Bioreactors cover. Two ports for feeding and ventilation were


Two similar Plexiglas bioreactors (each of approximately 15.7 L) were
constructed so that each bioreactor can accommodate 10 L of mixture of soil,
water and pesticide in addition to a head space. Each bioreactor was
constructed from 1.0-cm thick Plexiglas cylinder. The cylinder dimensions were 20
cm diameters and 50 cm length. The bottom of the cylinder was made of 20 cm
diameter circular Plexiglas plate (1.0-cm thick) and was glued to the cylinder.
The lid was made of 22 cm diameter circular plate (1.0 cm thick) and was fixed
into the cylinder by six stainless steel screws and wing nuts. The lid was
designed to accommodate one hole (1.5 cm diameter) in the center for the mixing
shaft and two other holes (1.5 cm diameter each) for the wastewater influent
and air exhaust. A 55 rpm induction motor (Japan Servo Company, Tokyo, Japan)
was used to mix the contents of the reactor. The motor was mounted on the
reactor lid and connected to a 1.5 cm diameter mixing shaft. A propeller (15 cm
diameter) was fixed to the shaft at 2 cm above the diffuser. An air inlet port
at the bottom of each cylinder was provided and a 20 mm PVC elbow was fitted to
it. The air diffuser (15 mm diameter and 25 mm height) (Dynamic Aqua-Supply
Ltd., Sydney, Nova Scotia, Canada) was fixed inside each cylinder to the elbow.
Tygon tubing was used to connected the diffuser to the air supply unit that was
consisted of air compressor (3/4 HP Shanborn model MCIFC 75-715, DV Systems
Maritimes, Halifax, Nova Scotia, Canada), pressure regulator (Model ZFMQ000PR,
Millipore Pressure Regulator, Etobicoke, Ontario, Canada) and flow meter (Model
60648, Cole Parmer, Chicago, Illinois, USA).

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The wastewater feeding system consisted of one tank for storing
wastewater and a manifold with three values for distribution. The storage tank
was made of plastic and holds 100 L plastic tank of the contaminated wastewater.
The tank was provided with a stirring shaft with paddles driven by a 1/12 HP electric
motor (Model: 5SCP10FG17AX, General Electric, Mississauga, Ontario, Canada) and
fixed to the tank cover. Two ports for feeding and ventilation were also provided
on the cover. A 5 cm ball valve was used to connect the feeding tank to the distribution
manifold. The other remaining two valves on the manifold were used to control
the flow to the bioreactors.


Two 25 L plastic carboys (Cat No.02-961B: Fishers Scientific,
Montreal, Quebec, Canada) were used for the collection of the overflow from the
two bioreactors.




The fungicide Captan 80-WP (C9H8ClNO2S)
was selected for this work since it is one of the most extensively used
pesticide. The structure as well as the properties of Captan 80-WP are illustrated
in Table (1).  Captan 80-WP is a
protectant eradicant fungicide and is one of the most popular pesticides nowadays
as a result of its effectiveness in treating a wide range of fungal infections
(Table 2). The pesticide is a microfine wettable powder containing 80% active
ingredient (75.5% N-trichloromethyl)thio-4-cyclohexene-1,2-dicarboaimide and
4.3% related derivatives). The balance (20%) is made of mineral dust and wetters
dispersants. This formulation is usually more acceptable by many growers because
it reduces visible residues and consequently provides perfect vegetable and
fruit end product.


The soil used in this study was obtained from an agricultural soil that
received fertilizers in the form of urea, NPK and raw liquid manure. The top
thin layer of the soil was removed and the soil was obtained by a shovel from
the top 30 cm layer and placed in a heavy duty Polyethylene bag (16 m thick) and
transferred to the laboratory for use in the experiments. The soil was the source
of mixed microbial culture.


Table 1. Chemical formula and structure for captan (Wolfram Alpha, 2011;
Sigma Aldrich, 2011).





