Biyolojik mücadele etmeni olarak Bacillus izolatlarının ekonomik bir prosesle üretim optimizasyonu
Küçük Resim Yok
Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Toprak dinamik bir yaşam alanıdır ve sadece tarım ve gıda güvenliğinde
kritik bir kaynak olmakla kalmayıp aynı zamanda tüm yaşam sürecinin
devamlılığını sağlamaktadır. Bitki sağlığını olumsuz etkileyen patojen
mikroorganizmalar, dünya çapında sürdürülebilir tarım için büyük ve kronik bir
tehdittir. Tarımsal üretimlerde verimi artırmak, patojenleri, zararlıları ve yabani
otları öldürmek için kullanılan tarım kimyasallarının ekosistem üzerinde büyük
zararlı etkileri vardır. Bu kimyasalların yan etkileriyle ilgili mevcut kamu
endişeleri nedeniyle, bitkiler ve rizosferdeki mikrobiyal popülasyon arasındaki
işbirliğinin anlaşılması önem kazanmıştır. Bu nedenle, dünya çapında kabul gören
biyolojik ajanların geliştirilmesi ihtiyacı doğmuştur. Bitki büyümesini teşvik eden
rizobakteri kullanımı bu sorunu çözmek için iyi bir alternatiftir. Çevre dostu
sürdürülebilir tarımın geliştirilmesi için toprak verimliliğinin artışında, bitki
büyümesinin teşvikinde ve fitopatojenin baskılanmasında önemli rol
oynamaktadırlar.
Bu tez çalışmasında temel amaç, yerli ve özgün Bacillus izolatlarının
fitopatojenik funguslara karşı antifungal etkilerinin belirlenmesi ve en etkili izolat
kullanılarak maksimum canlı hücre sayısı elde edecek şekilde ekonomik bir
üretim ortamının ve üretim parametrelerinin optimize edilmesidir. Bu amaçla,
1574 adet Bacillus izolatı Botrytis cinerea, Fusarium solani ve Rhizoctonia solani
gibi fitopatojenik funguslara karşı antifungal etki bakımından in vitro koşullarda
incelenmiştir. Her üç fitopatojene karşı da imhibisyon zonu oluşturduğu belirlenen
118 adet Bacillus izolatı için yüzde büyüme inhibisyon değerleri hesaplanmıştır.
İncelenen izolatların gösterdiği maksimum yüzde büyüme inhibisyon değerlerinin B.cinerea’ya karşı %65,6, F.solani’ye karşı %42,2 ve R.solani’ye karşı ise %55,6
olduğu belirlenmiştir. Hemolitik aktiviteleri de incelenen izolatlardan elde edilen
antifungal lipopeptid ekstraktları için ise, bu değerlerin B.cinerea’ya karşı %60,0,
F.solani’ye karşı %36,4 ve R.solani’ye karşı ise %37,9 olduğu belirlenmiştir.
İncelenen 77 Bacillus izolatından elde edilen proteaz, kitinaz ve kitosanaz
için spesifik enzim aktivite değerleri sırasıyla 9.7-6652 U/mg, 0.22-46.64 U/mg
ve 8,02-22.17 U/mg arasında belirlenmiştir. Ayrıca, bu izolatlardan 11 tanesinin
HCN üreticisi olduğu belirlenirken 70 tanesinin ise siderofor üreticisi olduğu
belirlenmiştir. Siderofor üreticisi izolatlar için siderofor indeks değerinin 1,22-
3,11 arasında değiştiği gösterilmiştir. Koagülaz aktivitesi de incelenen izolatlar
arasından iki tanesinin koagülaz pozitif olduğu belirlenmiştir. Biyokontrol
mekanizmaları incelenerek biyolojik kontrol ajanı olarak kullanılabilecek
antifungal etkisi en yüksek olan 19 izolat seçilmiş ve bu izolatlar için moleküler
tanımlama ile tür tayini yapılmıştır.
