Isolation, characterization and fingerprinting of some chlorpyrifos- degrading bacterial strains isolated from Egyptian pesticides-polluted soils

1 Faculty of Biotechnology, Misr University for Science and Technology, Egypt. 2 College of Medicine, Biotechnology and Genetic Engineering Unit, Taif University, Taif, Kingdom Saudi Arabia. 3 Department of Biological Sciences, Faculty of Science, King Abdul-Aziz University, Jeddah, Saudi Arabia. 4 Department of Biotechnology, Faculty of Science, Taif University, Taif (El-Khorma), Kingdom Saudi Arabia.


INTRODUCTION
Recently the use of microbes for effective detoxify, degrades and removal of toxic chlorinated compounds from contaminated soils has emerged as an efficient and cheap biotechnological approach to clean up polluted environments (Strong and Burgess, 2008). Chlorpyrifos (CP) is one of the most widely used organophosphorous insecticide. Chlorpyrifos-oxon and 3,5,6-trichloro-2pyridinol (TCP) are the two potent transformation products of chlorpyrifos (Bhagobaty et al., 2007). Unlike *Corresponding author. E-mail: nabilfaris151@yahoo.com or nabilfaris@hotmail.com. Tel: 00966 509891624. other organophosphorous compounds, CP has been reported to be resistant to enhanced degradation since its first use in 1965. Although CP has been reported to be degraded co-metabolically by bacteria in liquid media, several attempts to isolate CP-degrading bacteria from agricultural soil have not been successful (Mallick et al., 1999;Racke et al., 1990). Singh et al. (2004) isolated the first CP-degrading bacterium, it was Enterobacter B-14, which hydrolyzed CP to diethylthiophosphate DETP and TCP, and utilized DETP for growth and energy. Two recently isolated CP-degrading bacteria, Stenotrophomonas sp. and Sphingomonas sp., could also utilize CP as the sole source of carbon and phosphorous, but they did not degrade TCP (Yang et al., 2006;Li et al., 2007). DNA-based analyses can contribute significantly to the characterization of bacteria that have been successfully isolated from the polluted environments. RAPD-PCR approach has been shown to be useful in fingerprinting and distinguish between microbial strains within a species (Nowrouzian et al., 2001). An excellent target for bacterial identification and phylogenetic characterization is 16S rRNA gene, the 16S rRNA gene is universally distributed and highly conserved (Woese, 1987). Due to its conserved nature and ease of manipulation, it has been extensively used to establish accurate identification of bacterial isolates. Cleavage of PCR-generated 16S rRNA gene amplicons by a restriction enzyme(s) (RE) results in differentiation by 16S rRNA PCR-RFLP technique. This procedure has been used extensively as a method for bacterial species identification (Wilson et al., 1995;Marshall et al., 1999;Conville et al., 2000;Steinhauserova et al., 2001;Bayoumi et al., 2010). The present study aims to isolate, characterize and fingerprinting of chlorpyrifos degrading bacteria from Egyptian pesticides-polluted soils as well as obtain specific molecular markers of the most potent chlorpyrifos degrading bacterial isolates. In addition examine the effects of various environmental factors which affect the bacterial biodegradation potentiality of chlorpyrifos.

Study site
Four Chlorpyrifos polluted soil samples were collected from various agricultural fields belong to Cairo and Giza Egyptian governorates.

Isolation of cp-degrading bacteria by enrichment and screening
One gram of each soil sample was added to 99 ml MS medium supplemented with yeast extract and glycerol containing 0.1 ml CP in 250 ml flasks. The suspension was incubated at 30°C with shaking at 100 rpm for 10 days; 10 ml of the culture broth was transferred after 10 days to 90 ml of the fresh media. After the fourth enrichment transfer to fresh medium containing CP as sole source of carbon and energy, the media was supplemented with 1.5% agar and incubated at 30°C for 7 days. Bacterial isolates grown on chlorpyrifos-containing agar were subjected to morphological, cultural and biochemical studies by the aid of Berge's Manual of Determinative Bacteriology (Holt et al., 1994). All bacterial isolates were screened based on the formation of degrading haloes around the bacterial colonies and potential isolates were obtained and tested for their potential degrading ability of CP.

