Research Article | Open Access
Slamet Isworo1 and Poerna Sri Oetari2
1Department of Environmental Health, Universitas Dian Nuswantoro, Jl. Imam Bonjol 207,
Semarang 50131, Central Java, Indonesia.
2Environmental Impact Assessment. PT. Mitra Adhi Pranata. Jl. Cemara IV No.45, Padangsari, Banyumanik, Semarang, Indonesia.
J Pure Appl Microbiol. 2021;15(2):897-914 | Article Number: 6993
https://doi.org/10.22207/JPAM.15.2.47 | © The Author(s). 2021
Received: 18/04/2021 | Accepted: 22/05/2021 | Published: 01/06/2021
Abstract

The Indonesian Pesticide Regulations state that Malathion and Profenofos have been restricted in their use for agriculture because of is bioaccumulative in ecological systems. Cleaning technology using microorganisms is an effective solution for cleaning pesticide residues. This study aims to identify the bacteria that degrade and the degradation process of Malathion and Profenophos into non-toxic compounds. The research method was experimental, identification of bacteria by 16S-rRNA gene analysis, degradation ability by GC MS. The results of phylogenetic tree analysis showed that the tested bacteria were closely related to Oceanobacillus iheyenis (RPL1) and Exiquobacterium profundum (RPL5) with a similarity level of 87% and 99%. The two bacteria are used as a consortium of test bacteria. The results of degradation based on the observation chromatogram T = 0 showed that the Malathion compound C10H19O6PS2 or butanedioic acid [(dimethoxyphosphinothioyl) thio]) was detected at peak 4, real-time = 19,675, area% = 7.37 and Profenofos compound C11H15BrClO3PSO-(4-Bromo-2-chlorophenyl)o-ethyl s-propyl thiophosphate, peak 8, real-time = 23,957, area% = 6.91. Likewise, the chromatogram results at T = 96 were still detected Malathion ((dimethoxyphosphinothioyl) thio) at peak 14, real-time = 19,675, area% = 2.25, and Profenofos (o- (4-Bromo-2-chlorophenyl)) o – ethyl. s – propyl thiophosphate) peak = 22 real-time = 23,951, area% = 2.2. However, the observation of T = 192 hours, Malathion and Profenofos compounds were not detected. The conclusion showed that the consortium bacteria were able to completely degrade Malathion and Profenophos within 192 hours.

Keywords

Consortium bacteria, Exiquobacterium profundum, Oceanobacillus iheyenis, Biodegradation, Malathion, Profenofos

Introduction

Pesticides are widely used to increase agricultural yields, plantations, forestry production, but pesticides can have a negative impact on the non-target environment. Pesticide pollution needs to be controlled because it can damage the ecological balance 1.  Malathion and  Profenofos are types of pesticides that are widely used by farmers around Lake Rawa Pening in Central Java, Indonesia, even though these pesticides have been banned especially for rice cultivation based on Regulation of the Minister of Agriculture of the Republic of Indonesia Number 39 / Regulation of the Minister of Agriculture / SR.330 / 7 / 2015 2. It is proven that Profenofos is widely used by farmers around Rawa Pening (50%), then Carbamate (16%), Deltamethrin (8%), Imidacloprid (6%), Fentoat (5%), Carbosulfan (5%), Carbofuran (5% ) and Lamda Sihalotrin (4%).  The results of the Profenofos residue analysis in the waters of Rawa Pening ranged from 0.021 ppm – 0.08 ppm, the sediments ranged from 0.12 ppm – 0.28 ppm while the Malathion residue in the waters ranged from 0.0366 ppm – 0.0521 ppm and in sediments ; 0.0567 ppm – 0.12 ppm 3, is above the specified threshold e” of 0.01 ppm 4.   Therefore, it is necessary to make efforts to clean the residue of Malathion and Profenofos which have long been exposed to the environment. Indigenous bacteria are developed as biological agents in modifying toxic residues into non-toxic compounds 5

The degradation process of Malathion – Diethyl (dimethoxythiophosphorylthio)succinate (C10H19O6PS2) in aquatic systems will be degraded to monocarboxylic acid-dimethyl monocarboxylic acid – dicarboxylic acid – dimethyl dicarboxylic acid- dicarboxylic acid-CO2 6.  Malathion fragmentation in the environment will become maloxon (C10H19O7PS), malathion monocarboxylic acid (C8H15O6PS2), and 2-mercaptosuccinic acid (C6H5NO2S) are derivative compounds that are no more toxic than the initial compound, Malathion 7.

Malathion-degrading bacteria produce catabolic enzymes-malathion carboxyl esterase and malathion dicarboxy latoxy reductase which are able to convert malathionic compounds into thiophosphates and phosphates8.

Profenofos pesticides will be broken down by bacteria into mono metabolite compounds and divalent acids through the enzymatic activity of oxidative desulfurization carboxylesterase 6 9 and demethylation processes in mineralization mechanisms that cause minor routes of metabolism, including oxidation, reduction of sulfur and methyl. 10.  The result of enzymatic degradation by consortium bacteria is able to degardate Profenofos into simpler compounds, namely 4-Bromo-2-chlorophenol and 1-phenyl-3-hydroxy-1, 2,4-triazole 11

This article discusses critical areas regarding the degradation of Malathion and Profenophos residues contained in the water and sediments of the Rawa Pening lake by a consortium of indigenous bacteria Exiquobacterium profundumOceanobacillus iheyenis which are expected to produce simpler and non-toxic final compounds.

