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Bacillus megaterium Biodegradation Glyphosate

By Nibal Khaleel Mousa, Abdul-Jabbar Ali and Maha Hussein

Submitted: February 9th 2021Reviewed: March 1st 2021Published: August 3rd 2021

DOI: 10.5772/intechopen.96919

Downloaded: 28

Abstract

The Bacillus megaterium ability was evaluated in this paper to degrade the Glyphosate. organophosphorus pesticides, The bacteria re-cultured that isolated from other researches of Baghdad soils and morphological identification and biochemical tests besides by selectivity media. The (5 and 25) ppm showed the highest growth results were within two days to two months on mineral salt media. The highest glyphosate degradation ratio % were (70) % per 25 ppm/two months. Incubation period Increasing led to highest glyphosate degradation ratio% at (25) ppm led to conclusion that bacteria digestive the pesticides as carbon and nitrogen sources and will be well harvest it form contaminated areas.

Keywords

  • bacterium
  • bioremediation
  • glyphosate
  • HPLC

1. Introduction

Glyphosate is an organophosphate which is a heterogeneous compound, utilized on the grasses leaves and broadleaf plants due its non-selectivity pesticides. In the seventies was the first registration in the United State by Monsanto (Roundup) [1]. Glyphosate stops a shikimic acid enzyme pathway which is essential for some microorganisms and plants. In trace analysis, Glyphosate is a difficult herbicide, has a good water solubility that causes difficulty in determination its physical and chemical properties [2].

The microorganism ability to eliminate pollutants is one of the bioremediation methods [3]. Bioremediation is applied as an auxiliary strategy due to its inoffensive to ecology, economic worth, reduce environmental poison, and validation [4, 5, 6, 7, 8]. Metabolic is one of the microorganism degradation methods across pesticides in soils, also catabolism strategies and the enzymes of co-metabolism [9]. Organic-pesticides fate in the ecology system can be marked by utilizing biodegradation as a major agent. The study purpose is to achieve and inspect the domestic bacterial separated on broken down different glyphosate concentrations that can be remaindered in soil and detect residues concentration from bacteria digestive via HPLC after extraction.

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2. Material and methods

2.1 Materials

Chemosate. The trade name of “Glyphosate”, bought from the local market. Materials were available and supply in Remediation Pollutants Center. The Mineral Salt Media (MSM), was used in growing B. megateriumto investigate glyphosate degradation, Table 1 [10]. As the only carbon source, Flasks (125 ml) were supplemented with Glyphosate. The Final Concentration of Glyphosate with 0.5 were (5 to 25) ppm from bacteria re- culture in comparison with control.

Weight (g)CompoundsNote
0.2KH2PO4Sterilized separately at 125 °C/25 min (part A)
0.5K2HPO4
1(NH4)2SO4All mixed and added to part A of 1-liter flask, and adjust (pH 7.0 ± 0.3)
0.2MgSO4.7H2O
0.2NaCl
0.05CaCl2.2H2O
0.025FeSO4.7H2O
0.005Na2MoO4
0.005MnSO4

Table 1.

The mineral salt media.

2.2 Re-growth and identification of Bacillus megaterium

Bacillus megateriumwas growth and kept in incubation of our laboratory from other studies [3] and to re-identification by selective media, was by Sperber’s Medium [10, 11], Table 2, By the spectrophotometer OD 600, then the hydrolysis capacity measured for different period (2,5,7,14,21,30,60) days [12].

Weight (g)CompoundsNote
10Glucose1liter distillate water
adjust (pH 7.0 ± 0.3)
incubate28-30 °C/48 h.
0.5Yeast extract
0.25MgSO4.7H2O
15Agar
0.1CaCl2.2H2O

Table 2.

Sperber media structure.

2.3 Glyphosate degradation ratio via Bacillus megaterium

The degradation ratio % investigated for (1-2) months, extracted through added equal volume each ethyl acetate and MSM which utilized as a reagent, for twice time, centrifuged at 3000 rpm/10 minutes, filtered then anhydrous sodium sulfate utilized as dry factor followed glass-fiber paper (Whatman GF/B) [10]. In Eq. (1), measured the degradation ratio:

P=1C1C0x100%E1

P = the rate of degradation of Glyphosate,

C1 = Glyphosate dose in sample.

C0 = control [13].

2.4 Metabolite analysis

Each ethyl acetate extraction was analyzed by HPLC condition, Table 3 [14], calculates the final concentration [15] of glyphosate used Eq. (2):

HPLC condition analysis
UV–Vis detector254 nm
Manual Injector Equipped20-μL loop
Column/Stationary phaseC-18*
Mobile PhaseAcetic acid (1%) + methanol (6:4 (v/v).
Flow Rate1.0 ml /min
Temperature23–25 °C

Table 3.

HPLC conditions.

ZORBAX (5 μm, 150 mm × 4.6 mmi.d.)


Pest.con=AsaxCs/AsxCsaE2

Pest.con = pesticide concentration in sample (mg/L)

Asa = sample peak area

Csa = sample concentration, mg/L

As = standard. peak.area

Cs = standard concentration, mg/L

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3. Results and discussion

3.1 Re-growth and identification of Bacillus megaterium

Besides using selective media, Sperber Medium, Table 4 shows the Test of Morphological, and Table 5 represents the biochemical tests.