Genomic DNA extraction
Genomic DNA was extracted from five studied isolates using Easy Quick DNA extraction kit (Genomix) following the manufacturer's instructions.

16S RRNA PCR-RFLP analysis
The amplification of 16s rRNA gene was performed with universal primer 968F AAC GCGAAGAACCCTAC and 1401R GCGTGTGTACAAGACCC. The reaction mixture was (10 Pmol. of each primer, 50 ng of DNA template and 12.5 µl of 2x superhot PCR Master Mix). The PCR program was as follow; 1 cycle at 95°C, 5 min; 35 additional cycles consisting of 95°C 1 min, 63°C 1 min, and 72°C 1 min and 72°C 10 min as post PCR reaction time. The amplified DNA fragments were digested with two restriction enzymes (EcoRI and. AluI) separately as described by the manufacturer (Jena Bioscience, Germany). The digested DNA fragments were separated on 2.5% agarose gel electrophoresis with DNA ladder standard 100 bp (Jena Bioscience, Germany), stained with 0.5 µg/ml ethidium bromide, visualized on a UV Transilluminator, photographed by Gel Doc. System and analyzed with software data analysis for Bio-Rad Model 620 USA.

Statistical analysis
The presence / absence RAPD and 16S rRNA PCR-RFLP data were analyzed using the SPSS-PC programs of (Nie et al., 1975). Pair-wise comparisons between strains were used to calculate the genetic similarity values (F) derived from Dice similarity coefficient.

RESULTS
Five different bacterial isolates were selected on the basis of production of halo zone around the bacterial colonies and growth in high concentrations of CP (100-300 mg /L). Morphological, cultural and biochemical parameters were determined and illustrated in the Table  2. Degrading bacterial isolates were characterized and identified as Pseudomonas stuzeri (B-CP5), Enterobacter aerogenes (B-CP6), Pseudomonas pseudoalcaligenes (B-CP7), Pseudomonas maltophila (B-CP8) and Pseudomonas vesicularis (B-CP9), by consulting Bergey's Manual of Systematic Bacteriology (Krieg and Holt, 1984) and Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). Pseudomonas stuzeri (B-CP5) was selected as the most potent CP-utilizing bacterial isolate. Table 3 showed that, when the Pseudomonas stutzeri (B-CP5) was grown on MS medium supplemented with 0.3 ml/L Chlorpyrifos as sole source of carbon and energy proved to be capable of grown with 7 days as optimum incubation period, preferred Chlorpyrifos concentration 0.1-0.35 ml/L, maximum inoculum size (0.5 ml), optimum temperature (30°C), optimum pH (7), favorite sugar (fructose) and preferred nitrogen source (ammonium nitrate) under shaking rotation of (100 rpm). Below and above optimal incubation period, Chlorpyrifos concentrations, inoculum size, temperature, pH, the phenolic compounds production decreased gradually. Fructose only was induced in biodegradation of Chlorpyrifos whereas all tested carbon sources failed to induce the production of phenolic compounds. Ammonium nitrate was exhibited the most preferd nitrogen source whereas other tested nitrogen sources failed in biodegradation of Chlorpyrifos.