Materials and Methods

Genomic DNA (Promega) Wizard Extraction Kit: EDTA, Lytic enzyme, nuclei lysis solution, RNAase solution, protein precipitation solution, DNA rehydration solution. Bact-FI primer. 5’AGAGTT TGATCMTGGCTCAG3 ‘/UniB1.5’GGTTACSTTGTTACGACTT3′ (Eurogentec AIT), agarose (Vivantis), ethanol 70%, Ethidium bromide, isopropanol, loading dye (Vivantis), marker (Vivantis), and HgCl2. Sediment from Rawa Pening Lake, Profenofos and Malathion Pro Analisys (PA), Sigma Aldrich Laborochemikallen GmbH, Malathion Pestanal Bath SEBC132XV, Profenofos pestanal Bath SZBC132XV, Malathon 96% and Profenofos Curacron 500 EC

Microbial Identification based on 16S-rRNA Gene Analysis
Bacterial identification was carried out using the 16S-rRNA gene analysis method which included DNA extraction, DNA amplification, purification of DNA amplification results, DNA sequencing, and subsequent construction of phylogenetic trees to obtain genetic diversity.

DNA extraction
DNA extraction using the Chelex 100 Kit. Bacterial cells that have been grown for 24 hours are put into a 1.5 ml Eppendorf tube containing 100 µl of aquadest, then add 0.5% saponins and let stand for 24 hours at 4 0 C. The samples were centrifuged at 12,000 rpm for 10 minutes, the supernatant from the centrifuge was discarded. A total of 1 ml of Phosphate Buffer Saline (PBS 1x) was added to the Eppendorf tube, then centrifuged again at 12,000 rpm for 15 minutes, the supernatant was removed, 100 µl aquadest and 50 µl Chelex 100 were added to the tube. The samples were boiled for 10 minutes (samples were vortexed in the first 5 minutes), then centrifuged again at 12,000 rpm for 10 minutes. The DNA containing the supernatant is transferred to a new Eppendorf tube which is ready for the DNA amplification process. 12

DNA amplification
Amplification is a molecular marker using the 16s rDNA Polymerase Chain Reaction (PCR) method. The temperature treatment used in the DNA amplification process is initial denaturation at 95 0C for 3 minutes, then 30 cycles (denaturation at 95 0C for 1 minute, annealing process at 55 0C for 1 minute and extension at 72 0C for 1 minute ), then extension at 72 0C for 7 minutes 13. The primers used for PCR 16S rDNA were universal primers for 27F bacteria (5′-AGAGTTTGATCMTGGCTCAG-3′) and eubacteria specific primers 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) 14. The mixture of materials used were Promega kit (25 µl) primer 270 F (2.5 µl), primer 1492 R (2.5 µl),  DNA template (2.5 µl) and aquabides (17.5 µl) so that total volume 50 µl. The ingredients were mixed in a 0.2 ml PCR tube.15

Visualization of DNA Amplification Results
Visualization of the results of DNA amplification was carried out through electrophoresis by inserting 5 µl of PCR products into 1% agarose gel wells. Making 1% agarose gel by dissolving 1 gram of agarose in 100 ml of TAE 1x buffer solution, then heating it in an oven until homogeneous. A total of 5.33 µl Ethidium Bromide was put into the gel solution and shaken so that it was homogeneous. The gel solution is poured into a comb-shaped mold that is placed in an upright position so that it passes through the comb to the desired thickness. Then the gel was allowed to stand for a while until it hardened, then the gel was immersed in a 1x TAE buffer solution, the gel was electrophoresed with a voltage of 100 V for ± 30 minutes. The amplified DNA bands were observed using the Gel Documentation tool. 16

Purification of DNA Amplification Result
Purification was carried out to obtain pure DNA from PCR 16S rDNA amplification. The PCR results were centrifuged at a speed of 12,000 rpm for 7 minutes. The supernatant was removed using a micropipette until the DNA was completely pure. A total of 50 µl of sterile aquadest was added to the DNA pellet and the results of the pure DNA were sequenced to determine the sequence of DNA bases.17

DNA sequencing
Sequencing was carried out according to the PCR sequencing cycle using Big Dye Terminator v.3.1. The formula for sequencing PCR reactions are  2 µl big dye, 2 µl 10x buffer, 4 µl DNA template, 1 µl primer with a concentration of 3.2 pmol, ddH2O to a final volume of 10 µl. DNA amplification carried out by cycles were initial denaturation (96 °C for 2 minutes), denaturation (96 °C for 10 seconds); annealing (50 °C for 5 seconds); and extension (60 °C for 4 minutes) by 25 cycles. PCR results were purified and sequenced using 27F primer. The sequences were analyzed automatically (ABI 3130XL, Applied Biosystem). 18

Phylogenetic Tree Construction
The pesticide-degrading bacteria that had successfully amplified their 16S rRNA gene could be seen from their relationship with other prokaryotes in the database based on their 16S-rRNA gene sequences. The results of partial sequences are edited using the Bioedit program. After obtaining data on the results of nucleotide sequence contigs, the homology will be compared with other prokaryotes in the Gene Bank database. 19 Cluster analysis was carried out using a database from the RDP website (Ribosomal Database Project) with the website (http://www.rdp.com). while making phylogenetic trees using the MEGA 5 program 20

Biodegradation Test of Malathion and Profenofos
The quantitative data analysis was carried out by determining the levels of Malathion and Profenofos which could be obtained based on the area of the chromatogram produced on Gas chromatography-Mass Spectrometry (GC-MS).21 The analysis was performed using a Gas Chromatography-Mass Spectrometry (GS-MS) instrument. The GS-MS conditions at the time of the study were injector temperature 250°C, oven temperature 80°C, column temperature 280°C, detector temperature 250 °C, helium gas flow rate 1ml / min, constant rate, sample constant rate 1 ›l splitless,  standard mix 1 ›l 100 ppm.22    To determine the degradation results of the specimens that had been refused were analyzed using GC MS at 0 hours, 96 hours and 192 hours observations.

