ISSN 0003-6838, Applied Biochemistry and Microbiology, 2021, Vol. 57, Suppl. 1, pp. S46–S53. © Pleiades Publishing, Inc.

, 2021.

Biodegradation of the Insecticide Bendiocarb by Bacillus thuringiensis

in a Packed Biofilm Reactor
S. Muñoz-Martíneza, D. Ahuatzi-Chacóna, *, F. Santoyo-Tepoleb, N. Ruiz-Ordaza,
J. Galíndez-Mayera, and C. Juárez-Ramíreza
a Departamento de Ingeniería Bioquímica, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas,
Av. Wilfrido Massieu s/n, Zacatenco, 07738 México
b
Central de Instrumentación de Espectroscopía, Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas,
Carpio y Plan de Ayala s/N, Casco de Sto. Tomás, 11340 México
*e-mail: dahuatzi@hotmail.com
Received May 25, 2021; revised July 6, 2021; accepted August 30, 2021

Abstract—Carbamates are among the most commonly used xenobiotics as insecticides, even though these
compounds are highly toxic to humans and animals. Within carbamates, bendiocarb has been used against a
vast range of nuisance and disease vector insects and in agriculture on sugar beet and maize crops. Since 1999,
the Environmental Protection Agency of the United States of America has banned bendiocarb use in the
United States, although the World Health Organization continues to recommend it for the control of malaria
in Africa. In Mexico, bendiocarb effectiveness for the control of disease vector insects transmitting dengue,
Zika and chikungunya has been demonstrated. One of the remediation technologies to confront the problem
of contamination by xenobiotic compounds is applying microorganisms that are able to use these compounds
as substrates. In this context, the isolation of microorganisms capable of efficiently degrading bendiocarb is
especially important. In this work, Bacillus thuringiensis was isolated from a microbial community isolated
from soil samples collected of a composting plant at the northeast of Mexico City and tested for its ability to
degrade bendiocarb. This bacterium immobilized in a biofilm reactor, using fragments of volcanic rock
(tezontle) as a support and operated on a continuous basis, degraded more than 90% of the insecticide with
high removal rates.

Keywords: bendiocarb, biodegradation, biofilm reactor, Bacillus thuringiensis, insecticide

DOI: 10.1134/S0003683821100070

The indiscriminate use of xenobiotic compounds mental Protection Agency of the United States of
as fertilizers and pesticides in agriculture has become a America (EPA) classifies bendiocarb in the category of
serious pollution problem because these compounds highest toxicity (category I) in the case of oral inges-
do not degrade easily and are displaced through the tion, category II for dermal exposure and inhalation,
soil to water bodies [1]. Moreover, these kinds of prod- category III for dermal irritation, and category IV for
ucts also have an environmental impact due to their primary eye irritation [7]. Bendiocarb also presents
ecotoxicity. In other words, the toxicity of these com- severe and chronic risk to freshwater invertebrates, and
pounds affects organisms that are important to the it has a lethal concentration (LC50 of 96 h) for fish
food chain. Among the most commonly used xenobi- that varies between 0.4 and 1.8 mg/L.
otics are insecticides, and many of them include car- Bendiocarb is also toxic to some beneficial organ-
bamates in their chemical composition. Carbamates isms, such as honey bees, earthworms and seed preda-
are compounds that inhibit the activity of the enzyme tors [6]. Since 1999, the EPA has banned bendiocarb
acetylcholinesterase. Additionally, these pesticides use in the United States; however, the WHO recom-
have been reported as very toxic to humans and ani- mends it for malaria control in Africa. In addition, in
mals and are highly persistent in the environment due recent years, in Mexico, the efficacy of bendiocarb was
to their significant mobility in the ground [2]. demonstrated for the control of infection vectors of
Bendiocarb is a carbamate insecticide widely used diseases such as dengue, Zika and chikungunya [8]. In
for the control of domestic pests and corn and beet some kinds of soil and pH conditions, humidity, and
crops [3–5]. The World Health Organization (WHO) temperature, bendiocarb presents mobility enough to
classifies bendiocarb as belonging to class II pesticide pollute underground and superficial water. For exam-
[6], indicating that it is moderately toxic to humans if ple, in year 2009, bendiocarb at concentration varied
ingested or absorbed through the skin. The Environ- from 0.15 to 5.68 μg/L was detected in superficial

