Enhancement of biohydrogen production from sustainable orange peel wastes using Enterobacter species isolated from domestic wastewater

Five facultative anaerobic bacterial isolates were recovered from domestic wastewater. These isolates were identified based on the 16S rRNA as Enterobacter aerogenes (one isolate), Enterobacter cloacae (two isolates), and Cronobacter sakazakii (three isolates). These isolates were examined for their potential to evolve hydrogen on a glucose medium. The most potent hydrogen‐producing isolates, E aerogenes (KY549389) and E cloacae (KY524293), were examined for their capacity to generate hydrogen, acetone, butanol, and ethanol using orange peel (OP) hydrolysate. OP powder was pretreated with n‐hexane to remove the toxicity of d ‐limonene. Different concentrations (4%, 6%, and 8% w/v) of limonene‐free OP were subjected to the boiling water (temperature of 100°C) or acid (HCl) treatments. The maximum fermentative H₂ production of 1700 and 1620 mL/L was obtained from 6% OP hydrolysate extracted with boiling water using facultative anaerobic E aerogenes (KY549389) and E cloacae (KY524293), respectively. Hydrogen production efficiency was 0.99 and 1.19 mol H₂/mol glucose for E aerogenes and E cloacae, respectively. The total fermentative acetone, butanol, and ethanol (ABE) generated by E aerogenes and E cloacae were 0.78 and 0.38 g/L including acetone (0.05 and 0.04 g/L), butanol (0.011 and 0.013 g/L), and ethanol (0.71 and 0.32 g/L), respectively. The maximum ABE productivity was 0.01 and 0.005 g/L/h generated at 60 g/L OP hydrolysate by E aerogenes and E cloacae, respectively. These strains were positive for nitrogen fixation (nitrogenase) capability estimated by the acetylene reduction assay. Application of OP hydrolysate without the addition of any nutritional components or reducing agent is considered an eco‐friendly, economical, and commercial substrate for desired biofuel production.     

Optimization of culture conditions for biohydrogen production from sago wastewater by Enterobacter aerogenes using Response Surface Methodology

Sago wastewater (SWW) causes pollution to the environment due to its high organic content. Annually, about 2.5 million tons of SWW is produced in Malaysia. In this study, the potential of SWW as a substrate for biohydrogen production by Enterobacter aerogenes (E. aerogenes) was evaluated. Response Surface Methodology (RSM) was employed to find the optimum conditions. From preliminary optimization, it was found that the most significant factors were yeast extract, temperature, and inoculum size. According to Face Centered Central Composite Design (FCCCD), the maximum hydrogen concentration and yield were 630.67 μmol/L and 7.42 mmol H₂/mol glucose, respectively, which is obtained from the sample supplemented with 4.8 g/L yeast extract concentration, 5% inoculum, and incubated at the temperature of 31 °C. Cumulative hydrogen production curve fitted by the modified Gompertz equation suggested that H_max, R_max, and λ from this study were 15.10 mL, 2.18 mL/h, and 9.84 h, respectively.     

色氨酸酶重组基因表达及其酶法合成L-色氨酸

为实现色氨酸酶高效、低成本催化合成L-色氨酸,利用p ET30a为载体在宿主细胞E.coli BL21(DE3)中重组表达了产气肠杆菌(Enterobacter aerogenes)来源的色氨酸酶,以丙酮酸、吲哚和氨为底物,探究其酶学性质,考察了反应温度、起始p H、底物摩尔比对酶促反应的影响,并利用丙酮酸发酵液为底物酶法合成L-色氨酸。结果表明,色氨酸酶重组表达成功,色氨酸酶最佳反应条件为:温度35℃,起始p H=9.0,底物摩尔比n(吲哚)∶n(丙酮酸)=0.6∶1,底物丙酮酸浓度为0.17 mol/L。利用重组色氨酸酶全细胞催化100 m L浓度为0.57 mol/L丙酮酸发酵液,流加浓度为4.27 mol/L吲哚酒精溶液6.5 m L,反应28 h后,L-色氨酸浓度达0.25 mol/L,吲哚摩尔转化率达91.8%。     

细菌Enterobacter dissolvens的直流电解刺激过程

以葡萄糖为唯一碳源,研究了细菌Enterobacter dissolvens在直流电条件下的生长和代谢过程. 实验结果表明,当使用盐桥屏蔽电极反应时,细胞生长曲线和葡萄糖代谢速率基本不受电流强度的影响,而当采用铂丝电极时,反应体系中发生水解反应,采用10 mA电流通电12 h后,菌液中细胞的脱氢酶比活力和葡萄糖降解率分别为对照的1.98倍和1.48倍,细胞生长亦有所加快,但在其活力下降时出现大量死亡. 扫描电镜照片显示细胞结构受到明显破坏,并发生细胞膜穿孔现象,这表明可能受到了过氧化氢等阳极水解中间产物的刺激作用.     

