Optimization of Culture Conditionsand Analysis of Fermentation Process for Plant Vaccine-producing Bacterium, FJAT-1458
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摘要: 对番茄青枯病植物疫苗菌株FJAT-1458进行培养条件优化和发酵过程状态参数研究。采用摇瓶培养和单因素试验研究菌株FJAT-1458的培养条件，确定最适培养基为SPA（蛋白胨5 g·L-1，蔗糖20 g·L-1，KH2PO4 0.5 g·L-1，MgSO4 0.25 g·L-1），最适培养温度为30℃，250 mL三角瓶最适装液量为35 mL。用50 L发酵罐发酵FJAT-1458，发酵过程中菌体数量呈现先升后降的变化趋势，FJAT-1458的生长可分为4个时期：0～6 h为发酵适应期、6～36 h为对数生长期、36～60 h为稳定生长期，72 h后为消亡期。发酵过程中，菌体的致病性指标-弱化指数变化小，介于0.832～0.855，方差分析表明其差异不显著，P>0.05；发酵液pH值从7.23上升至8.78；菌体细胞为短杆状，大小为（1.0～1.5）μm×（0.5～0.8）μm，随着发酵进程，细胞颜色变深，发酵后期细胞开始变形、裂解。Abstract: Conditions to culture FJAT-1458, a bacterium that produces plant vaccine against the tomato bacterial wilt caused by Ralstonia solanacearum, were optimized by asingle factor experiment. They were determined to be the use of an SPA medium containing peptone 5 g·L-1, sugar 20 g·L-1, KH2PO40.5 g·L-1, and MgSO40.25 g·L-1 at 30℃ with 35 mL of broth in a 250 mL shaking flask. Subsequently, in a 50 L fermentation tank, the fermentation was carried out and the process monitored. During the fermentation, the cell count on FJAT-1458 increased initially and followed by a gradual decline with a 4-stage growth period that included the adaptive (0-6 h), logarithmic (6-36 h), stationary (36-60 h), and decline phases (after 72 h). In addition, it was observed that(a) the pathogenicity indicator, attenuation index, of FJAT-1458 ranged from 0.832 to 0.855 showing no significant difference (P>0.05) throughout the entire process; (b) the pH of the fermentation broth increased from 7.23 to 8.78; (c) morphologically, the bacteria appeared to be(1.0-1.5)μm×(0.5-0.8)μm rods with a darkening color as the process proceeded; (d) the cellular secretion from the bacteria increased with time; and, (e) some of the bacterial cells deformed and lysed near the end of fermentation.
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图 1 菌株FJAT-1458发酵过程菌体形态变化
Figure 1. Morphological changes on FJAT-1458 during fermentation process
表 1 植物疫苗菌株FJAT-1458在不同培养条件下的生长量
Table 1. Growth of avirulent strain, FJAT-1458, under varied culture conditions
测定项目 FJAT-1458菌体浓度/
(×108cfu·mL-1)培养基 SPA 1.56±0.12 a NA 0.93±0.05 c LB 1.39±0.11 a 温度 20℃ 1.14±0.06 bc 25℃ 1.27±0.12 b 30℃ 1.77±0.10 a 35℃ 0.91±0.06 c 40℃ 0 装液量 15mL 1.82±0.07 a 25 mL 1.75±0.07 a 35 mL 1.87±0.11a 45 mL 1.77±0.04 a 55 mL 1.71±0.06a 注：同例不同小写字母表示差异达显著水平(P < 0.05)。表 2同。 
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表 2 植物疫苗菌株FJAT-1458发酵过程各参数变化
Table 2. Changes on parameters during fermentationprocess of avirulent strain, FJAT-1458
时间
/hFJAT-1458菌体浓度
/(×108cfu·mL-1)菌株弱
化指数pH值 0 1.12±0.07 a 0.84±0.03 a 7.23±0.01 h 6 1.37±0.15 a 0.84±0.01 a 7.51±0.01 g 12 3.05±1.56 b 0.84±0.03 a 8.27±0.01 f 24 9.37±1.02 c 0.85±0.01 a 8.53±0.03 d 36 12.48±2.55 d 0.85±0.01 a 8.38±0.02 e 48 11.72±1.82 e 0.85±0.01 a 8.72±0.01 b 60 11.05±1.22 f 0.85±0.02 a 8.67±0.01 c 72 8.32±0.43 g 0.83±0.03 a 8.78±0.03 a
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