التوصيف الوراثي للبكتريا المحللة للبايفينيل المعزولة من مصادر بيئية وسريرية في مدينة الموصل

الباحث

رغدة محمد صبحي طه الزبيدي

المشرف

ريان مازن فيصل العبيدي

الجامعة

جامعة الموصل

الكلية / القسم

كلية العلوم / قسم علوم الحياة

الشهادة

ماجستير

السنة

—

لغة الرسالة

انكليزي

الكلمات المفتاحية

Biphenyl ; Extensimonas ; DSMZ ; Growth Curve

This study involved the isolation and molecular identification of biphenyl degrading bacteria isolated from 100 stool samples and 55 soil samples collected from different sources in Nineveh governorate during the period September 2021 to February 2022. For this purpose, biphenyl degraders were isolated from soil samples incubated in DSMZ minimal media containing biphenyl as a sole source for carbon and energy. Results showed that 16.4% of the samples showed positive results by forming turbidity in DSMZ broth medium and forming colonies on DSMZ agar containing biphenyl and not on negative control plates, while 83.6% of the samples did not contain biphenyl degrading organisms. Results also showed that the samples with the highest source of biphenyl degraders was Mishraq sulfur station soil (33.33%) followed by sewage soil (28.57%), on the other hand no biphenyl degraders were isolated from oil refinery soils, agricultural soils and garden soils. Isolates were diagnosed based on their cultural characteristics and 16S rRNA gene sequence, submitted to NCBI and given the initials RM. A total of 9 biphenyl degrading isolates were identified from both Gram positive and Gram negative bacteria. Five isolates were Gram positive that included two Rhodococcus pyridinivorans strains, Rhodococcus rhodochrous, Rhodococcus sp. and Microbacterium barkeri and three isolates were Gram negative that included Extensimonas perlucida, Pseudomonas luteola and Achromobacter sp. Our results introduced for the first time worldwide the ability of E. perlucida to use biphenyl as a sole source for carbon and energy. Throughout analysis of 100 stool sample we did not detect the ability of an Enrerobacteriaceae member to degrade biphenyl. The growth rate of the nine biphenyl degraders understudy was identified by growing the strains individually in DSMZ broth medium and measuring the absorbance OD600 at different time points. Growth rates varied with different strains reflecting the efficiency of their enzymes in degrading biphenyl. The growth rate (doubling time) was 24.79 hours for E. perlucida RM1, 17.05 hours for Rh. pyridinivorans RM2, 17.69 hours for Rh. rhodochrous RM3, 16.80 hours for Rhodococcus spp. RM4, 19.43 for Rhodococcus spp. RMS, 19.65 for Achromobacter sp. RM6, 16.48 hours for M. barker RM7, 16.93 for RA. pyridinovorans RM8 and 16.99 for P. /uteola RM9. Duplication of the strains on DSMZ containing biphenyl as a sole source for carbon and energy provides another proof for the ability of the strains understudy to metabolize biphenyl. Six different primers were used to identify the presence of biphenyl degrading genes in the isolates understudy. The primer B PHD-f3 and BPHD-r1 was used to amplify a 524bp DNA fragment from the Rieske non-heme iron dioxygenase gene expected to be used in the initial step of biphenyl degradation. Our results showed that 5/9 (55.55%) of our biphenyl degrading strains amplified this fragment indicating the presence of this gene in these biphenyl degrading strains. The second set of primers (Rieske_f and ARHD2R) was used to target the 382bp fragment that represents the Rieske cofactor of the biphenyl dioxygenase. Throughout nine isolates only E. perlucida amplified the 382bp target providing a second proof that the bipheny! dioxygenase used by this strain is a Rieske type dioxygenase. The third set of primers (C230-f and C230-r ) was used for amplifying the gene encoding the ring cleavage enzyme (meta cleavage dioxygenase) used to convert 2,3-dihydroxybiphenyl to the yellow intermediate, HOPDA. This primer was capable of amplifying a 469bp fragment from this gene in 2/9 (22.22%) indicating the presence of this gene as a component of the biphenyl degradation pathway genes. The fourth set of primers (DOalpha-2 and DOalpha-3) was designed to target angular dioxygenase genes involved in the first step of aromatic hydrocarbon degradation other than biphenyl. Interestingly, our results showed that E. perlucida and Pseudomonas luteola RM9 contains an angular dioxygenase gene that is probably involved in degrading other aromatic compounds besides biphenyl. تأييد المشرف ا.م.د. سيناء عبد الله علي مسؤول الدراسات العليا — كلية العلوم

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