Fermentation of maltose and starch by Klebsiella oxytoca P2

Klebsiella oxytoca P2, which has genes from Zymomonas mobilis encoding the alcohol dehydrogenase and pyruvate decarboxylase integrated in its chromosome, fermented 50 g maltose/l to 25.4 g of ethanol/l. It also fermented 10, 20 and 40 g starch/l yielding 4, 8.4, and 17.7 g ethanol/l, respectively, representing 72, 75 and 78% of the theoretical yield. © Rapid Science Ltd. 1998     

Involvement of a plasmid in exopolysaccharide production by Klebsiella oxytoca

A Klebsiella oxytoca isolate which can produce significant levels of an exopolysaccharide using whey as a growth substrate has been reported. The plasmid profile of this isolate was shown to be different from that of the non-exopolysaccharide-producing K. oxytoca ATCC 43863. Irreversible curing of the single plasmid in the K. oxytoca isolate was achieved using 30 g acriflavin/ml. The ability to produce the exopolysaccharide was lost with the curing of the plasmid (parent strain produced a medium viscosity of 1260 cP at 1 s^–1 compared to 1.6 cP at 200 s^–1 produced by the cured strain). However, the ability to metabolize lactose was not significantly affected by curing, and both the parent and the cured strain produced similar levels of viable cells (~10⁹ cfu/ml) after 62 h growth on lactose-rich medium. The exopolysaccharide-producing ability of the isolate was stable for at least 139 generations.     

Biodegradation of Phenol by Actinobacillus Sp.: Mathematical Interpretation and Effect of Some Growth Conditions

Models of the cultivation process of Actinobacillus sp. cells in two media, rich (NB) and minimal (M9) that includes phenol as a sole carbon source, have not been described in the available literature. For these reasons, several single-substrate inhibition models (Monod, Andrew, and Tesseir) were investigated in order to determine the mathematical expression of Actinobacillus sp. growth rate. The experimental data for both nutrient broth and M9 media were fitted to the above models mentioning that Andrews' model best fits these data adequately for both media with regression coefficient of 0.973 and 0.962, respectively. The maximum predicted growth rate by this model is 0.37 h^{- 1} for both media obtained when the initial concentration of phenol is 100 mg/L. The half-saturation concentration constant, K_P, is 1.00 mg/L, which represents the phenol concentration when μ is equal to half μ_max. On the other hand, the inhibition constant, K_p is 13,000.00 mg/L for broth medium and 12,000 mg/L for M9 medium, which is a measure of sensitivity to inhibition by inhibitory substances. When cells are grown in nutrient broth and minimal media, the rate of cell production with time can be expressed by the Reccati and Voltera models. Voltera model better fits in the case of M9 minimal medium plus phenol as sole carbon source. The pH of 7, the incubation temperature of 35°C to 37°C, and the agitation rate of 150 rpm are the optimal conditions for achieving the higher percentage of phenol degradation by Actinobacillus sp. Succinic acid and glycine as carbon and nitrogen source, respectively, were the most efficient of the cosubstrates (out of 10 substrates tested) for removal of phenol on an mg/L basis.     

Short Communication: Optimization of bioprocess conditions for exopolysaccharide production by Klebsiella oxytoca

Optimization of bioprocess conditions increased exopolysaccharide production by a strain of Klebsiella oxytoca from 6g/l to 15g/l; this corresponded to an increase in medium viscosity from 36cP at 12s–1 to 20,000 cP at 0.6 s–1. A combination of equal proportions of tryptone nitrogen and urea nitrogen proved to be the best nitrogen source. Lactose was shown to be the preferred carbon source. At an optimum temperature of 25°C, a pH of 7 was found to be the best for exopolysaccharide production. The concentration of exopolysaccharide produced on whey, enriched whey, enriched whey permeate and lactose-rich medium was comparable.     