S46
 BIODEGRADATION OF THE INSECTICIDE BENDIOCARB S47

water in Japan and at concentration of 0.181 mg/L in Standard count agar medium containing (g/L):
some underground water samples from 15 regions in agar—9.0; dextrose—1.0; tryptone—5.0; yeast
the east, north and south of Saudi Arabia [9]. extract—2.5. The medium was used to determine via-
One of the remediation technologies to solve the ble cell counts (CFU/mL) and to identify the micro-
problem of contamination by xenobiotic compounds organisms by their colonial morphology. Luria–Ber-
is biodegradation using microorganisms able to use tani medium was used to grow the isolated microor-
contaminants as a substrate. Biodegradation is an eco- ganisms to perform DNA extraction. The medium
nomical, efficient and environmentally friendly pro- contained (g/L): tryptone, 10.0; yeast extract, 5.0; and
cess that has emerged as a very promising alternative NaCl, 5.0 (pH 7.0).
for pesticide removal [10]. Isolation and characteriza- Biofilm reactor. The bioreactor used in this work
tion of microorganisms that are able to degrade pesti- was supplemented with a sealed glass lid with neo-
cides makes it possible to develop technologies for the prene gaskets at the base of a glass column. The col-
restoration of contaminated environments or for the umn dimensions were 5 cm in diameter and 15 cm in
treatment of waste before final disposal [11]. height with an operating volume of 275 mL. The col-
umn had two inputs at the bottom, one for the entrance
A wide variety of microorganisms with the ability to of liquid medium and another for air supply. An output
degrade pesticides have been isolated, but biodegrada- of culture medium was located at the top of the column.
tion depends not only on the presence of microorgan- The bioreactor was provided with a porous glass diffuser
isms with the appropriate enzymes but also on a wide with a pore diameter from 40 to 100 μm.
range of environmental conditions. In this context, the
use of immobilized cells represents a promising alter- Fragments of volcanic stones (called tezontle in
native because the cells have resistance to toxic com- Mexico) were used as the support and characterized by
pounds and the possibility of maintaining the catalytic determining the particle size and porosity of the bed.
activity for long time periods [12, 13]. The bacterial To determine the average size of the packing material,
biofilm is composed of an extracellular matrix that 50 fragments were taken, and considering them as
contains exopolysaccharides, proteins, DNA, surfac- ellipsoidal bodies, 3 characteristic radii were mea-
sured: a, b, and c [15]. With the three measurements,
tants, lipids, glycolipids, and ions such as Ca2+. This the volume was obtained, and subsequently, the aver-
matrix keeps the cells associated with each other and age particle diameter, dp, was calculated as 12.22 ±
with a solid surface [14]. The use of biofilm reactors
1.28 mm.
increases because the microorganisms used in this
type of bioreactor have resistance to the toxicity of Microorganism. The microorganism, able to degrade
pollutants. Moreover, biofilm reactors are easy to the insecticide, was selected from the microbial com-
work with at high dilution rates. The purpose of this munity isolated for its ability to degrade the commer-
work was to evaluate the biodegradation of the bendi- cial insecticide bendiocarb. The microbial community
ocarb insecticide in an aerobic biofilm reactor, packed was obtained used culture enrichment by successive
with volcanic porous rock fragments as support, at transferences. Soil samples collected of a composting
laboratory scale imitating the behavior of a permeable plant at the northeast of Mexico City, were used in
biological biobarrier. B. thuringiensis was selected from Erlenmeyer flasks with 100 mL of MS medium con-
soil samples collected of a composting plant at the taining the insecticide (20 mg/L) and fragments of
northeast of Mexico City and tested for efficiently volcanic rocks. After 2 weeks for incubation under stir-
degrading bendiocarb with potential use for bioaug- ring at room temperature, 90% of the medium was
mentation in a biobarrier. drained and it was substituted with fresh MS
medium. The bendiocarb concentration was moni-
tored every 24 h by UV spectrophotometry for several
MATERIALS AND METHODS weeks until after 4 refills of the liquid medium.
Isolation of predominant bacterial strains and iden-
Chemicals. The commercial insecticide “Ficam W” tification of cultivable microorganisms. Appropriated
distributed by Bayer AG (Germany) was purchased.
The bendiocarb standard with a purity of 99.8% was dilutions (103, 104, 105) of the collected cell suspension
acquired from Chem. Service (USA). were made and inoculated as 200 μL aliquots on stan-
dard plate count agar medium. The colonies were iso-
The minimum mineral (MS) medium contained lated by differences in their colonial morphology.
(g/L): K2HPO4, 0.4; KH2PO4, 0.2; MgSO4, 0.1; and The identification was made with DNA extraction,
CaCl2, 0.04. The medium was supplemented with the 16S rDNA PCR amplification and sequencing. For
following trace elements, obtaining in final concentra- DNA extraction, the Go Taq® Flexi DNA poly-
tion, mg/L: FeSO4⋅7H2O, 0.55; ZnSO4⋅7H2O, 0.23; merase kit (Promega Corp., USA) was used. The
MnSO4⋅7H2O, 0.34; and Na2MoO4, 0.034. The com- amplification of the 16S rDNA was performed in a
mercial insecticide Ficam W was added at a concen- GeneMate thermocycler (BioExpress, USA) using the
tration of 20 mg/L to the MS medium as the sole universal initiators FD1 and R6 [16]. Sequencing was
source of carbon and nitrogen to reach. carried out by Macrogen Inc. Laboratory, South