RESULTS AND DISCUSSION

Identification of indigenous bacteria
Molecular genetic identification of indigenous bacteria by using genomic-DNA isolation as a template, then the results of genomic-DNA isolation are shown based on the DNA-bands resulting from the 16S-rRNA gene amplification electrophoresis process, shown in the following figure (Figure 1).

Fig. 1. The Results from the Gel Electrophoresis process – 16S-rRNA Amplification. (M) Marker; (1) Bacteria Identification Code = RPL1 and (2) Bacteria Identification Code = RPL5

The species identification by polymerase chain reaction technology (PCR product) using gene-16S rDNA / 16S rRNA (PCR-amplified 16S rRNA) of bacterial species, was carried out using agarose gel electrophoresis method23. The DNA fragments with a size of 50-20,000 bp are the best sizes that agarose gel can separate 24  Analysis using the 16S rDNA/16S rRNA gene has been carried out experimentally in the laboratory because the 16S rDNA/16 rRNA gene is universal and is part of the ribosomal structural RNA which plays an important role in protein synthesis. Therefore the 16 rRNA gene is always present in prokaryotic organisms, is immortal, and almost never is transferred horizontally. This makes the 16S rRNA gene ideal for the reconstruction of the phylogenetic tree and the identification of prokaryotic organisms 25

The isolation process of the tested bacterial genome with the code RPL1 and RPL5 was marked by the formation of one band for each genome of the tested bacteria after being observed using Ultra Violete Transluminator, then it was described by the 16S rRNA gene coding band 1.5 kb, then compared with a Marker (1kb DNA ladder). The results of 16 rRNA DNA amplification were sequenced to obtain the nucleotide sequence and analyzed for similarities using the Gen Bank with the BLAST-N (Basic Local Alignment Search Toll-Nucleotode) program so that the homology and species of bacteria tested could be determined. 26, to determine the phylogeny relationship / relationship with other organisms, the 16S rDNA sequencing results of RPL1 and RPL5 isolates were compared with 16S rDNA sequence data from several species obtained from the data bank. The 16S rDNA sequence data was then synchronized with the ClustalX ver 2.0 program27  The next process is the creation of a phylogenetic tree using the MEGA version 5.03 program with the Neighbor-Joining Tree statistical method, 1000 bootstrap level p-distance models 28   The PCR results of the 16S rDNA gene were shown with a single band on the gel electrophoresis with a size of about 1500 bp.

The results of sequencing using forward and reverse primers to determine the sequence of bacterial nucleotide bases are as follows: (Table 1, fig 2 and fig 3)

Table (1):
Sequencing results (primary forward and reverse).

No
Code
Nucleotide base (bp)
Species name
Homology
No accession
1
RPL 1
1071
Oceanobacillus iheyensis
87 %
LC10790
2
RPL 5
1238
Exuquobacterium profundum
99  %
LC19791

Fig. 2. Exiguobacterium profundum gene for 16S rRNA, partial sequence, strain: RP-L-5   1,238 bp linear DNA GenBank: LC019791.1, species, firmicutes

Fig. 3.  Oceanobacillus iheyensis gene for 16S rRNA, partial sequence, strain: RP- L-1 1,071 bp linear DNA.  species, firmicutes

Results of 16S-rRNA Gene Sequence of RPL-1 Bacterial GGGGTATTGCATCATAATGCAGTC GAGCGCAGGAAGCTATCTGATCCTCTTTTAGAGGTGACGATAATGGAATGAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTAAGACTGGGATAACTCGTGGAAACGCGAGCTAATACCGGATAACACTTTTCATCTCCTGATGAGAAGTTG AAAGGCGGCTTTTGCTGTCACTTACAGATGGGCCTGCGGCGCATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGAC GAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCCATAGTAACTATGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGC GCGTAAAGCGCTCGCAGGCGGTTCTTTAAGTCTGATGTGAAATCTTACGGCTCAACCGTAAACGTGCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGCAATTCCACGTGTAGCGGTGAAATGCGTATAGATGTGGAGGAACACCAGTGGCGAACGCGACTCTCTGG TCTGTAACTGACGCTGAGTAGCCAAGCGTCGGGAGCGACAGGATTAGATACCCTGGTAGCCCCTGCCGTAGACGATGAGCGCTAGTCGTCAGGGGTTTCCGCCCCTTATGCTGAAGTTACTCATTAAGCACTCCACCTGTGACGTCAGACGCAAGCATCAACTCAAAGGATTTACGCGGAC CACTCAAGCGATGATCACTCGTTTAATTACAGCACCGCGAGAACTTACCAGGCTTGGATTCCTCTGAACATCTAAAATAGCCTTTCCTTCAGGGAAGAGTTCTCCCGACAAAGATTTTTCAACCCANACCTAAATTTCAGTAAGCCCGCACGAAGAAATCTTGA

