Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30-40 g/L) was greater than that of freely suspended cells (8-19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ∼52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 °C, with NH4Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 °C with (NH4)2 SO4 as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.

中文翻译：

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新型酵母分离物固定化细胞柠檬酸生产的优化。

柠檬酸是一种商业上有价值的有机酸，广泛用于食品，制药和饮料行业。在这项研究中，从土壤，面包，果汁和水果废物中分离了260种酵母菌株，并使用溴甲酚绿琼脂平板初步筛选了它们产生有机酸的能力。总体而言，有251种酵母菌分离株显示阳性结果，菌落周围有黄色光晕。使用游离和固定细胞技术评估了20种有希望的分离株的柠檬酸生产。结果显示固定化细胞的柠檬酸产量（30-40 g / L）大于自由悬浮细胞的柠檬酸产量（8-19 g / L）。在20种分离物中，根据柠檬酸的生产水平，选择了2种（KKU-L42和KKU-L53）进行进一步分析。固定化的KKU-L42细胞具有较高的柠檬酸产生率（62.5％），而固定的KKU-L53细胞与游离细胞相比，柠檬酸产量增加了约52.2％。通过对26S rRNA基因D1 / D2结构域进行扩增和序列分析，可以准确鉴定出这两种分离株，并基于GenBank进行序列比较，确认分离株KKU-L42和KKU-L53分别为热带假丝酵母和克鲁维酵母。优化了两个因素，包括发酵时间，pH，温度以及碳和氮源，以通过两种分离物提高柠檬酸的产量。两种分离物在葡萄糖为碳源的pH 5.0的发酵条件下，在5天的发酵过程中达到了最大产量。热带假丝酵母生产柠檬酸的最佳温育温度为32°C，NH4Cl是最佳氮源，而P. 在27°C下以（NH4）2 SO4为氮源观察到kluyveri。在20天的时间内，柠檬酸的生产可保持约四批重复。我们的结果表明，苹果和香蕉废物是新型酵母菌株的潜在来源。可用于商业柠檬酸生产的热带假丝酵母和克鲁维假单胞菌。