Powerful protectant fungicide Solid (yellow amphous powder)
Insoluble in water
Molecular Weight= 300.59 g/mol
Boiling Point = 314°C
Melting Point = 172°C
Density = 1.74g/mL
Flash Point = 143°C
Moderate eye irritant
Skin sensitizer
Toxic by inhalation
No evidence of phototoxicity


Table 2.
Crops and fungal diseases registered for Captan 80-WP.


Diseases Treated


Scab, Sooty Bloch, Fly Speck, Brook’s Spot, Bitter Rot, Black
Rot, Bull’s Eye Rot


Brown Rot


Brown Rot, Leaf Spot


Brown Rot, Scab


Scab, Sooty Bloch


Black Rot, Brown Rot


Dead Arm, Dawny Mildew, Black Rot


Fruit Rot


Fruit Rot


Cane Spot, Fruit Rot, Leaf Spot, Spur Blight


Fruit Rot, Mummy Berry


Gray Mold Rot, Leaf Spot


Leaf Rot


Anthracnose, Scab


Anthracnose, Septoria, Leaf Spot



experiments were performed in this study. The first one was a batch experiment
which was run for 15 days and the second one was a continuous experiment which
was run with 15 days retention time. A Captan concentration of 144 mg/L in the
wastewater was used in the experiments. This was achieved by mixing 18 g of Captan
dust (14.4 g active material) to 100L of water. The initial ratio of soil:water
in the bioreactors was 1:3. This was achieved by placing 0.75 L of soil and 2.25
L of water in the bioreactors. For the batch experiment the bioreactor was
filled up to the 10 L final volume. For the continuous experiment, the feeding
pump was adjusted to introduce a flow rate of 0.46 mL/minute in order to
achieve 15 days retention time. The mixing motor and the air compressor were
operated. The air flow rate was adjusted to 10 L/min (1v/v/min) by the use of a
meter. Samples were collected from each bioreactor every day and filtered under
vacuum using a coarse filter paper (P8 Grade, FisherScientific, Montreal,
Quebec, Canada) to separate any soil particles. The filterate was then used for
microbial (plate count) and pesticide analyses.

Plate Count

plate count was determined for the initial mixture of soil and water before the
addition of captan and on the samples collected daily from the bioreactors. For
each sample, 5 test tubes were used and filled with 9.9 ml of peptone solution.
A 1:100 dilution was prepared by pipetting 0.1mL of the initial dilution into
the first tube. This was then mixed well and 0.1 mL was placed on an agar lined
Petri dish and spread over the entire surface using a glass wand. Before each
use it was made sure that he wand was clean by using reagent alcohol and
flaming. From the diluted tube, 0.1 mL was transferred to another tube contains
9.9 mL peptone solution to produce a dilution of 1:1000. 0.1 mL of the mixture was
transferred into a new Petri dish after mixing it well. The same procedure was
repeated to achieve different final dilutions (1: 100, 1:1000, 1:10000, 1:10000,
1:100000 and 1:1000000). Three plates for each dilution were performed. All the
inoculated Petri dishes were then placed at 35°C in a temperature control
incubator (Model 2020, Sheldon Manufacturing Inc, Cornelius, Oregon, USA). The
plates were tested after 24h of incubation in order to select the dilution that
produced reasonable countable number of colonies. The total colonies on each Petri
dish were determined with the aid of a plate counter (Model 7-901, Fisher
Scientific, Montreal, Quebec, Canada). The population was then determined by
multiplying the number of colonies by the dilution factor.

Pesticide Analysis

ml of the sample were mixed with 3 mL of the hexane/ether solvent (95:5) in a
test tube. The test tube was then caped and heated at 115 °C
for 30 min followed by a cooling period to a temperature below 0°C. When the
sample was frozen, the liquid solvent was withdrawn from the top. A volume of 2 µL of this sample was
then used for analyses using a gas chromatographer (5890 series II, Hewlett
Packard, California, USA). The initial and final concentrations of captan in
each unit were determined. The chromatograph was calibrated by injecting 1.0 µL
of the extracted captan mixture into the 25 m ´ 0.2 mm
capillary column.1.0 µL of the extracted sample was then injected into the
column. First, the temperature of the column was maintained at 40°C for 3
minutes and then the temperature increased at the rate of 10 °C
per minute until a temperature of 270°C was reached. The column was then
maintained at highest temperature (270 °C) for 5
minutes. The injection port was adjusted to 25°C while the flame ionization
detector was adjusted for 250
°C. Helium was used as a
carrier gas at a flow rate 1.2 mL/min.