Results of 16S-rRNA Gene Sequence of RPL-5 Bacterial Samples CAATTGCGCGGCTATAATGCAGTCGAGCGCAGGAAACCGTCTGAACCCTTCGGGGGGACGACGGCGGA ATGAGCGGGGGACGGGTGAGTAACACGTAAAGAACCTGCCCATAGGTCTGGGATAACCACAAGAAATCCGGGCTAATACCGGATGTGTCATCGGACCGCATGGTCCGCTGATGAAAGGGGCTCCGGCGTCTCCCATGGATGGCTTTGCGGTGCATTAGC TAGGTGGTGGGGTAAAGGCCCACCAAGGCGACGATGCATAGCCCAGCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGGCCAGACTCCTACGGGAGGGGGCAGTAGGGAATCTTCCCCAATGGACGAAAGTCTGATGGAGCAACG CCGCGTGAACGATGAAAGCTTTCGGGGCGTAAAGTTCTGTTGTAAGGGAAGAACAAGTGCCGCACGCAATGGCGGCGCCTTGACGGTACCTTGCGAGAAAGCCACGGCTAACTACATGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGG CGTAAAGCGCGCGCAGGCGGCCTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGCCATTGGAAACTGGGAGGCTTGAGTATATGAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTACAGATGTGAAGGAACACCCTTGTCGAAAGCGACTCTTTGGCCTATA TCTGACGCTGAGGCGCGAAAACGTGGGGAGCAACACGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGAGCTAA GTGTTGGAGGGTTCCGCCCTTTGTGCT CAGCTAAGCATTAACACTCCCCTGGGGA GACAGTCGCAGGCTCAACTCAAGGATTG ACGGGACCCCACACCAGTGGAGCATGTGGTTTATTTGAGCACACGGAAAACTTTCCACTCTTGAATCCCCTGACCGGAAAAAATGTACCTTCCCTCTGGGGCAGGGTGACAAGTGTGGATGGTTGCGTCAGCCCCGTCCGAGAGATGCGTTAATCCCCAACAAGGCAACCTTGTCTTTTTTGC ACATTCGTTGGCCCCCTAGGAAATGCCGTGACAACCGAAGAAGGGGGATAACCAAATTCATGCCCTTAAAGTGGGTACACGTGTCAATGGAGGGCAAGGGACCCAACCCCAGTGGACCATCCCAAACGTTTCNTTGGATGGGGGGCACCCCCGTAGACCGAATCTGGCGGGTGC TATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGAAGTCAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAATTCTTTCCCTCACATGAGGGAAAGCTGAAAGATGGTTTCGGCTATCACTT ACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCA ACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCA GGCGGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTC TGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATT GACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTCCTGACAACCCTAGAGATAGGGCGTTCCCCTTCGGGGGACAGGATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAAGGAAGGTGGGGGATGACGGTCAAATCATCATGGCCCCTTAAGGACCTGGGGCTAACNCACGTGCTACAATGGGATGGGAACAAAGGGGTTCGAAGACCC GCAAGGTTAANCGGAATCCCCATAAAACATTTTTCAAGTTCNGAATTGCAGGGTTGAAACTCTCCTTGTTTGAAACCCGGATT

Based on the phylogenetic tree analysis, the test bacterial isolate with code RPL1 has the closest relationship with the  Oceanobacillus iheyenis bacteria with a maximum similarity rate of 87%, while the tested bacterial isolates with code RPL5 had the closest relationship with Exiquobacterium profundum with a maximum similarity level of 99%.  as follows (Fig 4)

Fig. 4. Phylogenetic tree reconstruction results

Biodegradation of Profenofos and Malathion by indigenous  bacterial consortium
Oceanobacillus iheyenis and Exiquobacterium profundum are indigenous bacteria isolated from the Rawa Pening lake. Both of these bacteria have the ability to degrade against Malathion and profenofos, therefore these bacteria are used as consortium bacteria for research on the biodegradation process of Malathion and Profenofos. The results of Isworo, Purwanto and Sabdono (2016) The results of the test of the degradation ability of selected bacteria in the form of a consortium showed a better ability than the degradation ability of a single isolate. The bacterial consortium Exuquobacterium profundum and Oceanobacillus iheyensis had the best degradation ability of 83.23% while the bacteria consortium Exuquobacterium profundum and bacillus formis had the best degradation ability with a value of 68.75% on the Profenofos substrate 29.  The detected biodegradation chemical compounds will be translated into a chromatogram that represents the compound being analyzed. Analysis of the test sample was carried out by observing the retention time and chemical structure of Malathion and Profenofos due to degradation from the bacterial consortium.30 Observations and sampling were carried out at 0 hours, 96 hours, and 192 hours.  The chromatogram of chemical compounds biodegradation of the bacterial consortium Exiquobacterium profundum – Oceanobacillus iheyenis at T = 0 hours then detected the malathion compound C10H19O6PS2 or Butanedioic acid, ((dimethoxyphosphinothioyl) thio) -, monoethyl esterer 31, detected on peak 4 with real time = 19,675, area% = 7.37, is follow :  (fig 5)

Fig. 5.  The chromatogram of the chemical compound Malathion (C10H19O6PS2 or Butanedioic acid, ((dimethoxyphosphinothioyl) thio) -, monoethyl ester

While the cromatogram for the chemical compound Profenofos with the chemical formula C11H15BrClO3PSO- (4-bromo-2-chlorophenyl) o-ethyl s-propyl thiophosphate 32  was detected at peak 8, real time = 23,957, area% = 6.91, as follow : (fig 6)

Fig. 6. Chromatogram and chemical structure of Profenofos (O- (4-Bromo-2-Chlorophenyl) O- Ethyl S- Propyl Thiophosphate))

The complete data on the chemical compounds resulting from degradation is explained based on observations on GC MS with the parameters Peak, Real-Time, Area%, as follows (table 2).

Table (2):
Chemical compounds from the biodegradation of the consortium bacteria (observation t = 0 hours).

