Biodegradation of Organophosphonates by Aspergillus Specie

The utilization of organophosphonates as sole source of phosphorus by Aspergillusniger was studied. Aspergillusniger was found to utilize the phosphorus in diethylethylphosphonate, 2aminoethylphosphonate and potassium hydrogen phosphate for its growth. Aspergillusnigerwas isolated from rotten pap and identified using standard manuals.The growth of the fungus was monitored by weighing while the phosphate ion released was monitored by UV-Visible spectrophotometer (Genesys IOS VI) at 660nm. The release of phosphate ion was an indication that the first step in organophosphonates degradation by fungus was cleavage of the CarbonPhosphorus (C-P)bond. There was an inverse relationship between the fungus growth and the amount of phosphate ion in the broth culture medium. The products of degradation were determined by Thin Layer Chromatographic method. The results showed that the phosphate ion in potassium hydrogen phosphate was released into the culture medium faster than that of diethylethylphosphonate and 2-aminoethylphosphonate. Key word: Organophosphonates, biodegradation,Aspergillusniger. Bacteria and fungi play a major role in phosphonate biodegradation in soil4,5,6. The first phosphonate to be identified to occur naturally was 2aminoethylphosphonic acid, found in membranes of plants and many animals3.Organophosphonates are used in large quantities as pesticides, flame retardants, corrosion inhibitors and plasticizers [7]. The Carbon-Phosphorus bond is resistant to chemical degradation[8]. However, there exists a good number of micro-organisms possessing the ability to biodegrade Carbon-Phosphorus bond of 166 ADELOWO et al., Orient. J. Chem., Vol. 31(Spl Edn.), 165-171 (2015) phosphonates through enzymatic hydrolysis [9]. These are widespread among bacteria [10, 5] and many strains of fungi11,12. Bacillus and Arthrobacterand yeasts were able to grow on phosphonates as the sole source of phosphorus, nitrogen or carbon1. Some organophosphorus insecticides, such as diazinon, chlorpyrifos, ethion, parathion, fonofos, malathion and gusathion, are susceptible to microbial hydrolysis and serve as phosphorus source for growth of pure and mixed cultures Flavobacterium sp., Pseudomonas sp. and Arthrobacter sp.13-14 reported that Streptomyces pilosus is capable of growing on several insecticides such as carbofuran, cloethocarb, trimethacarb, isofenphos, fonofos and phorate. Several methods such as titrametry, spectrophotometry, gas-liquid chromatography (GLC), High Performance Liquid Chromatography (HPLC), and photometry, have been used for the determination of organophosphorus compounds [15, 16, 17]. The analysis of orthophosphate ions (PO4 3-)in fermentation broth of microorganisms has been carried out with the use of Fiske-Subbarow method [18]or through the oxidation of organophosphorus pesticides with slight excess of NBromosuccinimide(NBS) and the unconsumed NBS determined with rhodamine-B through spectrophotometry [19]. Various analytical methods have been used to monitor the growth rate of microorganisms in biodegradation studies. The growth kinetics of Pseudomonas sp. Strain PG2982 was monitored by measuring the turbidity of culture broth medium using spectrophotometric and colorimetr ic methods20, 21. The growth of Penicilliumcitrinium was monitored by weighing bacteria dry mass12. The complete mineralization of 2aminoethylphosphonate, a biogenic organophosphonate of wide distribution occured via its conversion to phosphonoaceltaldehyde and subsequent cleavage of the C-P bond by phosphonoaceltaldehydephosphonohydrolase (phosphonatase). This pathway has been described in cells of Bacillus cereus and Pseudomonas aeruginosa, which utilized 2aminoethylphosphonate as a sole phosphorus source1, 23. The microbial mineralization of Diethylethylphosphonate to diethylhydroxyl phosphonate has also been previously reported24. In this study, we report the capability of fungal strain, Aspergillus niger to