Identification of heat stable protease of Klebsiella oxytoca isolated from raw milk

AIMS: The aim of this study was to identify and characterize heat stable proteinases of psychrotrophic proteolytic bacteria isolated from raw milk. METHODS AND RESULTS: A strain of Klebsiella oxytoca producing a high proteolytic activity when cultured on milk was isolated. Maximum proteolytic activity was observed at the stationary phase during growth on milk or casein-peptone broth. The bacterium demonstrated the capability to grow at 7 degrees C, classified as psychrotrophic. The crude enzyme showed optimum activity at 37 degrees C, and pH 5.0 and 7.0. The proteinase was very resistant to heat, maintaining 74% of initial activity after incubation at 142 degrees C. CONCLUSIONS: A heat stable protease of a psychrotrophic strain of K. oxytoca was identified and partially characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Thermal stable proteases may constitute a serious problem to ultra-high temperature (UHT) processed milk, leading to undesirable physical and sensory alterations.     

产酸克雷伯氏菌M5al普鲁兰酶的异源表达及酶学性质研究

普鲁兰酶(EC 3.2.1.41)是一类淀粉脱支酶,能够特异性水解淀粉中的α-1,6-糖苷键,从而提高淀粉的利用率,在以淀粉为原料的食品、纺织、生物燃料和洗涤剂等行业中具有重要的应用价值。本研究以产酸克雷伯氏菌Klebsiella oxytoca M5al基因组DNA为模板,将PCR扩增得到的普鲁兰酶基因pul A克隆至表达载体p ET28a(+),构建好的重组质粒转化大肠杆菌Escherichia coli BL21(DE3),在培养基中添加0.5 mmol/L异丙基硫代半乳糖苷(IPTG)的条件下对该酶基因进行诱导表达,经镍柱纯化获得重组普鲁兰酶用于酶学性质研究。SDS-PAGE及Western Blot检测显示普鲁兰酶基因pul A在上述大肠杆菌宿主中成功获得了表达。该重组酶最适反应p H5.5,最适温度60℃。金属离子对酶活性有一定影响。Mn2+对酶活促进作用显著;Fe3+、Mg2+、Fe2+对酶活只有微弱的促进作用,而Cu2+对酶活造成强烈抑制。来源于Klebsiella oxytoca M5al的普鲁兰酶最适催化条件符合工业生产中淀粉糖化工艺的要求,具有应用于淀粉工业的潜力。     

Production of 2,3‐butanediol by Klebsiella oxytoca from various sugars in microalgal hydrolysate

A new fermentation process using a mixed sugar medium is proposed in this study for 2,3‐butanediol (2,3‐BDO) production. The medium contained seven different monosugars known to be present in Nannochloropsis oceanica hydrolysate. The performance of each sugar when existing alone or together with glucose was evaluated. All the sugars except fucose were successfully metabolized for 2,3‐BDO production. A 2,3‐BDO yield of 0.31g/g was achieved with the mixed sugar medium, which was very close to that with the glucose‐only medium. However, the 2,3‐BDO productivity (0.28 g L^?1 h^?1) was found to be about 30% lower than that with glucose, implying, as expected, the existence of glucose repression on the uptake of other sugars. Strain development is in need to remove such negative effect of glucose for improved process efficiency. Fucose with the lowest uptake rate and no contribution to 2,3‐BDO production can be a high value‐added byproduct, once recovered and purified. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1669–1675, 2015     

Klebsiella oxytoca: Resistance to aztreonam by overproduction of the chromosomally encoded β-lactamase

Abstract Aztreonam-resistant Klebsiella oxytoca strain SL7811 was selected on agar containing 1 μg of aztreonam per ml from a susceptible strain SL781. The MICs for the resistant mutant towards penicillins, aztreonam and ceftriaxone were much higher, to cefotaxime slightly higher and to ceftazidime unchanged. Synthesis of β-lactamase was 223-fold greater in the mutant compared with the susceptible strain. SL781 and its resistant mutant SL7811 produced β-lactamase with the same isoelectric point and substrate profile. The β-lactamase genes from SL781 and SL7811 were cloned in plasmid pBGS18 giving pBOF-1 and pBOF-4 respectively. The sequences of the two putative promoters indicated two modifications in the resistant plasmid pBOF-4: a transversion (G → T) in the first base of the − 10 consensus sequence and a deletion of one C residue four base pairs upstream of the − 10 hexamer.     

