Optimizing the Effect of pH and Temperature on Atrazine Degradation by Bacillus safensis strain BUK_BCH_BTE6 an Efficient Atrazine Tolerating Bacteria from an Agricultural Soil in Kura Local Government Area of Kano State, Nigeria

The widespread and long term use of atrazine results in high residue levels of atrazine in soil, which further causes water contamination, it is considered as an endocrine disruptor and is potentially carcinogenic. Microbial degradation of herbicide represents a time cost effective way of eco-restoration. This research was aimed at isolating and characterizing bacteria capable of degrading and utilizing atrazine as a sole carbon source. An enrichment method was used to isolate the bacteria on mineral salt media (MSM) following serial dilution. The isolate was identified morphologically, biochemically and molecularly as Bacillus safensis strain BUK_BCH_BTE6 based on 16S rRNA gene sequence and molecular phylogenetic analysis. The effect of pH and temperature on the degradation of atrazine was studied in MSM medium supplemented with atrazine as sole carbon source. Growth and degradation of atrazine in this isolate was optimal at pH 7.5 and temperature of 35 °C. Bacillus safensis is highly efficient in atrazine degradation with an optimum range of pH and temperature. Bacillus safensis could be a suitable candidate for bioremediation of atrazine polluted sites.


Introduction
Worldwide, several anthropogenic activities have resulted in high increase of environmental pollution, and its effect has affect almost all components of the ecosystem (Izabel et al., 2020). Soil, as an important constituent of the terrestrial ecosystem is exposed to pollution from various sources, including industrialization and various agricultural practices. Different forms of anthropogenic pollutant entering the soil causing a very serious impact and risk to human health and terrestrial ecosystem (Sayara and Sánchez, 2020). High growth in demand and use of agricultural and industrial products have lead to rapid increases in polluted soils and waters (Zhao et al., 2018).

Atrazine
(2-chloro-4-ethylamino-6isopropylamino-1, 3, 5-triazine) is a s-triazine herbicide with a molecular formula C8H14ClN5 (He et al., 2019). It is a nonpolar, nonvolatile and low soluble compound (Olu-arotiowa et al., 2019). It is powdery white substance that is unstable at high temperature. It has a melting point of 173 -175 °C, and a boiling point of 200 °C. it solubility in water is 33 mgLˉ¹ at 20 °C, it is very soluble in organic solvents (Ariole and Abubakar, 2015). Atrazine is usually detected in the earth surface water and groundwater system because of it long half-life that ranges from 13 -261 days (Li et al., 2019). Atrazine can restrain and remove broadleaf weeds and some grass weeds that affect crop growth, and also inhibit some perennial weeds (He et al., 2019). It is also applied to soil as herbicide on crops such as sorghum, maize, sugarcane, ornamental plants and also applied to forests as well. It's considered among the top used herbicide in the world due to its low cost and effectiveness (Shamsedini, 2015). Atrazine is considered as a precedence pollutant that is toxic and carcinogenic. It is toxic when inhale, taken orally or excess exposure with the skin. likewise many studies on it toxicity reported atrazine to exhibit long term effect on reproductive and endocrine system, epidemiological effect to decrease sperm counts in men and is assume to be human carcinogen that lead to breast and ovarian cancer (Oluarotiowa et al., 2019).
Previous research reported various bacteria capable of atrazine biodegradation. isolation of microorganisms with high atrazine tolerance and degradation is critically needed to reduce it adverse effects on human health and ecosystem (Shiri, 2016). This paper reports the efficiency of Bacillus safensis strain BUK_BCH_BTE6 in the degradation of atrazine in MSM media.

Samples Collection
Soil profiles of 1 -30 cm under the surface were sampled using a sterile scoop after removal of the top layer of the soil up to 1 -5 cm. Three soil samples were collected from agricultural soil in Kura Local Government Area of Kano state located between latitudes 1003´ and 120 37´ North and longitudes 703´ and 90 5´ East of the Greenwich meridian (Ayodele, 2016). All samples were transferred to sterile polyethene bags, transported to the Microbiology laboratory in Bayero University Kano for analyses. The samples were dried, homogenized and passed through a 2 mm sieve. Atrazine used was of analytical grade with 90% purity.
A total of 10 g of each sample was initially inoculated and homogenized in 250 ml Erlenmeyer flasks containing 100 ml of mineral salt media and was supplemented with 10 ml of atrazine as sole carbon source at a final concentration of 100 mg L -1 . The flasks were then incubated under orbital agitation (150 rpm) at 37 °C. After 7 days, a 10 ml aliquot of each culture was transferred to new MSM broth (100 mL) with same atrazine volume.
Subsequently, each culture was serially diluted from 10 -1 to 10 -6 in sterile distilled water and proper dilutions 10 -6 was plated on MSM agar, supplemented with 0.1 mL of atrazine (100 mgL -1 ). The Petri dish was then incubated at 37 °C for 48 hours (Wang et al., 2015).

Effect of Initial pH on Atrazine Degradation
The effect of pH ranges: 5.5, 6.5, 7.0, 7.5, 8.0, and 8.5 was assessed. Mineral salt medium (50 ml) was introduced into conical flask, 100 µL of bacterial culture was used and supplemented with 100 µL of atrazine with initial concentration of 100 mgL -1 and incubated at 37 °C for 120 hours. Bacterial aliquot (1 ml) was taken after every 24 hours to measure the OD600 for bacterial growth (Mansee et al., 2017).

Effect of Temperature on Atrazine Degradation
The effect of temperature ranges was assessed. 50 ml of Mineral salt medium was introduced into conical flask, 100 µL of bacterial culture was used and supplemented with 100 µL of atrazine with initial concentration of 100 mgL -1 and incubated at different temperature ranges: 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C for 120 hours. Bacterial aliquot (1 ml) was taken after every 24 hours to measure the OD600 for bacterial growth (Ariole and Abubakar, 2015).