Peak
R.Time
Area
Area%
Height
Name
1
4.034
163.635
6,7
21025
(R,S)-2-Butanol, (3R)-3-[(Benzyloxycarbonyl)Amino]-
2
4.240
57.279
2,4
12654
T-Butyl (R)-3-(Benzyloxy)-Butanoate
3
7.957
59.397
2,4
14450
4,6-Dimethyl-4-Hydroxyhept-5-Enoic Acid
4
13.113
98.397
4,0
39793
Pentadecanenitrile(CAS)
5
17.833
427.673
17,6
141327
Hexadecanenitrile(CAS)
6
18.384
171.142
7,0
61700
Hexadecanoic Acid,Methyl Ester(CAS)
7
19.675
179.194
7,4
61739
Malathion E50
8
21.927
145.766
6,0
45806
9-Octadecenal,(Z)-(CAS)
9
22.345
85.071
3,5
30227
9-Octadecenoic Acid (Z)-,Methyl Ester(CAS)
10
22.481
126.591
5,2
42298
Hexadecanenitrile(CAS)
11
22.958
65.369
2,7
21510
Heptadecanoic Acid,16-Methyl-,Methyl Ester(CAS)
12
23.914
87.158
3,6
43370
Hexadecanamide(CAS)
13
23.957
168.200
6,9
72947
O-(4-Bromo-2-Chlorophenyl)O-Ethyl S-Propyl Thiophosphate #
14
25.481
79.450
3,3
27975
9-Octadecenamide(CAS)
15
25.635
147.922
6,1
29660
1,1,3,3,5,5,7,7,9,9,11,11,13,13-Tetradecamethylheptasiloxane
16
26.930
91.252
3,8
17443
1-Piperazinepropanamide, N-(4-Fluorophenyl)-4-Methyl-
17
27.164
60.743
2,5
17767
3-Octadecene-1,2-Diol(CAS)
18
27.258
58.346
2,4
20271
14.Alpha-Cheilanth-12-Enic Methyl Ester
19
28.801
82.966
3,4
15704
Silicone Grease,Siliconfett
20
29.705
77.285
3,2
19635
Silicone Grease,Siliconfett
2432836
100
757301

In the observation time of t = 0 hours that the tested Malathion and Profenofos compounds were still detected, this indicates that the Malathion and Profenofos compounds have not been completely degraded into simpler compounds.

The chromatogram data of chemical compounds biodegradation results from the bacterial consortium Exiquobacterium profundum-Oceanobacillus iheyenis at T = 96 hours observations, are completely shown in table 3, which is the result of staging on GC MS with parameters peak, real-time, area%, as follow :

Table (3):
Chemical compounds from the biodegradation of the consortium bacteria (observation t = 96 hours).

Peak R.Time Area Area% Height Name
1 3.975 44718 1.25 9359 Phenol,3,5-Dimethyl-(CAS)
2 4.477 31093 0.87 10563 2-Butenedioic Acid (E)-,Diethyl Ester
3 6.706 36954 1.04 20894 Phenol,2-methoxy-4-(2-propenyl)-(CAS)
4 7.965 72138 2.02 16400 1-(3,3-dimethyl-bicyclo[2.2.1]hept-2-yl)pentan-2-one
5 10.485 34744 0.97 8757 Pentanedioic acid,2,2-dimethyl-,bis(1-methylpropyl)ester (CAS)
6 12.989 54577 1.53 23184 Acethydrazide,2-(2-naphthylamino)-N2-(2,6-dichlorobenzylideno)-
7 13.105 121792 3.42 43652 Dodecanenitrile(CAS)
8 15.995 31813 0.89 13964 Cyclo(-L-Pro-L-Val-)
9 16.575 62669 1.76 17072 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane
10 17.827 401025 11.25 140342 Hexadecanenitrile(CAS)
11 18.384 139427 3.91 47978 Hexadecanoic acid, methyl ester (CAS)
12 18.891 72297 2.03 15832 5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a
13 19.493 57249 1.61 18294 Tetradecanamide
14 19.670 80070 2.25 30221 Malathion E50
15 21.914 121504 3.41 37368 Hexadecenenitrile
16 22.025 46680 1.31 15990 9-Octadecenal, (Z)-(Cas)
17 22.336 98016 2.75 34032 14-Octadecenoic Acid,Methyl Ester(CAS)
18 22.469 150614 4.22 42639 Hexadecanenitrile (CAS)
19 22.946 75605 2.12 23171 Octadecanoic Acid,Methyl Ester(CAS)
20 23.770 26632 0.75 7619 4-(4-Bromo-3-Nitro-Benzylidene)-1-(4-Chloro-Phenyl)-Pyrazolidine-3,5-Dione
21 23.913 90583 2.54 42273 Hexadecanamide(CAS)
22 23.951 78877 2.21 34703 O-(4-Bromo-2-Chlorophenyl)-O’-Ethyl Ester of Propylthio-Phosphoric Acid
23 24.527 41166 1.15 10099 Acetamide,N,N’-[(3.beta.)-18-hydroxypregn-5-ene-3,20-diyl]bis- (CAS)
24 24.615 33053 0.93 11041 3-(4-Hydroxy-3-methoxyphenyl)-2-isothiocyanatopropionic acid, ethyl ester,TMS
25 25.225 65384 1.83 13189 1,3,5,7,9-Pentaethyl-1,9-Dibutoxypentasiloxane
26 25.475 28709 0.81 13917 1-(Cyanomethyl)-3-Piperidinecarboxamide
27 25.550 34399 0.96 14017 N-(2-Adamantan-1-Yl-Ethyl)-4-(Piperidine-1-Sulfonyl)-Benzamide
28 25.620 33752 0.95 7609 (3E)-4-(1,2-Methoxycarbonylepimino-2,6,6-Trimethylcyclohexyl)-3-Buten-2-One
29 25.863 32630 0.92 20625 Hexasiloxane, Tetradecamethyl-(CAS)
30 25.936 28321 0.79 14045 Silikonfett
31 26.010 58329 1.64 19251 Cyclotetrasiloxane, Octamethyl(CAS)
32 26.055 50380 1.41 19742 Pentasiloxane,1,1,3,3,5,5,7,7,9,9-Decamethyl-
33 26.120 26494 0.74 11753 (2,2-Dibromo-1-Propylcyclopropane)Carboxylic Acid
34 26.150 70222 1.97 17378 Pentasiloxane, Dodecamethyl- (CAS)
35 26.316 58923 1.65 17078 1-Pentene, 1,3-Diphenyl-1-(Trimethylsilyloxy)-
36 26.345 86119 2.42 17965 14.Alpha.-Cheilanth-12-Enic Methyl Ester
37 26.430 35483 1.00 16504 4-Acetyloxyimino-6,6-Dimethyl-3-Methylsulfanyl-4,5,6,7-Tetrahydro-Benzo
38 26.480 63149 1.77 18989 Tartronic Acid, 4-(Dimethylethylsilyl)Phenyl-, Dimethyl Ester
39 26.560 59074 1.66 15147 Cyclotetrasiloxane, Octamethyl- (CAS)
40 26.730 34307 0.96 14017 Cyclopentasiloxane, Decamethyl- (CAS)
41 26.786 31297 0.88 19358 Silikonfett
42 26.882 51321 1.44 13561 Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-Decamethyl-
43 26.920 25776 0.72 12505 Sarpagan-17-Ol, 16-[(Acetyloxy)Methyl]-, Acetate (Ester) (CAS)
44 26.985 30442 0.85 12869 1,2-Bis(Trimethylsilyl)Benzene
45 27.712 47255 1.33 17719 Silikonfett
46 27.760 30689 0.86 14958 Silane, [[4-(2-Isothiocyanatoethyl)-1,2-Phenylene]Bis(Oxy)]Bis[Trimethyl- (CAS)
47 28.085 39623 1.11 20936 3-Ethoxy-1,1,1,5,5,5-Hexamethyl-3-(Trimethylsilyloxy)Trisiloxane
48 28.268 28903 0.81 12857 Tetradecamethylcycloheptasiloxane
49 28.530 36573 1.03 16310 Silikonfett
50 28.585 96854 2.72 18658 Silane, Trimethyl[5-Methyl-2-(1-Methylethyl)Phenoxy]- (CAS)
51 28.690 40740 1.14 22872 Cyclotetrasiloxane, Octamethyl- (CAS)
52 28.747 38641 1.08 23116 Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-Decamethyl-
53 28.855 53826 1.51 16658 3,4-Isopropylenedioxy-10b(S)-Pancratistatin-1,2-Cyclic Sulfate
54 28.938 39942 1.12 15193 3,3-Diethoxy-1,1,1,5,5,5-Hexamethyltrisiloxane
55 29.022 35906 1.01 19894 Silikonfett
56 29.128 28055 0.79 15987 Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-Decamethyl-
57 29.375 31829 0.89 9676 N-(Cyclohexyl)-3-Ethyl-3-Methyl-1,3-Dihydropyrrol-2-One Alpha.Methyl Ester
58 29.610 32848 0.92 10645 14.Alpha.-Cheilanth-12-Enic Methyl Ester
59 29.755 29380 0.82 1E+06 1H-Pyrrole-2,4-Dicarboxylic Acid,3,5-Dimethyl-,Diethyl Ester (CAS)
60 29.853 45965 1.29 Cyclotetrasiloxane,Octamethyl-(CAS)
3564836 100.00