阴沟肠杆菌基因nifA经转座子Tn5整入催娩克氏杆菌NG13的基因组

根际固氮细菌与禾谷类作物如水稻、小麦、玉米等进行联合固氮,能使作物有不同程度的增产（APp等1980,Watanable和hn1984）。水稻是世界上主要粮食作物之一,因此,研究增强水稻根际细菌的联合固氮作用,吸引着科学家们的兴趣。催娩克氏杆菌（KMFithemp）NG13与水稻能进行有效的联合固氮（Yoo等1986）。但是在有氨的生长条件下,细菌的固氮活力受到阻遏。ZhU等将带有nifA的重组质粒引进阴沟肠杆菌EZ6后,观察到有氨存在条件下固氮酶组成型生成。我们实验室构建了具有广泛接合转移特性的阴沟肠杆菌Tns－nifA嵌合质粒PBF101,基因nif…     

Endophytic colonization of Typha australis by a plant growth-promoting bacterium Klebsiella oxytoca strain GR-3

AIMS: To isolate and characterize endophytic diazotrophic bacteria from a semi-aquatic grass (Typha australis) which grows luxuriantly with no addition of any nitrogen source. METHODS AND RESULTS: Ten endophytic diazotrophic bacteria from surface-sterilized roots and culm of T. australis were isolated and screened for plant growth-promoting activities employing standard methods. Based on the rate of nitrogenase activity, indole acetic acid (IAA) production and phosphate (P) solubilization, one root isolate namely GR-3 was found to be the most efficient one. This isolate was identified as Klebsiella oxytoca on the basis of 16S rDNA sequence analysis. Amplification of nifH by polymerase chain reaction (PCR) and detection of dinitrogenase reductase by western blot confirmed the diazotrophic nature of GR-3. It was tagged with gusA fused to a constitutive promoter and the resulting transconjugant was inoculated onto endophyte-free rice variety Malviya dhan-36 seedlings to express cross-infection ability which resulted in a significant increase in root/shoot length and chlorophyll a content. CONCLUSIONS: Roots and culm of T. australis harbour several endophytic diazotrophic bacteria. One root isolate, identified as K. oxytoca GR-3, seems to be an efficient plant growth-promoting bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant growth-promoting properties of GR-3 suggest that this promising isolate merits further investigations for potential application in agriculture.     

含hup基因的质粒在催娩克氏杆菌中的稳定性及吸氢酶的表达

以质粒pKT230为栽体,亚克隆大豆根瘤菌吸氢酶结构基因(hupSL)片段,构建成嵌合质粒pKH1。将质粒分配系统基因(parDE)片段和吸氢酶结构基因(hup)片段插入载体质粒pRK415,构建成质粒pRKBH。质粒pKH1、pRKBH和载体pRK415经转化和三亲本杂交,得到DH5α/pHR11、DH5α/pRKBH、E1201/pKH1、NG13/pKH1(NGH999)、NG1390/pRK415、NG1390/pHR11、NG1390/pRKBH和NG1390/pKH1(NGH982)等接合子。稳定性分析发现,质粒pKH1在催娩克氏杆茵中传80代后仍有92％以上的菌株含此质粒,说明质粒pKH1有较高的稳定性。吸氢酶活性分析表明,H     

Cadmium-Resistance Plasmid Affected Cd+2 Uptake More Than Cd+2 Adsorption in Klebsiella oxytoca

