levels –1 and +1.	55	1.25	2	0.5125	15
Unit	45	1.5	1	0.4875	5
Estimated coefficient (f_t)^b b Change in oxalic acid biosynthesis per unit, obtained in the screening experiment.	1.946	2.646	3.104	–1.877	5.134
Unit × f_t	87.57	3.97	3.10	–0.92	25.67
Increment (∆) relative to increment in time^c c Changes in factor levels were calculated based on the change in the level of the factor with the highest regression coefficient in the screening experiment, that is, incubation time.	17.1	0.77	0.60	–0.18	5

#	Glucose	KH₂PO₄	Yeast extract	MgSO₄.7H₂O	Incubation time	Oxalic acid observed	Oxalic acid estimated
	------------------------------------------------------------------------------ g L^–1 --------------------------------------------------------------------------------				d	--------------------------------------- mmol L^–1 ----------------------------------------
1	80 (–1)^a a Values in parentheses are the coded levels.	1.25 (–1)	2.25 (–1)	12.5 (–1)	22.5 (1)	25.50	23.40
2	120 (1)	1.25	2.25	12.5	17.5 (–1)	35.92	39.32
3	80	2.75 (1)	2.25	12.5	17.5	29.36	25.99
4	120	2.75	2.25	12.5	22.5	50.03	46.82
5	80	1.25	3.75 (1)	12.5	17.5	28.78	35.57
6	120	1.25	3.75	12.5	22.5	60.48	56.41
7	80	2.75	3.75	12.5	22.5	-	43.08
8	120	2.75	3.75	12.5	17.5	68.85	59.00
9	80	1.25	2.25	37.5 (1)	17.5	38.88	41.90
10	120	1.25	2.25	37.5	22.5	34.34	31.72
11	80	2.75	2.25	37.5	22.5	40.76	49.41
12	120	2.75	2.25	37.5	17.5	33.62	34.31
13	80	1.25	3.75	37.5	22.5	-	58.99
14	120	1.25	3.75	37.5	17.5	43.16	43.89
15	80	2.75	3.75	37.5	17.5	61.74	61.58
16	120	2.75	3.75	37.5	22.5	53.75	51.40
17	60 (–2)	2 (0)	3 (0)	25 (0)	20 (0)	54.85	48.20
18	140 (2)	2	3	25	20	45.53	53.93
19	100 (0)	0.5 (–2)	3	25	20	31.38	28.25
20	100	3.5 (2)	3	25	20	34.09	38.34
21	100	2	1.5 (–2)	25	20	48.33	45.55
22	100	2	4.5 (2)	25	20	70.92	74.82
23	100	2	3	0 (–2)	20	37.50	45.61
24	100	2	3	50 (2)	20	58.58	56.52
25	100	2	3	25	15 (–2)	29.87	28.69
26	100	2	3	25	25 (2)	31.30	33.60
27	100	2	3	25	20	56.24	51.07
28	100	2	3	25	20	55.81	51.07
29	100	2	3	25	20	60.90	51.07
30	100	2	3	25	20	55.44	51.07
31	100	2	3	25	20	46.93	51.07
32	100	2	3	25	20	37.75	51.07

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[1] *Corresponding author <gilbertomendes@ufu.br>