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S48 MUÑOZ-MARTÍNEZ et al.

Korea, and with the use of the BLAST program, the Samples were taken every 24 h to determine the con-
sequences were aligned with those in the GeneBank centration of bendiocarb and cell growth.
Database of the National Center for Biotechnological Analytical methods. The cell concentration was
Information (NCBI) to define their similarity with determined as viable cell count (CFU/mL). A rapid
those of the most similar microorganism. determination of the insecticide concentration was
Evaluation of the growth of each member of the bac- estimated by measuring the absorbance on a Beckman
terial community and their degradation of bendiocarb. DU®650 spectrophotometer at its maximum wave-
Microorganisms were grown in 25 mL flasks with 7 mL length of 226 nm [13]. Subsequently, the concentra-
of MS medium, and 20 mg/L bendiocarb was added as tion of the insecticide was determined by HPLC. The
the sole source of carbon and nitrogen to evaluate the chromatograph was performed using Agilent Technol-
ability of each isolate to grow in axenic cultures using ogies (USA) 1260 infinity instrument equipped with a
the insecticide as a substrate. diode-array detector and ZORBAX SB-phenyl C18
For the study with planktonic cells, 2 mL of the column (150 mm × 4.6 mm, 5 μm). The mobile phase
flask contents used to evaluate the growth of the iso- contained acetonitrile/water (60 : 40, vol/vol) and
lates was transferred to a 250 mL flask with 50 mL of used at a flow rate of 1 mL/min, and UV detection was
MS medium and 20 mg/L bendiocarb. Samples were implemented at 206 nm.
taken every 24 h, and the concentration of bendiocarb One of the first degradation products generated
in the medium was determined by UV spectropho- during the molecule’s conversion either by biotic or
tometry at OD226 [6] for one month to assess the effi- abiotic processes and observed in soil treated with
ciency of insecticide removal. A control without cells bendiocarb, is benzodioxol-4-ol [17]. Due to the
was also included. lack of the benzodioxol-4-ol standard, its concentra-
For the study with sessile cells, we proceeded in the tion was determined stoichiometrically when a sample
same way as in the study with planktonic cells but used of bendiocarb reached complete hydrolysis after
10 g of tezontle. Before inoculating the flasks with 30 days of incubation. Analysis of the areas of both
each of the isolates, the support was saturated. For sat- compounds showed that the area presenting benzodi-
uration, the flasks were kept under constant agitation oxol-4-ol was larger than the area of pure bendiocarb
at 60 rpm, and samples were taken from the culture at the same concentration. Therefore, a correction
medium every 24 h to determine the solution at OD226 factor was determined using the ratio of the molar
on a Beckman DU®650 spectrophotometer (USA). extinction coefficients of both molecules, which
Saturation was reached when absorbance values were turned out to be 0.74. The molar extinction coefficient
equal to those obtained with the initial sample. Once was determined using the relationship proposed by