Morphological tests
Spore shapeRod-like/ flagella spores
ColoniesRound to irregular /yellow to brown or black after prolonged incubation
Motility+
Gram stain+
Aerobic+
Temperature3-20 °C/ 35-45 °C,optimun30°C
pH5.7- 7

Table 4.

The tests of morphological.

Biochemical tests
Catalase+Nitrate reduction / Degradation of tyrosine+/−
Starch Hydrolysis+Casein hydrolysis+
Citrate utilization+Indol/ Methyl Red
Esculin hydrolysis+Arginine dihydrolase
Gelatin hydrolysis+Tryptophan deaminase
Oxidase+Hydrolysis Urea

Table 5.

The tests of biochemical.

3.2 Bacillus megateriumhydrolyzes and bacteria growth

3.2.1 Growth of B. megaterium

In Figure 1, the results show that the highest B. megateriumgrowth was in (two months) for both (5, 25) ppm (0.164, 0.167) sequentially, while the 15 ppm show in 60 days, the highest growth is (0.215) in comparison with others when used glyphosate as carbon sources.

Figure 1.

Culture ofB. megateriumon Mineral Salt Media with Glyphosate.

3.2.2 Degradation rate%

The highest degradation rate% for Glyphosate by B. megateriumin comparison among concentration was for both the 5-25 ppm in 60 days reached (70.01-70.9) %, Figure 2.

Figure 2.

The degradation rate of Glyphosate in MSM in Comparative with control.

3.2.3 Glyphosate residues by HPLC test

The B. megateriumgrowth on different concentration in MSM at 30 °C, in comparison with control in Figures 3 and 4. Decreasing in glyphosate in 30 days for concentration (5,10) ppm (7, 8)% is the best peak area, while the 25 ppm showed 28%, while the results glyphosate peak area two months incubation, led to (20, 15) ppm in comparative with control. When comparing among HPLC, Figures 5 and 6improved that the Bacteriadegradation ratio% positive results with all glyphosate concentrations via incubating two months incubation. However, the best was for (5, 25) ppm for each degradation ration% and the HPLC.

Figure 3.

Peak height, peak area and retention time of Glyphosate after incubation B.M. 30 days in MSM.

Figure 4.

Peak height, peak area and retention of Glyphosate after incubation B.M. 60 days in MSM.

Figure 5.

Comparative among the degradation ratio% and Glyphosate concentrations via HPLC in 30 days incubation on MSM.

Figure 6.

Comparative among the degradation ratio% and Glyphosate concentration via HPLC in 60 days incubation on MSM.

Organophosphorus pesticides microbial degradation and the bioremediation progress development for contaminated soils depend on the introduction of microbe’s biodegrading [16]. B. megateriumshows the degradation rate% and highest growth in two months cultivation time for both five and twenty five concentration. The degradation pesticide ability like Chlorpyrifos (600 mg-L1) via B. megateriumin ten days incubation was 81% [17] and 73% for 20 ppm during three weeks. On the other hand, Bacillus megaterium99% in seven days, degradation ratio towards atrazine (50 ppm) and Chlorpyrifos in (1-2) weeks [18, 19]. Monocrotophos (MCP), 83% degradation ratio reached led sub-products to CO2, NH4, and (HPO3) [20]. Each bacterium produces enzymes to analyze compounds and supplying its self with elements in need for re-build enzyme and cell body. One of Bacillus megateriumenzyme is β-Amylase [21] that consist from carbon and nitrogen mainly, so the bacteria most often organic-phosphors compounds, Glyphosate used to supply a single element (carbon, phosphorus or sulfur) and [22]. Due to that the concentration of glyphosate generally reduces along with bacteria growth. Phosphate ester and phosphonate are the analysis hydrolysis results of phosphorus. The esters groups have they have many vulnerable to hydrolysis sites. Beside hydrolysis, the oxidation also one of the major reactions, and the alkylation,dealkylation [23]. Detoxification is one of microbial degradation principles via hydrolysis phosphor bonds with oxygen (P-O-alkyl, P-O-aryl [24].

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4. Conclusion

In this study, Bacillus megateriumimproved the best results for growth was in 48 h while in two months, had the same growth each five and twenty five concentration. The degradation rate % ability was the best in (5,25) ppm/two months reached (70-71)%. The Glyphosate degradation ratio% increasing equally with the increasing the incubation to two months,the best was for five and twenty five ppm, each the HPLC and Degradation ratio%. The conclusion is the B. megateriumutilized Glyphosate as supplier for elements sulfur, carbon, nitrogen and phosphorus and cultivated highly from culture could be well exploited for biodegradation from its pollutants sites.

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Nibal Khaleel Mousa, Abdul-Jabbar Ali and Maha Hussein (August 3rd 2021). <em>Bacillus megaterium</em> Biodegradation Glyphosate [Online First], IntechOpen, DOI: 10.5772/intechopen.96919. Available from:

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