Microbial Growth

growth of the mixed microbial population in the batch and continuous bioreactors
is shown in Figure 1. The initial cell number in the soil water mixture in both
bioreactors (batch and continuous) was 30.1 ´ 106
cells/mL. The number of cells first declined with time during the first 24
hours reaching 15.6 ´ 106
cells/mL in the batch bioreactor and 11.1 ´ 106
cells/mL in the continuous bioreactor and then increase gradually in both
reactors. This was as a result of the inhibitory effect of captan (at initial
concentration of 144 mg/L) on some soil microbial species hat tare less
tolerant to the pesticide. The lower value (29% lower) observed in the
continuous bioreactor compared to that of the batch bioreactor was due to the
loss of microbes from that bioreactor with the effluent.

results were reported by several authors for captan. Wainwright and Pugh (1975)
reported a decreasing trend of microbial population (from 16.5 ´ 106
to 14 ´ 106cells)
during first 48 hours after application of 25µg/g captan to field soils. Agnihotri
(1971) achieved a significant decrease (from 2.3 ´ 105
to 0.4 ´ 105cells)
in the actinomycete population in fresh soil by day 7 after application of 125
ppm captan. Martínez-Toledo et al. (1998) studied the effect of different captan
concentrations (2.0-10.0 kg/ ha) on the microbial activities in four
agricultural soils under aerobic conditions and detected significant decreases
in total culturable fungal populations, nitrifying bacteria and nitrogen fixing
bacteria during the first 14 days. Piotrowska-Seget et al. (2008) reported 46%
reduction (from 7.8 ´ 105
to 4.2 ´ 105)
in bacterial population during the first 10 days after application of captan at
a rate of 8.5 mg/g of soil and observed increase (from 4.2 ´ 105 to 7.6 ´
105) in
population after 94 days which was about 97.43% of the original population.
Similar results were also reported for other pesticides such as malathion (Shan
et al., 2009), chlorpyrifos alone and in combination with chlorothalonil
(Xiaoqiang et al., 2008), dichlorvos (Ning et al., 2010),
2-(2-methyl-4-chlorophenoxy) propionic acid (MCPP) (de Lipthay et al., 2003;
Tuxen et al., 2006), phenoxy acids (Chilton et al., 2005), 2,4-D (Broholm et
al., 2001), 2-(2,4-dichlorophenoxy) propionic acid (de Lipthay et al., 2003).

1. Microbial growth

four normal microbial growth phases (lag, exponential growth, stationary growth
and death phases) that usually a in a achieved in batch modes of operation were
detected in the batch bioreactor’s growth curve. The microbial population in
the batch bioreactor started to increase with the increase in time after an
initial lag period reaching maximum value of 113.9 ´ 106
after 120 h from the beginning of the experiment. This maximum value remained
relatively constant till 240 h and then started to decrease. The microbial
population of the continuous bioreactor also increased but at a slower rate
than that of the batch bioreactor due to the continuous loss of microbes from
the bioreactor with the effluent. It reached a steady state condition (when the
rate of microbial growth in the bioreactor was equal to the rate of microbial
loss from the bioreactor with the effluent) after 288 h. The microbial
population in the continuous bioreactor at the steady state was 86.5 ´ 106
cells/mL (75.94% of that in the batch reactor).

lag period and specific growth rate were determined from the batch operation data
according to the procedure described by Ghaly et al. (1989) as shown in Figure
2. The lag period and the specific growth rate were 22 h and 0.096 h-1,
respectively. Radianingtyas et al. (2003) reported 18 h lag
period and 0.014 h-1 specific growth rate while degrading
4-choloroaniline (1 mM) in a batch reactor with a
bacterial consortium comprising of four different species isolated from an
Indonesian agricultural soil. Lappin et al. (1985) reported 18 h
lag period and 0.09 h-1 specific growth rate while degrading mecoprop (1 gm/L) in a batch reactor with a microbial community isolated
from wheat root systems. Rhee et al. (1997) studied pyridine degradation (3 mM)
using denitrifying bacteria isolated from industrial wastewater in a batch bioreactor
and obtained 13 hours lag period and 0.08 h-1 specific growth rate.