Based on  table  3, it  shows  that  the compound Malathion [(Dimethoxy-phosphinothioyl) Thio)] was detected at peak 14, real time = 19.675, area% = 2.25 while Profenofos (O-(4-Bromo-2-Chlorophenyl)O-Ethyl S-Propyl Thiophosphate) detected at peak = 22, real time = 23,951 and area% = 2.2, as follows (Fig 7 and fig 8).

Fig. 7. The chromatogram and chemical structure of Butanedioic Acid Malathion [(Dimethoxyphosphinothioyl) Thio] at observation t = 96 hours

Fig. 8.  The chromoatogram and chemical structure of Profenofos[O-(4-Bromo-2-Chlorophenyl) -O’-Ethyl Ester Of Propylthio-Phosphoric Acid] at observation t = 96 hours

Also detected a chemical compound (4-Bromo-3-Nitro-Benzylidene) -1- (4-Chloro-Phenyl) -Pyrazolidine-3,5-dione which is the result of degradation of the profenofos compound, at peak = 20, real-time = 23,770 , area% = 0.75, as follows: (Fig 9)

Fig. 9.  Chemical compounds (4 – Bromo – 3 -Nitro – Benzylidene) -1- (4- Chloro – Phenyl) -Pyrazolidine-3,5-dione

Likewise the chemical compound O- (4-Bromo-2-Chlorophenyl) -O’-Ethyl Ester from Propylthio-Phosphoric Acid resulted from the enzymatic degradation of Profenofos by bacteria, this compound was detected based on a chromatogram at peak = 22, real-time = 24.525 and area % = 2.21, as follow : (Fig 10)

Fig. 10. The chemical compounds O-(4–Bromo–2-Chlorophenyl)-O’-Ethyl Ester of Propylthio-Phosphoric Acid

The chemical compound resulting from the degradation of Profenofos (Profenofos O-  (4-Bromo-2-Chlorophenyl) O-Ethyl S-Propyl Thiophosphate) will become a compound of phosphorus and phosphate groups which are degradation compounds that are not toxic. 33

In table 3 also detected compounds resulting from enzymatic malathion degradation by the bacterial consortium, is Chemical compounds of Malathion degraded into Butanedioic Acid, [(Dimethoxyphosphinothioyl) Thio]) detected at peak =  2,  real-time =  4,477 , area %. = 0.87 (fig 11).