saturation was reached, the flasks were inoculated and Pelillo et al. [18].
left under constant agitation for 72 h to favor the A closed reflux method 8000 was used for chemical
immobilization of each of the isolates. After this time, oxygen demand (COD) determination [19]. The reac-
the culture medium was drained, and the same volume tive kit used, from Hach Co. (USA), containing sulfuric
of newly prepared culture medium was added to start acid, potassium dichromate, silver, and mercury ions,
the evaluation of the degradation in batch culture, for could determine COD levels from 0.7 to 40 mg/L.
which samples were taken every 24 h to evaluate the
concentration of bendiocarb and cell growth.
RESULTS AND DISCUSSION
Evaluation of the degradation of bendiocarb in a
packed bed reactor. Seventy percent of the volumetric Isolation of members of the microbial community. The
capacity of a glass column was packed with volcanic cultivable microorganisms that were members of the
stone (tezontle) as support. Both the column and the microbial community were isolated from soil samples
support were previously sterilized. Using a peristaltic collected of a composting plant at the northeast of Mex-
pump, sterile MS medium containing 20 mg/L bendi- ico City to evaluate their capacity for degrading bendio-
ocarb was supplied. To saturate the support material carb. Twelve microorganisms with the highest abun-
with the commercial insecticide, the abiotic test was dance in the microbial community were identified: iso-
carried out continuously at a flow feeding of 100 mL/h late 1: Pseudoxanthomonas spadix (NC_016147.2,
for MS medium containing bendiocarb (20 mg/L). 98%), isolate 2: Brevundimonas sp. (KU851032.1,
Saturation was reached when the concentration of the 97%), isolate 3: Agromyces sp. (NZ_LMKQ01000001.1,
insecticide at the exit of the bioreactor matched the 98%), isolate 4: Pseudomonas alkylphenolia
concentration at the entrance. Once saturated, the (NZ_CP009048.1, 98%), isolate 5: Aminobacter aminov-
system was inoculated with the selected microorgan- orans (LN995690.1, 96%), isolate 6: Pseudomonas
ism. After 7 days in batch culture to allow for coloniza- denitrificans (NC_020829.1, 99%), isolate 7: Ochrobac-
tion of the support, the medium was drained, and fresh trum anthropic (NC_009668.1, 97%), isolate 8: Bosea
MS medium with 20 mg/L bendiocarb was added to thiooxidans (NZ_LMAR01000067.1, 99%), isolate 9:
evaluate the biodegradation kinetics in batch culture. Ralstonia sp. (FJ774001.1, 92%), isolate 10: Brevibac-
Aeration was kept constant at a flow of 0.2 L/min. terium sp. (KM507608.2, 97%), isolate 11: Kokuria sp.

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 BIODEGRADATION OF THE INSECTICIDE BENDIOCARB S49

(a)
mg/L
40

35

30
2
25
11
1 34 5 9 12
6 78 10
20

15

10

0
0 144 312 480 570 607 672 696 h
(b)
mg/L
35

30

25
2 11
1 34567891012
20

15

10

0
0 144 312 480 570 607 672 696 h

Fig. 1. Batch biodegradation of bendiocarb by planktonic (a) and sessile (b) cells. 1—Isolate 1; 2—isolate 2; 3—isolate 3; 4—isolate 4;
5—isolate 5; 6—isolate 6; 7—isolate 7; 8—isolate 8; 9—isolate 9; 10—isolate 10; 11—isolate 11 and 12—isolate 12.