lag period and net specific growth rate (µnet) were also determined
for the continuous mode of operation during the initial unsteady-state phase using
the same procedures (Figure 3). The lag period and the net specific growth rate
for the continuous bioreactor were 26 h and 0.045 h-1, respectively.
Krishna and Philip (2009) performed investigation on the
biodegradation of carbofuran at 150 mg/L concentration under continuous mode of
operation and obtained 48 h lag period and 0.3928 d-1 specific
growth rate using carbofuran enriched cultures.

net specific growth rate (µnet) under the continuous mode of
operation is as follows:

= µ-kr                                                                                                                                                (1)


µ    = Specific growth rate (h-1)

= Net specific growth rate (h-1)

kr    = Cell removal rate with effluent (h-1)

results showed that the rate of microbial removal with the effluent (kr)
was 0.051 h-1.


Figure 2. Lag
period and specific growth rate for the batch bioreactor.

3. Lag period and net specific growth rate for the continuous bioreactor.

Pesticide Degradation

pesticide concentrations in the effluent samples taken from the batch and
continuous bioreactors overtime are shown in Figure 4. The pesticide
concentration started to decrease with time under the batch mode of operation
reaching zero after 120 hours from the start. The concentration of captan in
the effluent under the continuous mode of operation also decreased with time
reaching a constant value of 15 mg/L after 288 hours. Thus, a removal
efficiency of 89.6% was achieved after 10 days with the continuous bioreactor
compared to a removal efficiency of 100% after 5 d with the batch bioreactor. Megadi
et al. (2010) achieved 100% degradation of the fungicide captan after 6 days of
operation by the growth of Bacillus
circulans in mineral salt medium (MSM) containing 0.1% captan. Buyanovsky
et al. (1988) achieved 33% degradation efficiency of captan (at initial
concentration of 50 mg/L) after 2 weeks (including lag phase of 2 days) of
incubation with soil bacteria, no further degradation of captan was detected
after the 2 weeks operation. The maximum permissible
value for captan and metabolites in livestock water is set 13 µg/L. The batch
bioreactor used in the study achieved 100% removal of captan, while the
effluent from continuous bioreactor contained 15 mg/L which is not acceptable
for livestock water.

Figure 4. Concentration of captan in the batch and continuous

Biological degradation of pesticides can be defined as the use of
microorganisms to convert those pesticides either in solid or liquid wastes to
a harmless by product. The biological treatment mainly depends on the microbial
activity and aeration. In this study, microbes that naturally exist in soil
increased significantly in number and began to biodegrade pesticide. The microorganisms
used the pesticide as a carbon source for obtaining energy in addition to the synthesis
of new cells according to the following equations:


                          Organic matter + O2                 

+ H2O + other products +Heat  


                          Organic matter + NH4             

microbes                                            (3)

The proposed pathway for the degradation of captan is shown in
Figure 5. The soil microbial population used in this study contained
microorganisms that was able to oxidieze the carbon, chloride, nitrogen and sulphur
in captan (C9H8Cl3NO2S) and convert
them to carbon dioxide (CO2), water (H2O), chloride (Cl),
nitrate (NO3) and sulphate (SO4) and thus obtaining the
energy required for microbial synthesis according to the following equations (Swanner
and Templeton, 2011; Megadi et al., 2010; Munch et al., 1996).