Individual cell lag time distributions of Cronobacter (Enterobacter sakazakii) and impact of pooling samples on its detection in powdered infant formula

Cells of six strains of Cronobacter were subjected to dry stress and stored for 2.5 months at ambient temperature. The individual cell lag time distributions of recovered cells were characterized at 25 °C and 37 °C in non-selective broth. The individual cell lag times were deduced from the times taken by cultures from individual cells to reach an optical density threshold. In parallel, growth curves for each strain at high contamination levels were determined in the same growth conditions. In general, the extreme value type II distribution with a shape parameter fixed to 5 (EVIIb) was the most effective at describing the 12 observed distributions of individual cell lag times. Recently, a model for characterizing individual cell lag time distribution from population growth parameters was developed for other food-borne pathogenic bacteria such as Listeria monocytogenes. We confirmed this model’s applicability to Cronobacter by comparing the mean and the standard deviation of individual cell lag times to populational lag times observed with high initial concentration experiments. We also validated the model in realistic conditions by studying growth in powdered infant formula decimally diluted in Buffered Peptone Water, which represents the first enrichment step of the standard detection method for Cronobacter. Individual lag times and the pooling of samples significantly affect detection performances.     

Inactivation of Enterobacter sakazakii in infant milk formula by gamma irradiation: determination of D10-value

ABSTRACT:  Enterobacter sakazakii is an emerging foodborne pathogen that has caused several cases of meningitis and necrotizing entercolitis in infants and has been associated with infant formulas. Five strains of E. sakazakii were inoculated individually into brain heart infusion broth and rehydrated or dehydrated infant milk formula and exposed to ionizing radiation. E. sakazakii strains in brain heart infusion broth and rehydrated infant milk formula (RIMF) were exposed to irradiation dose of up to 1 kGy while strains in dehydrated infant milk formula (DIMF) were exposed to irradiation dose of up to 9 kGy. The D₁₀-values were determined by using a linear regression model. Average calculated D₁₀-values ranged from 0.21 to 0.29 kGy, 0.24 to 0.37 kGy, and 1.06 to 1.71 kGy in brain heart infusion broth, RIMF, and DIMF, respectively. The results obtained from this study will be useful for powdered infant milk formula industries to reduce the risk associated with E. sakazakii .     

羊奶粉生产环节阪崎肠杆菌分离鉴定及其毒力基因和药敏检测

目的:分析羊奶粉生产环节阪崎肠杆菌的污染状况,分离株毒力基因携带情况以及耐药性。方法:采自某羊奶粉加工厂空气过滤车间、液态奶车间、流化床车间、喷雾干燥车间和包装车间的生产样品及环境样品共180份,按国标GB4789.40-2010和PCR方法进行阪崎肠杆菌的分离鉴定;采用PCR方法检测cpa、hly、sip和omp X毒力基因;采用琼脂稀释法测定药敏性。结果:27个采样点中有14个阪崎肠杆菌阳性点,检出率为51.9%;180份样品中有29份检出阪崎肠杆菌,污染率为16.1%;阳性样品主要为粉状和棉签涂抹样品,污染率分别为23.5%和16.9%。毒力基因检出率为100%,毒力基因类型有cpa-omp X和cpa-hly-omp X,检出率分别为79.3%,20.7%。29株菌对利福平、甲氧苄啶/磺胺甲恶唑、头孢西丁钠、阿莫西林、阿莫西林/克拉维酸的耐药率依次为100%,75.9%,6.9%,3.4%和3.4%;对其它11种抗生素均敏感,多重耐药率为6.9%。结论:羊奶粉加工厂存在阪崎肠杆菌的污染。空气流动、粉尘污染、操作人员交叉污染及生产设备清洗消毒不彻底可能是加工过程中该菌的污染途径。分离株毒力基因携带率较高,对大多数供试抗生素敏感,出现多重耐药现象。     

Inhibitory Activity of Paenibacillus polymyxa on the Biofilm Formation of Cronobacter spp. on Stainless Steel Surfaces

The objective of this study was to control the survival or biofilm formation of Cronobacter spp. on stainless steel surfaces using Paenibacillus polymyxa. The antibacterial activity of a cell‐free culture supernatant (CFCS) of P. polymyxa against Cronobacter spp. was found to vary with P. polymyxa incubation time. Maximum activity occurred when P. polymyxa was incubated at 25 or 30 °C for 96 h. When the CFCS was introduced to Cronobacter spp. adhered to stainless steel strips at 25 °C for up to 72 h, the CFCS successfully inhibited Cronobacter biofilm formation. Additionally, stainless steel surfaces with a preformed P. polymyxa biofilm were exposed to Cronobacter spp. suspensions in PBS or 0.1% peptone water at 3, 5, or 7 log CFU/mL to facilitate its attachment. The Cronobacter population significantly decreased on this surface, regardless of inoculum level or carrier, when the P. polymyxa biofilm was present. However, the microbial population decreased within 6 h and remained unchanged thereafter when the surface was immersed in an inoculum suspended in 0.1% peptone water at 5 or 7 log CFU/mL. These results indicate that P. polymyxa is able to use a promising candidate competitive‐exclusion microorganism to control Cronobacter spp.     

Enterobacter sakazakii survives spray drying

Four strains of Enterobacter sakazakii were inoculated into 35% reconstituted skim milk at 10⁷ and 10² cfu/g dry wt. After spray-drying in a Buchi mini spray drier (inlet 160°C and outlet 90°C), the resulting powders were analysed for E. sakazakii by enrichment and enumeration. In all cases, E. sakazakii survived the spray drying process and were detected in the powders with a low inoculum and enumerated in all the powders with the high inoculum for at least 12 weeks. The results emphasize that the controls in place to prevent E. sakazakii from getting to the spray drier are essential.