Tuğba KILIÇ, Başar KARACA, Arzu ÇÖLERİ CİHAN

Abiyotik yüzeylerde termofilik Anoxybacillus rupiensis DSM 17127T suşunun biyofilm oluşumu ve polistiren yüzeyler üzerindeki biyofilm yapısının giderimi

Bu çalışmanın amacı, Anoxybacillus rupiensis DSM 17127T’nin endüstride kullanılmakta olan abiyotik yüzeyler üzerinde biyofilm oluşumunu incelemek ve polistiren yüzeyler üzerinde oluşan biyofilm yapısının sanitasyon ajanları ile giderimini sağlamaktır. Bakterinin genomik DNA (gDNA)’sı ve biyofilm matriksi yapısında bulunan ekstraselüler DNA (eDNA)’sı spektrofotometrik analiz ve agaroz jel görüntülemesi ile tespit edilmiştir. Polistiren yüzeylerde biyofilm oluşumu ve biyofilm yapısının giderimi kristal viyole bağlanma yöntemi ile belirlenmiştir. Bakterinin ideal planktonik gelişimi için optimum sıcaklık, pH ve tuzluluk istekleri sırasıyla 55 oC, 8.0, % 1 ve ideal biyofilm üretimi için 60 oC, 8.0, % 0 olarak saptanmıştır. gDNA’nın (27.6 kb) molekül ağırlığının, eDNA’dan (20.9 kb) daha büyük olduğu belirlenmiştir. gDNA ve eDNA saflaştırıldıktan sonra DNaz I, RNaz A ve proteinaz K ile muamele edilmiştir. gDNA sadece DNaz I ile tamamen parçalanmıştır. Saflaştırılmış haldeki eDNA ise, üç enzime de direnç göstermiştir. Ancak, olgun biyofilmlerin DNaz I enzimi ile muamelesi sonucunda biyomasta 2 saat içerisinde % 80 oranında azalma gözlenmiştir. Bakterinin polikarbonat, polipropilen, polivinil klorür, paslanmaz çelik, polistiren ve cam yüzeylerinde biyofilm oluşturduğu gözlenmiş olup, en ideal yüzey polikarbonat (5.69 log kob/cm2) olarak belirlenmiştir. Biyofilm giderimi çalışmalarında, protein parçalayıcı sanitasyon ajanlarının polisakkarit parçalayıcı ajanlardan daha fazla etkili olduğu görülmüştür. Sonuç olarak, bakterinin eDNA’sının olgun biyofilm matriksinin bütünlüğü ve sağlamlığı adına önemli bir yapısal rol oynadığı belirlenmiştir. Ayrıca, bakterinin abiyotik yüzeylerde biyofilm oluşturma yeteneğine sahip olduğu görülmüştür. A. rupiensis’in biyofilminin giderimi için süt endüstrisinde kullanımı olan seçilmiş sanitasyon ajanlarının önemli ölçüde etki sağladığı saptanmıştır.

Biofilm formation of the thermophilic Anoxybacillus rupiensis strain DSM 17127T on abiotic surfaces and removal of its biofilm structure on polystyrene surfaces

The aim of this study was to examine the biofilm formation of Anoxybacillus rupiensis DSM 17127T on abiotic surfaces used in the industry and to remove the biofilm structure formed on polystyrene surfaces with sanitation agents. The genomic DNA (gDNA) and the extracellular DNA (eDNA) in the biofilm matrix structure of the bacteria were determined by spectrophotometric analysis and agarose gel imaging. For the biofilm formation on abiotic surfaces and the removal of the biofilm structure formed on polystyrene surfaces, crystal violet binding assay was applied. The optimum temperature, pH, and salinity for the growth and biofilm formation of the bacteria were 55 oC, 8.0, 1 % and, 60 oC, 8.0, 0 %, respectively. The molecular weight of gDNA (27.6 kb) was determined to be larger than eDNA (20.9 kb). gDNA and eDNA were partially purified and treated with DNase I, RNase A and proteinase K. The purified gDNA was completely disrupted only by DNase I., and the purified eDNA was resistant to all three enzymes. As a result of the treatment of biofilm containing eDNA with DNase I, it was observed that the eDNA was sensitive to DNase I and the biofilm mass decreased by 80 % within 2 hours. The bacterium was observed to form biofilms on polycarbonate, polypropylene, polyvinyl chloride, stainless steel, polystyrene and glass surfaces, and the most ideal surface determined as polycarbonate (5.69 log cfu/cm2). In the biofilm removal studies, protein degrading sanitation agents were found to be more effective than polysaccharide degrading agents. In conclusion, it was determined that the eDNA of the bacteria plays an important structural role in the integrity and robustness of the mature biofilm matrix. In addition, it was showed that the bacterium is capable of forming biofilms on abiotic surfaces. Dairy industrial sanitation agents had a significant effect on the removal of the biofilm mass of Anoxybacillus rupiensis.

Keywords:

Abiotic surfaces Anoxybacillus rupiensis, biofilm removal, sanitation agents,

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