Biodegradation of thiocyanate using co-culture of Klebsiella pneumoniae and Ralstonia sp.

Thiocyanate-degrading microbial co-culture was isolated from thiocyanate-contaminated site and tested for thiocyanate degradation potential and thiocyanate-toxicity tolerance and identified as Klebsiella pneumoniae and Ralstonia sp. by 16S rDNA sequencing. The co-culture was able to degrade thiocyanate with degradation rate of 500 mg L⁻¹d⁻¹ at 2,500 mg L⁻¹ thiocyanate concentration at pH 6.0 and 37oC following thiocyanate hydrolase pathway. The Haldane kinetic model elucidates the growth and thiocyanate biodegradation kinetics of the co-culture with Ki value of 1,876 mg L⁻¹. The thiocyanate biodegradation kinetics was not affected by the additional supply of glucose. The very high activities of thiocyanate hydrolase, cyanide oxygenase, and cytochrome P-450 content during growth on thiocyanate were observed, showing the induction mechanism.     

Biodegradation of tributyl phosphate by novel bacteria isolated from enrichment cultures

Tributyl phosphate (TBP) is an organophosphorous compound, used extensively (3000–5000 tonnes/annum) as a solvent for nuclear fuel processing and as a base stock in the formulation of fire-resistant aircraft hydraulic fluids and other applications. Because of its wide applications and relative stability in the natural environment TBP poses the problem of pollution and health hazards. In the present study, fifteen potent bacterial strains capable of using tributyl phosphate (TBP) as sole carbon and phosphorus source were isolated from enrichment cultures. These isolates were identified on the basis of biochemical and morphological characteristics and 16S rRNA gene sequence analysis. Phylogenetic analysis of 16S rRNA gene sequences revealed that two isolates belonged to class Bacilli and thirteen to β and γ-Proteobacteria. All these isolates were found to be members of genera Alcaligenes, Providencia, Delftia, Ralstonia, and Bacillus. These isolates were able to tolerate and degrade up to 5 mM TBP, the highest concentration reported to date. The GC–MS method was developed to monitor TBP degradation. Two strains, Providencia sp. BGW4 and Delftia sp. BGW1 showed respectively, 61.0 ± 2.8% and 57.0 ± 2.0% TBP degradation within 4 days. The degradation rate constants, calculated by first order kinetic model were between 0.0024 and 0.0099 h⁻¹. These bacterial strains are novel for TBP degradation and could be used as an important bioresource for efficient decontamination of TBP polluted waste streams.     

Biodegradation of epoxyconazole and piraclostrobin fungicides by Klebsiella sp. from soil

Three bacterial strains have been isolated from soil in which soybean had been continuously cropped and treated with Opera^®, a fungicide containing epoxyconazole and pyraclostrobin. The three strains (1,805, 2,801 and 3,803), obtained from soil at 80–100 cm depth, were selected on medium containing 0.03% Opera^®. Morphological examination revealed that the strains were Gram-negative, and two of them (1,805 and 2,801) exhibited polymorphism. The growth profiles demonstrated that 1,805 and 3,803 were more efficient growing in the presence of Opera^® than 2,801. Maximum growth was reached between 24 and 48 h, however, 2,801 was not able to survive after this period. The total protein content produced by 1,805, 2,801 and 3,803 in liquid selective medium containing Opera^® were 111.0 ± 0.02, 80.0 ± 0.05 and 130.5 ± 0.07 μg/ml, respectively. According to its biochemical and molecular features, strain 1,805 was identified as Klebsiella sp. On the basis of the characteristics presented (facultative anaerobic nature, polymorphic character and capacity of growing in the presence of Opera^®) strain 1,805 seems to be able to degrade the epoxyconazole and pyraclostrobin.     