Fig. 11. Chemical compounds of Butanedioic Acid, [(Dimethyl Phosphinothioyl) Thio])

The chemical compound Butanedioic Acid, [(Dimethoxyphosphinothioyl) Thio] -, Diethyl Ester is a synonym for Malathion Dicarboxylic Acid or Mercapto-O, O-Dimethyl Phosphorodithioate Succinic Acid which is the result of aerobic degradation of Malathion. Butanedioic Acid, [(Dimethoxyphosphinothioyl) Thio] -, Diethyl Ester will be degraded into a compound with this carboxylate group, namely 4-Acetyloxyimino-6,6-Dimethyl-3-Methylsulfanyl-4,5,6,7-Tetra hydro-Benzo [ C] Thiophene-1-Carboxylic Acid. the compound was detected at peak = 37. real time == 26.430, area% = 1.00, as follows:  34 35. (fig 12)

Fig. 12. The chemical compounds 4-Acetyloxyimino-6,6-Dimethyl-3-Methylsulfanyl – 4,5,6,7-Tetra hydro-Benzo [C] Thiophene-1-Carboxylic Acid

Based on these data, Malathion and Profenofos compounds have been degraded into simpler compounds, this can be compared with the decrease in peak values, real time and% area of Malathion and Profenofos compounds.36

The chromatogram of chemical compounds degradation of Malathion and Profenofos by the bacterial consortium Exiquobacterium profundumOceanobacillus iheyenis at observation t = 192 hours (table 4), as follows:

Table (4):
Degradation results of chemical compounds (the observation t = 192 hours).

Peak R.Time Area Area% Height Name
1 4.989 48189 1.26 9539 Ethanamine,1-(2,4-Cyclopentadien-1-Ylidene)-N,N-Dimethyl-(CAS)
2 5.866 63142 1.65 14298 5h-1-Pyrindine
3 8.732 64651 1.69 16861 1,3,3-Trideuterio-Endo-6-Hydroxy-9-Oxabicyclo(3.3.1)Nonan-2-One
4 12.985 36128 0.94 14142 Acethydrazide,2-(2-Naphthylamino)-N2-(2,6-Dichlorobenzylideno)-
5 13.101 81069 2.11 36269 Tetradecanenitrile
6 15.998 57762 1.51 17709 1,4-Diaza-2,5-Dioxo-3-Isobutyl Bicyclo[4.3.0]Nonane
7 17.826 353665 9.22 122031 Hexadecanenitrile(CAS)
8 18.372 98932 2.58 42608 Hexadecanoic Acid,Methyl Ester(CAS)
9 18.606 35870 0.94 12399 1,4-Diaza-2,5-Dioxo-3-Isobutyl Bicyclo[4.3.0]Nonane
10 19.486 38666 1.01 13905 9-Octadecenamide,(Z)-(Cas)
11 21.917 119642 3.12 40310 Hexadecenenitrile
12 22.02 57378 1.5 17685 1H-Fluorene,Dodecahydro-(CAS)
13 22.329 57101 1.49 22640 6-Octadecenoic Acid,Methyl Ester,(Z)-(CAS)
14 22.472 106596 2.78 37955 Hexadecanenitrile(CAS)
15 22.941 51171 1.33 20237 Tetradecanoic Acid,Methyl Ester(CAS)
16 23.914 63356 1.65 26794 N-Tetradecanoic Acid Amide
17 24.915 30091 0.78 9222 Sydnone, 4-Bromo-3-(Dimethylamino)-(CAS)
18 24.975 34098 0.89 12093 Caprolactone Oxime,(NB)-O-[(Diethylboryloxy)(Ethyl)Boryl]-
19 25.04 79169 2.06 12035 3,3-Diethoxy-1,1,1,5,5,5-Hexamethyltrisiloxane
20 25.19 50875 1.33 14698 1h-Indole-2-Carboxylic Acid,6-(4-Fluorophenyl)-3-Methyl-4-Oxo-4,5,6,7-Tetra
21 25.28 99698 2.6 20663 Pentasiloxane,1,1,3,3,5,5,7,7,9,9-Decamethyl
22 25.381 46788 1.22 28603 3-Isopropoxy-1,1,1,5,5,5-Hexamethyl-3-(Trimethylsiloxy)Trisiloxane
23 25.415 41195 1.07 22263 3,6-Dioxa-2,7-Disilaoctane,2,2,4,7,7-Pentamethyl-(CAS)
24 25.44 50152 1.31 21294 1,1,3,3,5,5,7,7,9,9,11,11-Dodecamethyl-Hexasiloxane
25 25.485 75004 1.96 32286 1,1,3,3,5,5,7,7,9,9,11,11-Dodecamethyl-Hexasiloxane
26 25.536 54901 1.43 30545 3-Ethoxy-1,1,1,5,5,5-Hexamethyl-3-(Trimethylsilyloxy)Trisiloxane
27 25.585 90159 2.35 21884 3,7-dibromo-6-ethyl-2-(pent-2′-en-4′-ynyl)octahydropyrano[3,2-b]pyran
28 25.641 58196 1.52 35794 Phenol,2-(4-diethylaminophenyliminomethyl)-
29 25.715 170080 4.43 29568 Silicone Grease,Siliconfett
30 25.854 130310 3.4 29240 (E)-1-[(1′,1′-Dimethylethyl)Diphenylsilyl]-2-(Trimethylsilyl)Ethylene
31 25.896 62480 1.63 26179 Silikonfett
32 25.947 121754 3.17 23743 Silikonfett
33 26.134 113921 2.97 20351 3-Ethoxy-1,1,1,5,5,5-Hexamethyl-3-(Trimethylsilyloxy)Trisiloxane
34 26.198 35139 0.92 23472 Silicone Grease,Siliconfett
35 26.303 50603 1.32 21563 1,5-Dimethyl-3-(4-Nitrophenyl)-1,3-Dihydro-2,1-Benzisothiazole 2,2-Dioxide
36 26.705 56983 1.49 15843 1,1,3,3,5,5,7,7-Octamethyl-Tetrasiloxane
37 27.031 55118 1.44 15602 Silikonfett
38 27.13 75329 1.96 14331 Cyclopentasiloxane, Decamethyl-(CAS)
39 27.21 39833 1.04 17390 Pentasiloxane,1,1,3,3,5,5,7,7,9,9-Decamethyl-
40 27.378 53927 1.41 15526 Hydroperoxide,9,10-Dihydro-9,10,10-Triphenyl-9-Anthryl(CAS)
41 27.44 32994 0.86 12840 Silikonfett
42 27.51 45836 1.2 20107 Tetracosamethylcyclododecasiloxane
43 27.54 41921 1.09 23431 1,3,5,7-Tetraethyl-1-Butoxycyclotetrasiloxane
44 27.674 59065 1.54 14410 Silikonfett
45 27.73 30912 0.81 13975 3-Isopropoxy-1,1,1,5,5,5-Hexamethyl-3-(Trimethylsiloxy)Trisiloxane
46 27.804 46229 1.21 14254 Silicone Grease,Siliconfett
47 27.858 39969 1.04 17706 1,1,3,3,5,5,7,7,9,9,11,11-Dodecamethyl-Hexasiloxane
48 28.599 32475 0.85 14537 Cyclotrisiloxane,Hexamethyl-(CAS)
49 28.741 82232 2.14 19951 Silicone Grease, Siliconfett
50 28.83 43445 1.13 19371 3-Isopropoxy-1,1,1,5,5,5-Hexamethyl-3-(Trimethylsiloxy)Trisiloxane
51 28.904 51224 1.34 16427 Tetrakis(Dimethylsilyl)-[18-O]-Dioxide
52 29.064 40249 1.05 19899 1h-Pyrrole-3,4-Diacetic Acid, 2-Acetoxymethyl-5-Methoxycarbonyl-,Dimethyl Ester
53 29.14 38332 1 15524 2-{4-[2-(4-Methoxymethylphenyl)Vinyl]Phenyl}Propan-2-Ol
54 29.285 30528 0.8 15811 Anthracene-9-Ol, 9,10-Dihydro-10-(4-Nitrobenzylideno)-
55 29.355 38555 1.01 16368  2,5-Dichloro-N,N-Diethyl-Benzenesulfonamide
56 29.44 44942 1.17 13947 Benzoic Acid, 3-[(Trimethylsilyl)Oxy]-,Trimethylsilyl Ester
57 29.565 34341 0.9 9740 Silane, Trimethyl[[1-[(Trimethylsilyl) Ethynyl] Cyclohexyl]Oxy]
58 29.69 30557 0.8 12757 1,1,1,3,5,7,9,9,9-Nonamethylpentasiloxane18591 Hexasiloxane,Tetradecamethyl-(CAS)
59 29.751 30337 0.79 17289 Hexasiloxane,Tetradecamethyl-(CAS)
60 29.8 31960 0.83 1306505 Pentasiloxane,1,1,3,3,5,5,7,7,9,9-Decamethyl-
3835224 100