utilize 2aminoethylphosphonate anddiethylethyl phosphonate as phosphorus source for its growth. MATERIALS AND METHODS Chemicals The chemicals used were of analytical grade (products of Sigma-Aldrich, USA). They included 2-aminoethylphosphonate, phosphon oacetaldehyde, diethylhydroxylphosphonate, potassium hydrogen phosphate(K2HPO4), acetic acid, acetone, potato dextrose agar, iodine, ethanol, calcium chloride, sodium chloride, sodium nitrate, magnesium sulphate, ferrous sulphate, sucrose,trisbuffer, tr ichloroacetic acid(TCA), 1,2,4aminonaphthosulphonic acid, sodium sulphite, sodium hydrogen sulphite, and ammonium molybdate.Diethylethylphosphonate was synthesized in our laboratory according to the procedure described previously25. Culture Media The culture media (Czapekdox broth) contained(g/l): sucrose, (30.0); magnesium sulphate, (0.5); calcium chloride, (0.5); ferrous sulphate, (0.1); sodium nitrate, (3.0);tris-buffer, (7.88);phosphorus source(0.0005 mol). 100cm3 of deionized water was added to the flask to dissolve the salts on a water bath (300C) with occasional swirling. The pH of the medium was adjusted to 7.2 with 1cm3 of 1.0 mol/L NaOH . The flask was finally made to mark with deionized water. 2-aminoethylphosphonate (0.0005mol, 0.11g/l) served as phosphorus source in culture medium A, diethylethylphosphonate (0.0005mol, 1.0 ml/l) served as phosphorus source in culture medium B while potassium hydrogen phosphate (0.0005mol, 0.087g/l) served as phosphorus source in culture medium C. Culture medium D contained all the nutrients except phosphorus source. Isolation of Aspergillus niger Microorganisms were collected from a rotten pap. Individual colonies of the microorganisms were picked up with sterilized 167 ADELOWO et al., Orient. J. Chem., Vol. 31(Spl Edn.), 165-171 (2015) inoculation loop and isolated. The isolated microorganisms were sub-cultured on potato dextrose agar slants. Aspergillusnigerfungus strain was isolated, purified and identified according to colony characters as described in standard manuals26-28. Biodegradation experiments and fungus growth kinetics The biodegradation experiments were carried out with pure isolated Aspergillusniger fungus colony for a period of twenty days. Glass bottles with covers were sterilized for the biodegradation experiments. Into each of the ten sterilized glass bottles were dispensed 47.5cm3 of culture medium A. This was followed by addition of 2.5cm3 of the innoculum. The mixture was incubated on a rotary shaker (Uniscope SM 101) at 120rpm at 300C and assayed for fungal growth by filtering the broth culture in each sample bottle at 48 hours intervals and the dry mass of the mycelia determined. The experiment was carried out in duplicates. Filtrates from the broth culture were used for the determination of the rate of utilization of the phosphorus source by the isolate through spectrophotometric analysis and the detection of the metabolites of organophosphonatesthrough Thin-Layer Chromatographic analysis. The biodegradation experiments were also carried out on culture media B, C, D and E. Culture medium E (Control) is culture medium A without the isolate. Determination of rate of utilization of organophosphonates by Aspergillus niger through spectrophotometric analysis of the filtrates. The spectrophotometric experiment was carried out on the filtrates of the ten sample bottles containing broth culture medium A. The following procedure was used for the preparation of the Test and Blank solutions: Filtrates of Broth culture medium A were used. The analysis of phosphorus in form of inorganic phosphate (PO4 3-) was carried out on UVVisible Spectrophotometer (Genesys IOS VI) at 690nm, using Fiske-Subbarow method [29, 30]. Trichloroacetic acid[5%(wt/vol) and 9.5cm3] was placed in a centrifuge tube, followed by addition of 1.0cm3 of filtrate of broth culture medium A from sample bottle 1. The mixture was stirred by stirring rod and allowed to stand for 5 minutes. The clear supernatant (5.0cm3) was