(KU199722.1, 97%), and isolate 12: Bacillus thuring- metabolic activity, withstanding high mechanical
iensis (CP032608.1, 99%). strength, high resistance to inhibition by toxic sub-
Evaluation of bendiocarb biodegradation by each of stances, and greater stability against temperature and
the isolates in batch culture with planktonic and sessile pH changes, when compared with the use of planktonic
cells. When planktonic cells were inoculated in MS cells. Sessile cell cultures also have the advantage of low
medium with 20 mg/L commercial insecticide, all iso- cell loss during continuous operation in bioreactors
lates showed growth after 48 or 72 h of incubation [20]. Therefore, the degradation of the insecticide was
under stirring at room temperature. Microbial growth also evaluated using sessile cells immobilized in volca-
seemed to be at the expense of adjuvants present in the nic stone (tezontle) as the support. The evaluation was
commercial formulation since none of the isolates carried out in batch culture for each of the 12 isolates,
were able to degrade the insecticide (Fig. 1a). and only two of the isolates (isolate 7, identified as
The use of sessile cell cultures in the treatment of O. anthropi, and isolate 12, identified as B. thuringien-
wastewater has important advantages such as high sis) were able to degrade the commercially available

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S50 MUÑOZ-MARTÍNEZ et al.

Table 1. Removal efficiencies (ηB), volumetric removal rate (RVB) of bendiocarb, removal efficiencies (ηCOD), volumetric
removal rate (RvCOD) of COD and cell concentration (UFC/mL) obtained with sessile cells of B. thuringiensis in batch cul-
tures
T, h CFU × 107/mL RvB, mg/L h η B, % RvCOD, mg/L h ηCOD, %
96 0.79 ± 0.20 0.091 ± 0.05 35.99 ± 0.05 0.103 ± 0.06 54.39 ± 0.75
360 1.00 ± 0.40 0.043 ± 0.01 64.49 ± 0.25 0.118 ± 0.04 78.02 ± 1.32
552 1.60 ± 0.30 0.037 ± 0.03 84.88 ± 1.21 0.027 ± 0.02 83.51 ± 0.93
672 6.82 ± 0.60 0.031 ± 0.02 87.27 ± 0.95 0.021 ± 0.03 80.76 ± 1.12