Based on table 4, at the observation t = 92 hours, the chemical compounds of Malathion and Profenofos were not detected. This shows that the concentration disturbance and Profenofos in the sample have broken down completely into simple compounds which are not contaminants. Prediction of Biodegradation of Malathion and Profenofos compounds according to the EAWAG-Biocatalysis and Biodegradation Pathway Prediction System that Malathion and Profenofos compounds will be degraded into simpler compounds, namely Hexadecanenitrile (CAS) chemical compounds Palmitonitrile, Palmitic acid nitrile, N-Hexadecanon 1- Cyanopentadecane which is the result of degradation of Profenofos. these compounds were detected at peak = 7 and real time = 17,826 and Hexadecanenitrile (CAS) at peak = 14, real-time = 22,472  (fig 13), chemical compound Anthracene-9-Ol,9,10-Dihydro-10-(4-Nitrobenzylideno)-(Functional Group-Ol/Alcohol) was detected at peak 54, real time = 29,285 (fig 14), whereas chemical compound 2- {4- [2- (4-Methoxymethylphenyl) vinyl] phenyl} propan-2-ol (functional group-ol / alhohol) with peak = 53 and real time = 29,140 (fig 15) and chemical compounds Acethydrazide compound, 2 – (2-naphthylamino) -N2- (2,6-dichloro benzylideno) is a decomposed benzyl aldehyde group, is a compound resulting from Malathion degradation, detected with peak 4 and real time = 12,985, as follow 33 (fig 16) :

Fig. 13.   The chromatogram of chemical compounds Hexadecanenitrile(CAS) Palmitonitrile, Palmitic acid nitrile, N-Hexadecanonitrile, 1-Cyanopentadecane

Fig. 14. Chromatogram of chemical compounds    Anthracene -9-Ol, 9,10-Dihydro-10- (4-Nitrobenzylideno) – (Functional Group – Ol / Alcohol)

Fig. 15.  The chromatogram of chemical compounds 2- {4-[2-(4-Methoxymethylphenyl) vinyl] phenyl} propan-2-ol (functional group –ol/alhohol)

Fig. 16. The chromatogram of chemical compounds Acethydrazide compound, 2- (2-naphthylamino) – N2 – (2 , 6 – dichloro benzylideno)

At the observation of t = 192 hours, the chemical compounds of Malathion and Profenofos have been degraded into simpler and non-toxic compounds..37

CONCLUSION

The indigenous bacterial consortium Exiquobacterium profundum – Oceanobacillus iheyenis was able to completely degrade Malathion and Profenofos at observation t = 4 (96 hours observation) based on a decrease in the area % of  Malathion from 7.37 to 2.25 and a decrease in area % of Profenofos from 6.91 to 2, 21. At the observation t = 8 (192 hours) Malathion and Profenofos compounds were not detected (area % = 0)

Declarations

ACKNOWLEDGMENTS
The researcher would like to thank all staff of the Integrated Laboratory of Diponegoro University, Semarang, and all staff of the Dian Nuswantoro University health laboratory, Semarang so that this research can be carried out well.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
SI does the research design, wrote the research results, wrote the initial draft of the manuscript. SI and PSO worked together to manage the research analysis. SI manages the literature and makes final draft corrections. Both authors read and approved the manuscript for publication.

FUNDING
None.

ETHICS STATEMENT
Not applicable.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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