极端嗜酸硫杆菌高效筛选、高密度发酵及保藏方法的研究

The study on efficient screening, high-density fermentation and preservation methods for extremely Acidithiobacillus sp.

摘要:

【目的】针对嗜酸硫杆菌极端特殊的生化特性，分别建立双层平板培养高效筛选方法和补料分批高密度发酵策略，并优选最佳保藏方法，以强化对该类菌种资源的利用和储备效率。【方法】分别采用以异养型微生物Sacchromyces ellipsoideu和Rhodotorula sp.为底层培养物的双层平板培养嗜酸硫杆菌，并结合透射电子显微镜技术(TEM)考察细胞形态差异。结合硫化矿培养基设计及单质硫补料培养策略，延长Acidithiobacillus thiooxidans对数期，提高比生长速率。分析不同保藏方法对嗜酸硫杆菌细胞存活率的影响。【结果】采用异养微生物——Rhodotorula sp.作为底层培养物的双层平板培养法在缩减1/3检出周期的同时将Acidithiobacillus ferrooxidans和Acidithiobacillus thiooxidans的检出率提高了3倍左右。TEM结果表明双层培养中细胞形态更为规则。采用基于Starkey-硫化矿培养基的补料分批发酵策略提高了Acidithiobacillus thiooxidans平均比生长速率，硫对生物量转化率和生产强度分别比分批培养提高31.1%和187.9%。4 °C低温保藏方式更适于嗜酸硫杆菌的保藏，有效保藏期1–3月。【结论】Rhodotorula sp.为辅助培养物的双层平板培养法可有效提高嗜酸硫杆菌的筛选效率。设计的Starkey-硫化矿培养基结合补料分批培养策略可实现Acidithiobacillus thiooxidans高密度培养。简单高效的4 °C低温保藏方式更适合于嗜酸硫杆菌的中短期保藏。

Abstract:

[Objective] Considering of the extremely special biochemical characteristics of Acidithiobacillus sp., the efficient double-layers screening, fed-batch high-density fermentation strategy and preservation method was respectively established and optimized for enhancing the utilization and reservation efficiency of these microbial resources. [Methods] The heterotrophic microorganisms such as Sacchromyces ellipsoideu and Rhodotorula sp. was employed as bottomed culture when the transmission electronic technology (TEM) was employed for observing the morphological differences. Based on the designed sulfide medium and elemental sulfur fed-batch strategy, the logarithmic phase of Acidithiobacillus thiooxidans was extended when the specific growth rate was improved. The effects of different preserved methods on cell survival rates were also investigated. [Results] Using heterotrophic microorganisms——Rhodotorula sp. as the underlying culture in the double-layers culture, the screening cycle was reduced by 1/3, while the plating efficiencies of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were improved by about 3 times. More regular cell morphology was shown by TEM in the double-layers culture. The average specific growth rate of Acidithiobacillus thiooxidans was improved via fed-batch fermentation strategy in the Starkey-sulfide medium. The biomass conversion rate of sulfur and productivity were improved by 31.1% and 187.9% respectively, compared to the batch fermentation. The simple and efficient 4 °C cryopreservation perseveration was more suitable for preserving Acidithiobacillus sp. with the validity period about 1-3 months. [Conclusion] The screening efficiency of Acidithiobacillus sp. could be effectively improved by the double-layers culture with the auxiliary culture-Rhodotorula sp.. High-density fermentation of Acidithiobacillus thiooxidans was achieved via fed-batch fermentation strategy in the Starkey-sulfide medium. Simple and efficient 4 °C cryopreservation preservation was more suitable for preserving Acidithiobacillus sp. in the short-term.