bendiocarb (Fig. 1b). O. anthropi had a bendiocarb hydrolyzed [17], forming benzodioxol-4-ol, however,
removal efficiency of 48.0 ± 1.2% and B. thuringiensis there are no precise data on the degradation of this
that of 60.0 ± 2.0% after 696 h of incubation. compound.
With both these isolates, the experiment was repeated When batch culture was carried out in the packed-
in batch culture with sessile cells in a flask to evaluate bed bioreactor, a degradation efficiency (ηB) of
insecticide biodegradation. Although O. anthropi has 92.60 ± 1.18% was obtained (Table 2). This value was
been reported to be involved in biodegradation processes slightly higher than that obtained in the batch culture
[21], no degradation of bendiocarb was observed when in a flask. It was also evident that the degradation time
cultured with O. anthropi. was considerably reduced, obtaining a volumetric
Degradation of bendiocarb in batch culture by B. thu- removal rate (RvB) up to 2.32 ± 0.18 mg/L h after 5 h
ringiensis using sessile cells in flasks. Unlike O. anthropi, of cultivation, more than double when compared with
B. thuringiensis showed continuous degradation of ben- the maximum RvB obtained in the flask experiment,
diocarb from the first day of cultivation. After 672 h, a which it was 0.09 ± 0.05 mg/L h. In terms of microbial
removal efficiency (ηB) of 87.27 ± 0.95% was obtained growth (measured in the effluent), an improvement
(Table 1). Likewise, microbial growth continued to was also observed, and a specific growth rate (μmax) of
increase with a specific maximum growth rate (μmax) 0.098 ± 0.01 1/h was obtained, a value that was greater
of 0.15 ± 0.01 1/d. than that detected with the same isolate in batch cul-
A complementary way to measure the degradation of ture in a flask.
the insecticide is by assessing COD. When COD was With these results, it can be said that constant and
evaluated, the maximum bendiocarb removal efficiency controlled aeration was required to increase the effi-
(ηCOD) of 83.51 ± 0.93% was obtained (Table 1). Upon ciency and removal rates, in addition to greater accli-
observing the high removal efficiencies of the insecti- matization of the microorganism and colonization of
cide and COD, the degradation evaluation was per- the support [22, 23].
formed in a constantly aerated packed-bed reactor.
When the degradation of bendiocarb in a continu-
Degradation of bendiocarb by B. thuringiensis in a ous regime was evaluated, the culture was started with
packed-bed reactor. The abiotic test was performed in a feed flow rate of 100 mL/h, and samples were taken
a packed-bed reactor to obtain information on the every 24 h to determine the bendiocarb concentration.
adsorption capacity of the support. Saturation of the Again, the chromatograms showed the peak corre-
support was reached after 18 days. Then, the reactor
was inoculated with a suspension of B. thuringiesis,
and after 7 days in the batch process, the evaluation of Table 2. Removal efficiencies (ηB), volumetric removal
insecticide biodegradation in the same system was rate (RvB) of bendiocarb and cell concentration (UFC/mL)
continued. in the biofilm reactor with B. thuringiensis
According to the results of the batch test using a T, h CFU × 107/mL RvB, mg/L h ηB, %
packed-bed biofilm reactor (Table 2), after 14 days,
bendiocarb was practically depleted and had been used 0 0.62 ± 0.10 1.61 ± 0.06 –
as a source of carbon and nitrogen by B. thuringiensis, 3 0.79 ± 0.30 1.61 ± 0.12 21.23 ± 0.24
as demonstrated by the increase in biomass concentra- 4 1.30 ± 0.20 1.68 ± 0.09 29.42 ± 0.35
tion. In the chromatograms obtained by HPLC, a
decrease in the peak corresponding to bendiocarb was 5 1.02 ± 0.40 2.32 ± 0.18 50.87 ± 0.75
observed; however, the peak corresponding to the 7 1.25 ± 0.50 1.97 ± 0.16 60.42 ± 0.38
benzodioxol-4-ol intermediate did not increase and 8 1.60 ± 0.30 2.02 ± 0.21 70.92 ± 0.92
instead showed a slight decrease (Fig. 2). It is import- 9 4.75 ± 0.20 2.05 ± 0.08 80.91 ± 0.83
ant to mention that there are no references about the
total mineralization of bendiocarb, and the vast 12 6.82 ± 0.40 1.72 ± 0.05 90.32 ± 1.23
majority of studies report that the compound is easily 14 7.20 ± 0.40 1.51 ± 0.04 92.60 ± 1.18

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 BIODEGRADATION OF THE INSECTICIDE BENDIOCARB S51

(a) (a)
100 mg/L
90 Retention times 1 25 1
80 Name

Bendiocarb
3.480
70 20
60
mAU

50 15 2
40
30 2 10
20

3.213
3.840

3.067
10 5
0
–10 0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
0 5 10 15 20
Minutes
Days
(b) (b)
150 mg/L
140 Retention times 25
130 1
120 Name 2
110 20
3.220

100
90
3.480 Bendiocarb
mAU

80 15
70
60
50 10 2
40
30 1
5
3.907

20
3.767

10
0
0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
Minutes 0 5 10 15 20
(с) Days
18 Retention times
Name Fig. 3. Bendiocarb biodegradation by sessile cells of
16 B. thuringiensis in the continuously operated packed-bed
14 reactor at different hydraulic retention times of (a) 1 and
(b) 0.5 h. 1—Influent, 2—effluent.
12
mAU

