Jishan LIU, Shuguang LI, Xiaojing XIE, Hongmei HU, Qiang FU, Yueqiang XIAO, Guanggang QU, Zhiqiang SHEN, Likun CHENG

Optimization of Culture Conditions for High Cell-Density Fermentation of Bovine Escherichia coli

Bovine Escherichia coli infection, which causes major economic losses to the cattle industry each year, can be prevented by administering formalin-inactivated vaccine. However, to enhance the application of this vaccine, the cell density of the formalin-inactivated E. coli should be boosted. This can be achieved by reducing the accumulation of acetate, a primary inhibitory metabolite in E. coli fermentation. To this end, the present study investigated the effect of pH, dissolved oxygen (DO) levels, and feeding methods on bovine E. coli fermentation, and developed two-stage pH and DO control strategies and a combined pH- and DO-mediated feeding strategy for the fermentation. The optimized conditions for Bovine E. coli were pH 7.0 at 0-10 h, 6.5 at 10-24 h; DO 50% at 0-10 h, 30% at 10–24 h; pH and DO feedback feeding at 0-10 h and 10-24 h, respectively. With Bovine E. coli fermentation under the optimized conditions, the acetate accumulation was 1.12 g/L and the cell density was 36.47 (OD600), which were 59.12% lower and 77.29% higher than these with the original conditions (pH 7.0; DO 20%; residual glucose concentration maintained at 2.0 g/L). After analyzing the main nodes of acetate synthesis, it was found that the lower carbon flux enters the Embden-Meyerhof pathway. Under the optimized conditions, the pyruvate flux and acetyl-CoA synthesis were low, and much of the acetyl-CoA participated in the tricarboxylic acid cycle. The extracellular acetate flux was 8.3%, which was 65.13% lower than in the original conditions.

Sığırlarda Escherchia coli’nin Yüksek Hücre Yoğunluğunda Fermentasyonu İçin En Uygun Kültür Koşullarının Sağlanması

Sığırlardaki Escherchia coli enfeksiyonları her yıl sığırcılık sektörünü büyük çaplı maddi zarara uğratmakla birlikte, formalinle etkisizleştirilmiş bakteriden yapılan aşıyla önlenebilmektedir. Ancak, bu aşının etkisini artırmak için formalinde etkisizleştirmiş E. coli hücre yoğunluğunun artırılması gerekmektedir. Bunu elde etmek için E. coli fermentasyonunu baskılayan birincil bir metabolik ürün olan asetatın birikimi azaltılmalıdır. Bu amaçla, mevcut çalışmada pH, çözünmüş oksijen (ÇO) düzeyleri ve beslenme şeklinin sığırlardaki E. coli fermentasyonuna etkileri araştırılmış ve iki aşamalı bir pH ve ÇO kontrolü stratejisi geliştirilerek pH ve ÇO düzeylerini birlikte değerlendiren bir beslenme planı ile fermentasyonun artırılması yoluna gidilmiştir. Sığır E. coli için optimize edimiş koşullar pH için 0-10 saat aralığında 7.0, 10-24 saat aralığında 6.5; ÇO için 0-10 saat aralığında %50, 10-24 saat aralığında %30, pH ve ÇO temelinde besleme için sırasıyla 0-10 saat ve 10-24 saat olarak belirlendi. Optimize koşullar altında sığır E. coli fermentasyonu ile birlikte asetat birikimi 1.12 g/L olup hücre yoğunluğu 36.47 (OD600) olarak tespit edildi. Bu değerler orjinal koşullardan (pH 7.0; ÇO %20; kalıntı glukoz derişimi 2.0 g/L’de tutulmuş) sırasıyla %59.12 daha düşük ve %77.29 daha yüksek bulunmuştur. Asetat sentezinin temel unsurları incelendiğinde, daha az olan karbon akışının EmbdenMeyerhof yolağına kaydığı saptanmıştır. En uygun koşullarda pirüvat akımı ve asetil CoA sentezi düşük iken, asetil CoA’nın büyük çoğunluğu trikarboksilik asit döngüsüne katılmıştır. Hücre dışı asetat akımı %8.3 olup, özgün koşullara göre %63.13 daha düşük çıkmıştır.

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