transferred into a test tube, followed by addition of 1.0cm3 of ammonium molybdate and 0.5cm3 mixture of 1,2,4aminonaphtholsulphonic acid, Na2SO3, and NaHSO3. The mixture was thoroughly mixed and allowed to stand for 10 minutes. The procedure was repeated for filtrates of broth culture A in sample bottles 2 to 10. The absorbance for each solution in a cuvet was determined at 690nm against distilled water. Analyses were carried out in duplicates. The experiment was repeated for the filtrates of broth culture of other phosphorus source (i.e. filtrates of broth culture media B and C). Detection of organophosphonates and metabolites through Thin Layer Chromatography (TLC) Filtrates from broth culture medium A (sample bottles 3, 6, and 9) were used for the Thinlayer chromatographic analysis. Broth culture filtrates were examined by TLC on hp-TLC (aluminium plastic sheets pre-coated with silicagel (20 × 20 cm) (Merck, Darmstady, Germany). Chromatograms were developed using n-butanolacetic acid-water (12:3:5) as a solvent system. Spots were visualized in an iodine chamber. Sample spots were identified by comparing with their corresponding standards. The R f value of each spot was determined. The TLC of filtrates of culture medium B was also carried out. RESULTS AND DISCUSSION The enrichment culture media were able to utilize organophosphonates as sole source of phosphorus. The growth rate of Aspergillusniger was observed to increase in all the culture media except the Control as shown in the Table 1 below: Aspergilllus niger was able to grow more in broth culture media containing phosphorus sources through enrichment procedure(Table 2). The results of the study showed gradual increase in dry mass of fungus, attaining the highest dry mass in few days and followed by gradual decrease in mass (Table 1 and Figure 1). These observations had been reported by other workers17,31.The decrease in dry mass of fungus was an indication that phosphorus source was being utilized by fungus 168 ADELOWO et al.,


INTRODUCTION
Phosphonates are a class of organophosphorus compounds characterized by Carbon-Phosphorus (C-P) bond 1 .Phosphonatesoccur widely among xenobiotics.Pollution of the environment by phosphonatesaroused interest in their pathways and mechanisms of biodegradation 2 .As a result of their structural similarity to some phosphate esters, phosphonatescould act as inhibitors of enzymes 3 .
Bacteria and fungi play a major role in phosphonate biodegradation in soil 4,5,6 .The first phosphonate to be identified to occur naturally was 2aminoethylphosphonic acid, found in membranes of plants and many animals 3 .Organophosphonates are used in large quantities as pesticides, flame retardants, corrosion inhibitors and plasticizers [7].The Carbon-Phosphorus bond is resistant to chemical degradation [8].However, there exists a good number of micro-organisms possessing the ability to biodegrade Carbon-Phosphorus bond of phosphonates through enzymatic hydrolysis [9].These are widespread among bacteria [10, 5] and many strains of fungi 11,12 .Bacillus and Arthrobacterand yeasts were able to grow on phosphonates as the sole source of phosphorus, nitrogen or carbon 1 .4] reported that Streptomyces pilosus is capable of growing on several insecticides such as carbofuran, cloethocarb, trimethacarb, isofenphos, fonofos and phorate.

3-
)in fermentation broth of microorganisms has been carried out with the use of Fiske-Subbarow method [18]or through the oxidation of organophosphorus pesticides with slight excess of N-Bromosuccinimide(NBS) and the unconsumed NBS determined with rhodamine-B through spectrophotometry [19].Various analytical methods have been used to monitor the growth rate of microorganisms in biodegradation studies.The growth kinetics of Pseudomonas sp.Strain PG2982 was monitored by measuring the turbidity of culture broth medium using spectrophotometric and colorimetric methods 20,21 .The growth of Penicilliumcitrinium was monitored by weighing bacteria dry mass 12 .