2
3.480 Bendiocarb

10
3.219

8 with both flows (Table 3). Biofilm reactors allow for

6 operation with low hydraulic residence times (HRTs),
3.7673.880

4 1 and xenobiotic biodegradation systems with HRTs of

2, 1 and 0.5 h have been reported [24–26]. In this
3.987

2
0 work, the tezontle-packed bed biofilm reactor allowed
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 for high insecticide removal efficiencies at low HRTs
Minutes of 1 and 0.5 h. The tezontle was used as the support
material that presents porosity and high roughness,
Fig. 2. HPLC chromatograms obtained during the batch allowing for the formation of a balanced biofilm [27].
process of bendiocarb’s degradation in the packed-bed
reactor after 0 (a), 8 (b) days and 14 (c) days of incubation Verification of B. thuringiensis in the biofilm reactor.
with sessile cells. 1—Bendiocarb, 2—benzodioxol. To verify that B. thuringiensis effectively remained an
axenic culture in the biofilm reactor, plate counting
was performed with biomass present in the effluent and
sponding to benzodioxol, but as in the batch culture, it in the packing material. The plates showed only one
was degraded by B. thuringiensis. Once a steady state was type of colonial morphology that matched isolate 12,
reached, after 16 days of incubation (Fig. 3a), an insec- identified as B. thuringiensis, and that was inoculated
ticide removal efficiency (ηB) of 93.21 ± 0.06% (Table 3) in the packed bed reactor. By sequence comparison of
was achieved. By observing such high removal efficiency, their 16S rDNA amplicons, the bacterial strain was
the feed flow was increased to 200 mL/h (Fig. 3b). The identified as B. thuringiensis (CP032608.1, 99%).
steady state was achieved after 8 days, and a removal The main application of B. thuringiensis in agricul-
efficiency (ηB) of 92.63 ± 0.08% was obtained. High ture is as a biological insecticide to control various
COD removal efficiencies (ηCOD) were also observed insects that affect crops and forests and that transmit

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S52 MUÑOZ-MARTÍNEZ et al.

Table 3. Operational characteristics of the packed-bed reactor, rates and efficiencies removal, bendiocarb and COD along
continuous operation
F, mL h HRT, h BvB, mg/L h RvB, mg/L h ηB, % BvCOD, mg/L h RvCOD, mg/L h ηCOD, %
100 1 23.13 ± 0.45 21.56 ± 0.19 93.21 ± 0.06 10.92 ± 0.04 9.90 ± 0.03 90.80 ± 0.03
200 0.5 44.02 ± 0.78 40.74 ± 0.13 92.63 ± 0.08 22.39 ± 0.07 20.42 ± 0.05 91.11 ± 0.06

diseases to humans and animals [28]. However, in the 5. Jankowska, M., Wisniewska, J., Faltynowicz, L.,
bioremediation area, it has been reported that B. thu- Lapied, B., and Stankiewicz, M., Molecules, 2019,
ringiensis is capable of degrading malathion, which is vol. 24, p. 3775.
an organophosphorus insecticide [29]. Recently, stud- 6. WHO Specifications and Evaluations for Public Health
ies have also found that this species degrades fipronil Pesticides. Bendiocarb. https://www.who.int/whopes/
[30], chlorpyrifos [31], cyalotrine [32], quinalfos [33], quality/Bendiocarb_eval_WHO_dec_2008.pdf.
and anthracene hydrazorbide [34]. 7. EPA. Prevention, Pesticides and Toxic Substances.
https://archive.epa.gov/pesticides/reregistration/web/
pdf/0409fact.pdf.
*** 8. Centro Nacional de Programas Preventivos y Control
The selection of microorganisms with a high de Enfermedades (CENAPRECE), Monitoreo de Resis-
capacity to degrade contaminants has vital importance tencia a Insecticidas (adulticidas) utilizados en el Pro-
grama de Enfermedades Transmitidas por Vectores en
in bioremediation processes. In this work, a microor- México, Secretaría de Salud, 2016.
ganism that was identified as B. thuringiensis was
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