Kitlesel üretim için uygun olduğu ve yüksek lipopeptid antibiyotik ürettiği
belirlenen Bacillus velezensis 4-Ka-22 suşu üretim optimizasyonu amacıyla
seçilmiştir. Bu izolat için melas, soya unu, CaCl2 ve gliserol içeren üretim ortamı
ve sıcaklık, çalkalayıcı hızı (rpm), pH ve inokülasyon oranı gibi üretim
parametreleri klasik yöntemler ve RSM kullanılarak optimize edilmiştir. Optimize
edilen üretim ortamı %0,1 melas, %1 soya unu, %0,5 CaCl2 ve %1 gliserol
içermektedir. Üretim sıcaklığı olarak 30°C belirlenmiştir. Ayrıca, üretim
parametreleri için RSM ile gerçekleştirilen optimizasyon sonucunda çalkalayıcı
hızı 195 rpm, pH 5,0 ve inokülasyon oranı ise %4,3 olarak belirlenmiştir.
Optimizasyon sonucunda Bacillus velezensis 4-Ka-22 suşu için elde edilen canlı
hücre sayısı 13,4 kat arttırılmıştır ve üretim ortamı bileşenlerinin maliyeti ise 467
kat azaltılmıştır.
Soil is a dynamic habitat and is not only a critical resource in agriculture and food security, but also ensures the continuity of the entire life process. Pathogenic microorganisms, which adversely affect plant health, are a major and chronic threat to sustainable agriculture worldwide. Agrochemicals used to increase productivity in agricultural production and to kill pathogens, pests and weeds have great harmful effects on the ecosystem. Due to current public concerns about the side effects of these chemicals, understanding the cooperation between plants and the microbial population in the rhizosphere has become important. Therefore, the need to develop biological agents that are accepted worldwide has arisen. The use of rhizobacteria that promote plant growth is a good alternative to solve this problem. They play an important role in increasing soil fertility, promoting plant growth and suppressing phytopathogen for the development of environmentally friendly sustainable agriculture. The main purpose of this thesis study is to determine the antifungal effects of native and original Bacillus isolates against phytopathogenic fungi and to optimize the production parameters and an economical production medium to obtain maximum viable cell count by using the most effective isolate. For this purpose, 1574 Bacillus isolates were investigated in vitro for their antifungal effects against phytopathogenic fungi such as Botrytis cinerea, Fusarium solani and Rhizoctonia solani. Percent growth inhibition values were calculated for 118 Bacillus isolates, which were determined to form inhibition zones against all three phytopathogens. It was determined that the maximum percent growth inhibition values of the examined isolates were 65.6% against B.cinerea, 42.2% against F.solani and 55.6% against R.solani. For antifungal lipopeptide extracts obtained from isolates whose hemolytic activities were also examined, these values were determined as 60.0% against B.cinerea, 36.4% against F.solani and 37.9% against R.solani. The specific enzyme activity values for protease, chitinase and chitosanase which obtained from the examined 77 Bacillus isolates were determined respectively between 9.7-6652 U/mg, 0.22-46.64 U/mg and 8, 02-22.17 U/mg. In addition, 11 of these isolates were determined to be HCN producers, while 70 of them were determined to be siderophore producers. It has been shown that the siderophore index value for siderophore producing isolates varies between 1.22 and 3.11. Among the isolates whose coagulase activity was also examined, two of them were found to be coagulase positive. By examining the biocontrol mechanisms, 19 isolates were selected among those with the highest antifungal effect which can ne used as a biological control agent and for these isolates species determination was made by molecular identification. Bacillus velezensis 4-Ka 22 strain, which was determined to be suitable for mass production and to produce high lipopeptide antibiotics, was selected for production optimization. For this isolate, the production medium containing molasses, soy flour, CaCl2 and glycerol, and production parameters such as temperature, shaker speed (rpm), pH and inoculation rate were optimized using conventional methods and RSM. The optimized production medium contains 0.1% molasses, 1% soy flour, 0.5% CaCl2 and 1% glycerol. The production temperature was determined as 30°C. In addition, as a result of the optimization performed with RSM for the production parameters, the shaker speed was determined as 195 rpm, pH 5.0 and the inoculation rate was 4.3%. As a result of the optimization, the number of viable cells obtained for the Bacillus velezensis 4- Ka-22 strain was increased by 13.4 times and the cost of the production media components was reduced by 467 times.