The complete mineralization of 2aminoethylphosphonate, a biogenic organophosphonate of wide distribution occured via its conversion to phosphonoaceltaldehyde and subsequent cleavage of the C-P bond by phosphonoaceltaldehydephosphonohydrolase (phosphonatase).This pathway has been described in cells of Bacillus cereus and Pseudomonas aeruginosa, which utilized 2aminoethylphosphonate as a sole phosphorus source 1,23 .The microbial mineralization of Diethylethylphosphonate to diethylhydroxyl phosphonate has also been previously reported 24 .
In this study, we report the capability of fungal strain, Aspergillus niger to utilize 2aminoethylphosphonate anddiethylethyl phosphonate as phosphorus source for its growth.

Isolation of Aspergillus niger
Microorganisms were collected from a rotten pap.Individual colonies of the microorganisms were picked up with sterilized inoculation loop and isolated.The isolated microorganisms were sub-cultured on potato dextrose agar slants.Aspergillusnigerfungus strain was isolated, purified and identified according to colony characters as described in standard manuals [26][27][28] .

Biodegradation experiments and fungus growth kinetics
The biodegradation experiments were carried out with pure isolated Aspergillusniger fungus colony for a period of twenty days.Glass bottles with covers were sterilized for the biodegradation experiments.Into each of the ten sterilized glass bottles were dispensed 47.5cm 3 of culture medium A. This was followed by addition of 2.5cm 3 of the innoculum.The mixture was incubated on a rotary shaker (Uniscope SM 101) at 120rpm at 30 0 C and assayed for fungal growth by filtering the broth culture in each sample bottle at 48 hours intervals and the dry mass of the mycelia determined.The experiment was carried out in duplicates.Filtrates from the broth culture were used for the determination of the rate of utilization of the phosphorus source by the isolate through spectrophotometric analysis and the detection of the metabolites of organophosphonatesthrough Thin-Layer Chromatographic analysis.The biodegradation experiments were also carried out on culture media B, C, D and E. Culture medium E (Control) is culture medium A without the isolate.Determination of rate of utilization of organophosphonates by Aspergillus niger through spectrophotometric analysis of the filtrates.
The spectrophotometric experiment was carried out on the filtrates of the ten sample bottles containing broth culture medium A.
The following procedure was used for the preparation of the Test and Blank solutions: Filtrates of Broth culture medium A were used.The analysis of phosphorus in form of inorganic phosphate (PO 4 3-) was carried out on UV-Visible Spectrophotometer (Genesys IOS VI) at 690nm, using Fiske-Subbarow method [29, 30].Trichloroacetic acid[5%(wt/vol) and 9.5cm 3 ] was placed in a centrifuge tube, followed by addition of 1.0cm 3 of filtrate of broth culture medium A from sample bottle 1.The mixture was stirred by stirring rod and allowed to stand for 5 minutes.The clear supernatant (5.0cm 3 ) was transferred into a test tube, followed by addition of 1.0cm 3 of ammonium molybdate and 0.5cm 3 mixture of 1,2,4aminonaphtholsulphonic acid, Na 2 SO 3 , and NaHSO 3 .The mixture was thoroughly mixed and allowed to stand for 10 minutes.The procedure was repeated for filtrates of broth culture A in sample bottles 2 to 10.The absorbance for each solution in a cuvet was determined at 690nm against distilled water.Analyses were carried out in duplicates.The experiment was repeated for the filtrates of broth culture of other phosphorus source (i.e.filtrates of broth culture media B and C).

Detection of organophosphonates and metabolites through Thin Layer Chromatography (TLC)
Filtrates from broth culture medium A (sample bottles 3, 6, and 9) were used for the Thinlayer chromatographic analysis.Broth culture filtrates were examined by TLC on hp-TLC (aluminium plastic sheets pre-coated with silicagel (20 × 20 cm) (Merck, Darmstady, Germany).Chromatograms were developed using n-butanolacetic acid-water (12:3:5) as a solvent system.Spots were visualized in an iodine chamber.Sample spots were identified by comparing with their corresponding standards.The R f value of each spot was determined.The TLC of filtrates of culture medium B was also carried out.