RAPD and 16S rRNA PCR-RFLP
RAPD and 16S rRNA PCR-RFLP techniques were used to conduct the genetic fingerprinting, construct the genetic relationship and determine genetic distance between the studied isolates and identify specific molecular markers for most potent isolate. An informative profile was obtained (Figures 1 and 2). The six used primers produced multiple band profiles with a variable number  and molecular weights of amplified DNA fragments (Table 1). Different polymorphic and monomorphic markers were obtained across RAPD profiles (Table 4 and 6). As regards genetic relationships, data presented in (Table 5) showed that, the highest genetic similarity was between CP8 and CP7& (66%), while the genetic similarity between CP8 and CP6 was the lowest (37%). All isolates gave the same pattern after digestion with two  restriction enzymes (EcoRI and AluI) separately except CP6 isolate (Figure 2).Two monomorphic bands were obtained (200 and 500 bp) among five bacterial isolates after digestion. Two specific bands for CP6 were identified (420 and 130 bp) with EcoRI and ALUI respectively ( Figure 2).

DISCUSSION
Obtained molecular genetic data documented that the studied isolates belongs to two different genera as showed in Figure 3. The dendrogram showed phylogenetic tree which divided into two main groups. The first main group consisted of two subgroups. The first subgroup contains CP7, CP8 and CP9 isolates. The closest genetic distance was found between CP7and CP8 isolates, which were first clustered together and then with CP9 isolate. The second subgroup of the first main group includes CP5 isolate. On the other hand, the second group includes CP6 isolate. These two genera have focused on its wide range of diverse degrading capabilities and potential application in bioremediation. This is might be due to the great genetic diversity among these two genera which confirmed by different pattern of amplification and restriction enzyme digestion based on RAPD and PCR-RFLP. Chlorpyrifos has been reported to be degraded co-metabolically by bacteria, which needs extra carbon sources (Richins et al., 1997;Mallick et al., 1999). (Singh et al., 2004) reported isolation of   Enterobacter sp. B14, which can use CP for the supply of carbon and phosphorous sources, which it stopped degrading CP in the presence of other carbon sources. However, strain Pseudomonas stuzeri (B-CP5) shows a more rapid degradation in the presence of an additional fructose as carbon source. This indicates that CP could also be degraded co-metabolically by Pseudomonas stutzeri (B-CP5), which might signify the environmental adaptation of this bacterium. Pseudomonas stutzeri (B-CP5) was distinguished and characterized by three random primers (B3, C3 and B5) among six used primers. Primer B3 generate two polymorphic bands (550 and 680 bp), C3 obtain two polymorphic bands (280 and 680 bp) while B5 release one polymorphic band (600 bp).These polymorphic bands considered as specific markers for Pseudomonas stutzeri with the three mentioned primers Table 4. On the other hand CP6 belongs to other genera so it was generate 26 polymorphic bands among six used random primers and two polymorphic bands (420 and 130 bp) with EcoRI and AluI, respectively. These polymorphic bands considered as specific markers for Enterobacter aerogenes. Chlorpyrifos has been reported to be degraded in liquid media by Flavobacterium sp. (Sethunathan et al., 1973), Pseudomonas diminuta (Serdar et al., 1982) and Arthrobacter sp. (Mallick et al., 1999), which were initially isolated to degrade other organophosphate compounds. However, these microorganisms do not utilize CP as a source of carbon but as a co-metabolite (Singh et al., 2003;Yang et al., 2006). Recently a Stenotrophomonas sp. has been isolated, which is capable of degrading both CP and TCP (Yang et al., 2006;Lakshmi et al., 2008) In conclusion, among the five studied isolates Pseudomonas stuzeri (B-CP5) was the most potent isolate to degrade the Chlorpyrifos. The results of this study demonstrate the usefulness of the RAPD-PCR analysis for detecting DNA polymorphism in bacterial isolates and establishing the relationships among different isolates. The majority of random primers used gave distinctly reproducible patterns in the entire isolates studied. However, primers are varied in the extent of information. Primers B5 and A2 revealed highly polymorphic patterns. Whereas, less polymorphic products were generated by C3 primer. Some DNA fragments were apparently similar in size among the studied isolates, whereas others were unique to a particular isolate. Therefore, different polymorphic and monomorphic bands were given and some of monomorphic considered as specific markers for identifying and tracking these bacterial isolates.