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%。     

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 by pulsed electric field in buffered peptone water and infant formula milk

The effect of high-intensity pulsed electric field (PEF) treatment on the survival of Enterobacter sakazakii suspended in buffered peptone water (BPW) and powdered infant formula milk (IFM) was evaluated. Reference medium and IFM samples were treated with PEF. Electric field intensity and treatment time were varied from 10 to 40 kV cm⁻¹ and from 60 to 3895 μs, respectively. Samples of buffered peptone water (3 g L⁻¹) and IFM were inoculated with E. sakazakii (CECT 858) (10⁹ cfu mL⁻¹) and then treated with PEF. The inactivation data were adjusted to the Weibull frequency distribution function and Bigelow model, and constants were calculated for both substrates. A maximum 2.7 log (cfu mL⁻¹) reduction was achieved in BPW after exposure of E. sakazakii to PEF for 360 μs (2.5 μs pulse width) at 40 kV cm⁻¹. In IFM, exposure of E. sakazakii to PEF, with the same conditions, led to a 1.2 log (cfu mL⁻¹) reduction. The greater the field strength and treatment time, the greater the inactivation achieved in both substrates. Even though further research will be necessary, according to the results, there are good prospects for the use of PEF in hospitals to achieve safe reconstituted infant formula before storage at refrigerated temperatures.     

应用DiversiLab分型系统对奶制品中的阪崎肠杆菌进行同源性分析

为研究奶制品污染中阪崎肠杆菌的同源性关系,并建立起阪崎肠杆菌基于重复序列的分型技术。对2005-2006年福建省出入境检验检疫局从各类奶制品中分离出的阪崎肠杆菌先进行API鉴定,然后进行DiversiLab分型。其中DiversiLab分型包括:DNA提取,rep-PCR和微电泳芯片分离检测。rep-PCR条件为:94℃预变性2 min;94℃变性30 s,55℃退火30 s,70℃延伸90 s,35个循环;最后70℃延伸3 min。结果表明:31株阪崎肠杆菌被API分成了5种不同的生物型,DiversiLab分型能将阪崎肠杆菌分为不同的亚型,但它们之间没有必然的联系。部分同一企业来源的菌株具有同源性,样品的同源性与采样地无关。DiversiLab分型系统是一种快速,高效,易于操作,高重复性,高分辨率的基因分型方法,可作为食源性污染的同源性分型工具。     

The presence of endotoxin in powdered infant formula milk and the influence of endotoxin and Enterobacter sakazakii on bacterial translocation in the infant rat

Lipopolysaccharide (LPS) is a heat stable endotoxin that persists during the processing of powdered infant formula milk (IFM). Upon ingestion it may increase the permeability of the neonatal intestinal epithelium and consequently bacterial translocation from the gut. To determine the level of endotoxin present in IFM, 75 samples were collected from seven countries (representing 31 brands) and analysed for endotoxin using the kinetic colorimetric Limulus amoebocyte lysate (LAL) assay. The endotoxin levels ranged from 40 to 5.5 x 10(4) endotoxin units (EU) per gram and did not correlate with the number of viable bacteria. The neonate rat model was used to address the risk of endotoxin-induced bacterial translocation from the gut. Purified Escherichia coli LPS was administered to rat pups followed by inoculation with Enterobacter sakazakii ATCC 12868. Bacteria were isolated from the mesentery, spleen, blood and cerebral spinal fluid (CSF) of endotoxin-treated rats due to enhanced gut and blood brain barrier penetration. Histological analysis of the colon showed marked distension of the mucosal and muscular layers. It is plausible that the risk of neonatal bacteraemia and endotoxemia, especially in neonates with immature innate immune systems, may be raised due to ingestion of IFM with high endotoxin levels.     

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。