+ H2O + 3HCl + NH3 + H2S+?E                                         (4)

The captan degradation
process takes place in several steps. In the first step, captan is converted
into cis-1,2,3,6-tetrahydro
phthalimide, thiocarbonyl chloride and hydrochloric acid. In the second, step cis-1,2,3,6-tetrahydro pthalimide is
converted into cis-1,2,3,6-tetrahydro
pthalimidic acid and thiocarbonyl chlorides converted into H2S, CO2
and H2O. In the third step, the cis-1,2,3,6-tetrahydro
pthalimidic acid is converted into O-phthalic acid and ammonia. In the forth
step, the O-phthalic acid is converted into protocatechuic acid. In the fifth
step, the protocatechuic acid is converted into 3- carboxy-cis, cis muconic acid which is oxidized to CO2 and H2O.
Nitrifying bacteria is responsible for the conversion of NH3 to NO3
under aerobic conditions while the H2S is converted into SO4
by hydrogen sulphide reducing bacteria.

+ 2O2        

+ H2O + H+ + ?E                                                                              (5)

 H2S + 2O2       

+ 2H+ + ?E                                                   (6)

The biodegradation of organic substrates such as pesticides in a
batch system can be described by the following equation.


Pt =Po e-kt                                                                     



Pt  = Concentration of pesticide at the time t

= Initial concentration of pesticide (mg/L)

k  =  Rate
constant (h-1)

t  =  Time


5. Pathway of fungicide captan degradation under aerobic condition (adopted
from Megadi et al., 2010).

plotting of ln (Pt/P0) versus time (t) yields straight
line with a slope equals k. However, a straight line could not be obtained for
continuous bioreactor when plotting the data for the entire period. The results
(Figure 6) indicated different degradation rates for the lag phase (0.0025 h-1)
and exponential growth phase (0.71 h-1). It is apparent from the
results that the microorganisms were able to use captan as a carbon source for
obtaining energy for maintenance during the lag period. Karpouzas et al. (2005)
reported 25% removal efficiency of cadusafos (12 mg/L initial concentration)
during 30 h lag period of 30 h using Flavobacterium
sp. and Sphingomonas sp. (isolated
from contaminated soil) followed by a gradual decline in bacterial populations (reaching
and 8´106
cells/mL for the Flavobacterium and
the Sphingomonas sp. in 72 h ,
respectively) and resulted in complete degradation of cdusafos by both isolated
bacteria after 78 h. Karpouzas and Walker (2000) reported a 30% degradation of ethoprophos (initial concentration of 100 mg/L) after inoculation of Pseudomonas
putida (isolated
from ethoprophos contaminated soil) with a mineral salts medium supplemented with nitrogen (MSMN) in the
first 33 h and
observed complete degradation after 50 h. In this study,
8.2% (12 mg/L) of the captan in the batch bioreactor was degraded during the
lag period of 22 h and complete degradation was achieved in 120 h.

6. Determination of rate constant k.

The captan half-life observed for the batch
bioreactor in this study was 52 h. Leoni et al. (1992) reported a captan half-life
of 3.6 days in an activated sludge system, Hermanutz et al. (1973) reported captan
half-life of 7 h at 12 ºC and 1 h at 25 ºC in Lake Superior. Ghaly et al. (2007)
reported 25 h in a composting system for the pesticide primiphos-methyle at a
temperature of 50-60ºC. In this study, a captan half life of 52 h was observed
for the batch bioreactor.



The initial cell number (30.1´106 cells/mL) in the soil water mixture was first decreased
as time increased for the first 24 h. The maximum cell number reached 15.6´106 and 11.1´106
cells/mL for the batch and continuous bioreactors, respectively. This was the
result of inhibitory effect of captan on some of the soil microbes that are
less tolerant to the pesticide at initial concentration of 144 mg/L. The
results showed maximum microbial population after 5 and 12 days of incubation from
the start of the experiment in batch and continuous bioreactors, respectively.
Lag period and specific growth rate of 22 h and 0.096 h-1, respectively
were achieved for the batch mode of operation. Captan degradation of 89.6% was obtained
after 10 days for the continuous mode of operation compared to complete removal
(100% degradation efficiency) after 5 days for the batch operation. A half life of 52 h was
observed in the batch bioreactor. This study showed that the batch mode of
operation completely removed captan while the effluent from the continuous
bioreactor had a captan concentration of 12 mg/L which is not acceptable for
livestock drinking water.