Soil is a dynamic habitat and is not only a critical resource in agriculture and food security, but also ensures the continuity of the entire life process. Pathogenic microorganisms, which adversely affect plant health, are a major and chronic threat to sustainable agriculture worldwide. Agrochemicals used to increase productivity in agricultural production and to kill pathogens, pests and weeds have great harmful effects on the ecosystem. Due to current public concerns about the side effects of these chemicals, understanding the cooperation between plants and the microbial population in the rhizosphere has become important. Therefore, the need to develop biological agents that are accepted worldwide has arisen. The use of rhizobacteria that promote plant growth is a good alternative to solve this problem. They play an important role in increasing soil fertility, promoting plant growth and suppressing phytopathogen for the development of environmentally friendly sustainable agriculture. The main purpose of this thesis study is to determine the antifungal effects of native and original Bacillus isolates against phytopathogenic fungi and to optimize the production parameters and an economical production medium to obtain maximum viable cell count by using the most effective isolate. For this purpose, 1574 Bacillus isolates were investigated in vitro for their antifungal effects against phytopathogenic fungi such as Botrytis cinerea, Fusarium solani and Rhizoctonia solani. Percent growth inhibition values were calculated for 118 Bacillus isolates, which were determined to form inhibition zones against all three phytopathogens. It was determined that the maximum percent growth inhibition values of the examined isolates were 65.6% against B.cinerea, 42.2% against F.solani and 55.6% against R.solani. For antifungal lipopeptide extracts obtained from isolates whose hemolytic activities were also examined, these values were determined as 60.0% against B.cinerea, 36.4% against F.solani and 37.9% against R.solani. The specific enzyme activity values for protease, chitinase and chitosanase which obtained from the examined 77 Bacillus isolates were determined respectively between 9.7-6652 U/mg, 0.22-46.64 U/mg and 8, 02-22.17 U/mg. In addition, 11 of these isolates were determined to be HCN producers, while 70 of them were determined to be siderophore producers. It has been shown that the siderophore index value for siderophore producing isolates varies between 1.22 and 3.11. Among the isolates whose coagulase activity was also examined, two of them were found to be coagulase positive. By examining the biocontrol mechanisms, 19 isolates were selected among those with the highest antifungal effect which can ne used as a biological control agent and for these isolates species determination was made by molecular identification. Bacillus velezensis 4-Ka 22 strain, which was determined to be suitable for mass production and to produce high lipopeptide antibiotics, was selected for production optimization. For this isolate, the production medium containing molasses, soy flour, CaCl2 and glycerol, and production parameters such as temperature, shaker speed (rpm), pH and inoculation rate were optimized using conventional methods and RSM. The optimized production medium contains 0.1% molasses, 1% soy flour, 0.5% CaCl2 and 1% glycerol. The production temperature was determined as 30°C. In addition, as a result of the optimization performed with RSM for the production parameters, the shaker speed was determined as 195 rpm, pH 5.0 and the inoculation rate was 4.3%. As a result of the optimization, the number of viable cells obtained for the Bacillus velezensis 4- Ka-22 strain was increased by 13.4 times and the cost of the production media components was reduced by 467 times.
Açıklama
Anahtar Kelimeler
Bacillus Velezensis, Biyokontrol Etmeni, Antifungal Etki, Lipopeptid Antibiyotikler, Üretim Optimizasyonu, RSM, Bacillus Velezensis, Biocontrol Agent, Antifungal Effect, Lipopeptid Antibiotics, Production Optimization, RSM