Isolation, Identification and Screening of Atrazine-Degrading Bacteria
After inoculation of the culture with MSM agar and incubating at 37 °C for 48 hours and further sub culturing for 24 hours at 37 °C resulted in the appearance of white, round, slimy shape colonies with raised elevation and rough edge which look like fried egg.
Based on Morphological identification (Table 1), it was found that after visualization under light microscope, the isolates were all gram positive and rod shape with one cocci. A total of six isolates were isolated from the atrazine contaminated soil samples, five were able to partially tolerate the varying concentrations (500 mgL -1 , 1000 mgL -1 and 1500 mgL -1 ) of atrazine (Table 2). Only one isolate tolerates all the concentrations of atrazine after 120 hours of incubation and therefore was used for the main study. The bacterium was identified to it generic level according to it characteristics, that may include morphological and physiological.

Keys: + =Positive, =Negative
Molecular identification of the isolate was presented in Figure 1. It was found that after subjecting the PCR product to sequencing using cycle sequencing kit genetic analyzer from both forward and reverse directions. The 16S rRNA gene sequences of the bacterium obtained was compared with GenBank database using Blast Server at NCBI.  Characterization of Atrazine-degrading Bacteria using One Factor at a Time (OFAT) Several factors arising from the environment are known to affect the growth of microorganism. A triplicate experimental run was conducted using one factor at a time (OFAT) to study the effect of pH and Temperature for maximum atrazine growth using the bacterial strain.
Effect of initial pH on the growth of atrazinedegrading bacteria The effect of various pH on the growth of atrazine-degrading Bacillus safensis on MSM media was presented in figure 2. It was found that growth of this bacterium tends to be optimum at pH 7.5 following 48 hours' incubation at 37 °C. A significant (p<0.05) decrease in growth was observed at pH higher than 7.5.
Effect of temperature on the growth of atrazinedegrading bacteria The effect of various temperature on the growth of atrazine-degrading Bacillus safensis strain BUKˍBCHˍBTE6 on MSM media was presented in figure 3. It was found that growth of this bacterium tend to be optimum at 35 °C following 48 hours' incubation. A significant (p<0.05) decrease in growth was observed at temperature higher than 40 °C. Statistical analysis showed a significant difference (p<0.05) between the temperature of 25 °C, 30 °C, 35 °C, 40 °C, 45 °C and 50 °C.

Discussion
There is a significant diversity in the genera of bacteria capable of degrading atrazine (Li et al., 2019). Based on this research, a bacterium with potential of atrazine degradation was isolated from agricultural soil and identified as Bacillus safensis strain BUKˍBCHˍBTE6 according to 16S rRNA gene sequencing and molecular phylogeny. The bacterial sequence was deposited in the GenBank with the accession number OK180490.1 pH is among the most important parameters influencing the effectiveness of many chemical and biological activities (Shamsedini et al., 2016).
The changes in values of pH affect both the structure and activity of protein, this also affects the activity of cellular enzymes. Drastic change in pH also damages the cell membrane and membrane proteins, which in turn debilitate permeability barrier of cell. The pH of the environment of the organism is frequently altered by metabolic activity of organism itself or by other activities in nature. This is implied when culture media are incorporated with buffers that can resist the change in pH (Cho et al., 2016). The effect of various pH on the growth of atrazinedegrading Bacillus safensis on MSM media was presented in Figure 2. It was found that growth of this bacterium tends to be optimum at pH 7.5 following 48 hours' incubation at 37 °C. A significant (p<0.05) decrease in growth was observed at pH higher than 7.5. Statistical analysis showed a significant difference (p<0.05) between the pH values of 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0.
This rate is consistent with findings from similar studies Abigail et al (2013) Khatoon and Rai (2020). This implies that pH plays a significant role in microbial metabolism and atrazine degradation is pH dependent (Andleeb et al., 2016).
All life processes leading to growth are catalyzed by the enzymes present in the cell. The activity of enzymes is influenced by temperature and therefore temperature is able to influence growth (Charpe et al., 2019). The effect of various temperature on the growth of atrazine-degrading Bacillus safensis on MSM media was presented in Figure 3. It was found that growth of this bacterium tend to be optimum at 37  (2017) and Zhao et al (2018). Growth and degradation abilities were both inhibited when the temperature was higher than 40 ºC or lower than 35 ºC. This result indicate that the bacterium is mesophilic and that temperature have significant effect in bacterial physiological process and biodegradation of atrazine herbicide. The results also revealed that the optimal temperature of 35 °C for atrazine degradation is close to the optimal growth temperature for Bacillus sp., which is 37 °C, this result is in agreement with that of Omotayo et al (2016). Results of this research prove the statement that the rate of microbial and chemical degradation are influenced by higher temperature and the rate at which pesticides dissipates is significantly faster under tropical conditions. As such, the overall degradation in tropical regions might be favorable than in temperate regions (Omotayo et al., 2016).

Conclusion
A bacterium with the ability to grow on and tolerate up to 1500 mgL -1 of atrazine herbicide (as sole carbon source) was isolated from agricultural soil in Kura Local Government Area of Kano State, Nigeria. Morphological and biochemical identifications revealed the isolate as gram positive, rod shaped, which was identified as Bacillus safensis strain BUKˍBCHˍBTE6 based on 16S rRNA gene sequencing and subsequent molecular phylogenetic analysis. Bacillus safensis has an optimum pH range of 7.5 and temperature of 35 °C. As such Bacillus safensis have great potential of atrazine-degradation and can be used as a suitable candidate for bioremediation of atrazine polluted sites.