A bacterial strain was isolated from Petra City Wastewater Treatment Plant. This isolate was identified as Klebsiella oxytoca based on 16S rDNA analysis. A single plasmid (> 23 kb) was detected in this strain and transformed into Esherichia coli JM83. The transformed E. coli cells exhibited elevated resistance to cadmium as compared to parental plasmid-free cells. The sodium dodecyl sulfate (SDS)-treated cells showed higher efficiency in plasmid curing than the ethidium bromide–treated cells. The ethidium bromide–cured cells grew only in a 10 μ g/ml Cd^{+ 2} minimal tolerable concentration, whereas the SDS-treated cells had no growth in any of the Cd concentrations tested (2, 5, 10, 20, 30, 40, and 50 ppm). Contrary to the Freundlich model, the Langmuir model gave a good fit to the Cd biosorption data by K. oxytoca cells. Plasmid curing caused 80%, 82%, and 70% inhibition in the Cd biosorption, adsorption, and uptake, respectively. Furthermore, the absence of lysine decarboxylase (LDC) activity in the cured strain strongly implies that the structural gene-encoding LDC in this bacterium is plasmid encoded. After curing of the plasmid, 100% of the antibiotic-resistant loci were observed as chromosomal encoded. All of the results shown above indicated that the Cd resistance is plasmid mediated.     

Induction of 4-Hydroxycinnamate Decarboxylase in Klebsiella oxytoca Cells Exposed to Substrates and Non-substrate 4-Hydroxycinnamate Analogs

The 4-hydroxycinnamate decarboxylase (4-HCD)-inducing activity of several substrate analogs toward Klebsiella oxytoca was investigated. Four E-cinnamateclass compounds, E-4-hydroxycinnamic acid (1), caffeic acid (2), ferulic acid (3) and E-2,4-dihydroxycinnamic acid (4), all of which were accepted as substrates, all of which were accepted as substrates of 4-HCD, enable K. oxytoca cells to induce the decarboxylase at a 2.0 mM concentration, while five non-substrate compounds of the E-cinnamate class so far tested were completely in-active. However, 6-hydroxy-2-naphthoic acid (11) and 7-hydroxycoumarin 3-carboxylic acid (14), both of which are non-cinnamate-class analogs of the substrate, acted as strong 4-HCD inducers, even at a 0.5 mM concentration. The 4-HCD-inducing activities of compounds 11 and 14 at 0.5 mM were 10-12-fold higher than that of substrate 1. Compound 11 maintained its 4-HCD-inducing activity toward cultured cells through the late-log and stationary phases, unlike 1 that induced 4-HCD only in the early log phase. SDS-PAGE electrophoresis of protein mixtures from the cultured cells exposed to any 4-HCD inducer indicated that the 21.5 kDa protein was always present.     

Effects of Magnetic Field on Phenol Biodegradation and Cell Physiochemical Properties of Rhodococcus erythropolis

Using phenol-degrading Rhodococcus erythropolis cells, the stimulative effect of a homogenous electromagnetic field (EMF) (magnetic induction 10–130 mT) on the growth and utilization of phenol (0.3–1.2 g/L) was investigated. Similarly, the EMF effect was tested on a R. erythropolis biofilm formation, which was found to increase the cell adhesion abilities significantly. Detected magnetic stimulation of cell adhesion disposition was supplemented with the results of cell surface hydrophobicity and chemical composition analysis.     

Biodegradation of Gossypol by Mixed Fungal Cultures in Minimal Medium

The fungal cultures, namely – Pleurotus sajor-caju MTCC 1806, Saccharomyces cerevisiae MTCC 6933 and Candida tropicalis MTCC 1406 and their combinations, C. tropicalis + S. cerevisiae, P. sajor-caju + S. cerevisiae and C. tropicalis + P. sajor-caju were grown in minimal medium containing 100 ppm of gossypol as the sole carbon and energy source. The culture supernatants of C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae had low residual gossypol levels of 29 and 25 ppm, respectively. In the present study, we attempted to isolate gossypol-degrading enzyme and biodegraded gossypol from the culture supernatants of C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae. The specific activity of laccase in the purified enzyme extracts of the C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae treated samples was 425 and 224 U/mg, respectively. In SDS-PAGE, the gossypol-degrading enzyme was revealed as 3 bands of molecular weights ranging from 45 to 66 kDa. The characterization of biodegraded gossypol by FTIR analysis showed a reduction in aldehydes (C-H) stretches in samples treated with fungi. Mass spectrometry analysis revealed that the monoisotopic mass of the biodegraded gossypol was 474 g/mol.     