RESULTS AND DISCUSSION
The enrichment culture media were able to utilize organophosphonates as sole source of phosphorus.The growth rate of Aspergillusniger was observed to increase in all the culture media except the Control as shown in the Table 1 below: Aspergilllus niger was able to grow more in broth culture media containing phosphorus sources through enrichment procedure(Table 2).The results of the study showed gradual increase in dry mass of fungus, attaining the highest dry mass in few days and followed by gradual decrease in mass (Table 1 and Figure 1).These observations had been reported by other workers 17,31 .The decrease in dry mass of fungus was an indication that phosphorus source was being utilized by fungus The fungal growth of Aspergillus niger was more in culture media A, B and C than that of culture medium D (Table 2).This showed that phosphorus played a vital role in the growth of the fungus.
Aspergillus niger would act on phosphorus source by enzymatic hydrolysis of Carbon-Phosphorus (C-P) bond thereby forming phosphate ions.The determination of rate of utilization of the released phosphate ions byAspergillusniger was carried out through spectrophotometric analysis using the method of Fiske -Subbarow.The  3.
The disappearance of phosphate ions with the corresponding increase in the growth rate of fungus was as a result of their utilization by the fungus for its growth (Figures 1 and 2).
The fungus was able to utilize potassium hydrogen phosphate, 2-aminoethylphosphonate and diethylethylphosphonate as sole phosphorus source.However, Aspergillus niger grew more significantly when potassium hydrogen phosphate served as sole phosphorus source.Potassium hydrogen phosphate being an inorganic compound, would release its phosphate ions more easily than the two organic compounds (2-aminoethyl phosphonate and diethyethylphosphonate).
Therate of growth of Aspergillusnigerwas more in 2-aminoethylphosphonate than diethylethyl phosphonate.This observation could be as a result of the presence of nitrogen in 2-aminoethyl phosphonate which would complement the effect of phosphorus( Table 1).
Certain essential ions are important for fungi growth and phosphate is one of them.With the absence of phosphate ions in culture medium D, there was a decrease in the growth rate of Aspergillusnigerwhen compared with the broth culture media A, B and C.The microbial activity and organophosphonates degradation followed an inverse relationship(Figures 1 and 2).
The biodegradation products of the organophosphonate compounds were determined by Thin Layer Chromatographic analysis.The organophosphonates and their degradation products were identified by comparing their Retention factor (R f ) with their corresponding standards.2-aminoethylphosphonate and its metabolite(phosphonoacetaldehyde) were not detected after the 12 th day (sample bottle 6) of the biodegradation experiment.This could be as a result of the quantitative release of their phosphate ions.On the other hand there was detection of Diethylethylphosphonate and its metabolite (diethylhydroxylphosphonate) even after the 18 th day of the biodegradation experiment.This showed that Aspergillus niger degraded 2-aminoethyl phosphonate faster than Diethylethylphosphonate.The present study demonstrates that the isolated Aspergillus niger possessed the ability to degrade organophosphonates.This findings could be applied in the bioremediation of soil contaminated with organophosphonate pesticides.

CONCLUSION
The enrichment culture study over a period of time gave indication that organophosphonates could serve as sole phosphorus source for microbial growth.Application of microorganisms could be used to remediate environments that have been contaminated with organophosphonate pesticides.The ability of this isolate to utilize organophosphonate pesticides effectively provides a means of removing them from the environment.The most promising approach for reducing the presence of pollutants in the environment is bioremediation by microbes since they exist in abundance in the environment.This approach is preferred to other methods since it is safer, cost effective and has recorded high degradation rate.