假单胞菌诱导筛选菌株PhA苯酚降解动力学及SDS对其影响

为提高苯酚降解速率,由假单胞菌(Pseudonomonas．sp)诱导筛选得到了一株能以苯酚为唯一碳源生长的新菌株Pha,并使其苯酚选择压力从400mg／L逐步提高到了700mg／L。且Pha菌株降解苯酚过程符合一级反应动力学方程。使用十二烷基磺酸钠(SDS)作为增溶剂来促进降解时,发现在SDS浓度为50～150mg／L时,降解苯酚的速率随SDS浓度增加而提高。SDS在低浓度时对其生长影响很小,但浓度达到300mg／L时,对其生长开始有了明显的抑制作用。结果标明PhA菌株有着较高的苯酚耐受浓度,SDS可以显著的提高苯酚的降解速率。SDS的理论最佳投放量为150mg／L。     

Phenol biodegradation by free and immobilized Acinetobacter

Acinetobacter sp. strain W-17, immobilized on porous sintered glass completely degraded 500 mg phenol l^–1 in 40 h, but free cells required 120 h for this to be achieved. Immobilized cells can be used 7 times without losing their activity.     

Saccharification and fermentation of Sugar Cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway

Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter papaer units (FPU) g(-1) acid-treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid-treated bagasse L(-1)), a combination of 20 FPU cellulase g(-1) bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L(-1). Alternatively, almost 40 g ethanol L(-1) was produced with 10 FPU cellulase g(-1) bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU(-1) of commercial cellulase. (c) 1994 John Wiley & Sons, Inc.     

一株耐高盐热带假丝酵母菌降解苯酚的研究

从含酚废水处理池污泥中驯化分离得到一株能以苯酚为唯一碳源的菌株FD-1。经18SrDNA和ITS序列的BLAST比对及系统发育分析,鉴定FD-1为热带假丝酵母(Candida tropicalis)的近缘种。FD-1对苯酚的降解能力较强,能够完全降解浓度为1 000mg·L-1的苯酚溶液。初步确定了FD-1在降解苯酚溶液时的最适温度为30~35℃,pH为6.0~7.0,并且通过探讨加入无机盐、培养基原料以及改变接种量三个因素对苯酚降解的影响,其耐受盐的浓度可达5%,对实践中应用微生物降解含酚废水具有积极的意义。     

Bioremoval of Cyanide and Phenol from Industrial Wastewater: An Update

In nature, phenols and cyanides are produced by certain microbes and plants. Phenols are antioxidants found in almost all plants, and cyanides are important components of lima beans, almonds, and cassava. Their presence in small amounts may not upset the environment, but their large-scale production, wide applicability, and unrestricted release by the industries makes them widespread and important pollutants. Phenols and cyanides can be recovered/removed from wastewater streams using various physicochemical techniques practiced commercially. Lack of complete mineralization, cost-effectiveness, and release of secondary by-products are amongst a few of the major considerations that limit the installation of such processes. Biological removal of such pollutants from industrial waste has gained momentum in recent years, as they promise to surpass the major drawbacks laid by the physicochemical methods and can be practically carried out in all conditions. Presence of either cyanide or phenol is highly dangerous, and in the presence of both, the effect is compounded. The present review illustrates the various industries involved in the release of phenols, cyanides, or both; it summarizes the available technologies for their treatment and emphasizes recent advances and advantages of biological abatement of these pollutants.