relA基因敲除对克雷伯氏菌CPK降解毒死蜱的影响

目的:敲除毒死蜱降解菌Klebsiella sp.CPK菌中的relA基凶,研究其对毒死蜱降解的影响.方法:采用同源重组技术敲除了Klebsiella sp.CPK菌中魔斑合成酶编码基因relA,通过PCR及RT-PCR扩增对其敲除进行了验证,分别采用分光光度法和气相色谱检测了该突变体对毒死蜱的耐受性和对毒死蜱的降解.结果:获得了一株relA基因敲除菌株△relA.在毒死蜱胁迫下,△ relA菌株的耐受能力和对毒死蜱的降解能力均弱丁野生型的CPK菌株.在初始降解的第一天,两者的降解速率相差最大,CPK菌株对毒死蜱的降解率高达50%以上,而△relA菌株对毒死蜱的降解率却低于15%.结论:relA基因的产物魔斑参与调控了Klebsiella sp.CPK菌在毒死蜱胁迫下的严谨反应,可能以此增强了该菌株对毒死蜱的耐受性并促进了它对毒死蜱的降解.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

Improved process for exopolysaccharide production by Klebsiella pneumoniae sp. pneumoniae by a fed-batch strategy

Exopolysaccharide (EPS) was produced by Klebsiella pneumoniae K63 grown in fed-batch cultures using different procedures of the supply of carbon or nitrogen (N) source, or both. Cultures grown with excess of glucose and limitation or exhaustion of N produced 54.8 and 47.4 g(EPS) l(-1), respectively. These cultures also led to an accumulation of 'overflow' metabolites representing more than 16% of carbon conversion. The consistency indexes ( K ) obtained to the end of the cultures, characteristic of the rheological property of the biopolymer, were 16.4 Pa s(n) for N deficiency and 5.2 Pa s(n) for N limitation conditions. The simultaneous limitation of glucose and N decreased the excretion of co-metabolites (6.4% of carbon conversion) and the EPS production (18.1 g(EPS) l(-1)), while improving the quality of the polysaccharide, characterized by the highest K of 126.2 Pa s(n) and the highest pseudoplasticity degree (flow behaviour index, n=0.2).     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.     

Biodegradation of tert-butylphenyl diphenyl phosphate.

The biodegradation of tert-butylphenyl diphenyl phosphate (BPDP) was examined in microcosms containing sediment and water from five different ecosystems as part of our studies to elucidate the environmental fate of phosphate ester flame retardants. Biodegradation of [14C]BPDP was monitored in the environmental microcosms by measuring the evolution of 14CO2. Over 37% of BPDP was mineralized after 8 weeks in microcosms from an ecosystem which had chronic exposure to agricultural chemicals. In contrast, only 1.7% of BPDP was degraded to 14CO2 in samples collected from a noncontaminated site. The exposure concentration of BPDP affected the percentage which was degraded to 14CO2 in microcosms from the two most active ecosystems. Mineralization was highest at a concentration of 0.1 mg of BPDP and was inhibited with 10- and 100-fold higher concentrations of BPDP in these microcosms. Indigenous heterotrophic and BPDP-utilizing microbial populations and phosphoesterase enzyme activities were highest in sediments which had the highest biodegradation of BPDP. We observed adaptive increases in both microbial populations and phosphoesterase enzymes in some sediments acclimated to BPDP. Chemical analyses of the residues in the microcosms indicated undegraded BPDP and minor amounts of phenol, tert-butylphenol, diphenyl phosphate, and triphenyl phosphate as biodegradation products. These data suggest that the microbial degradation of BPDP results from at least three catabolic processes and is highest when low concentrations of BPDP are exposed to sediment microorganisms of eutrophic ecosystems which have high phosphotri- and diesterase activities and previous exposure to anthropogenic chemicals.     

节杆菌PJ3降解咔唑和联苯

菌株PJ3经16SrDNA鉴定为节杆菌(Arthrobacter sp.),同源树表明,该菌株与已报道咔唑降解菌株IC177同源关系最近,其次是联苯降解菌株K37和R04。为了明确PJ3菌株对咔唑和联苯的降解能力,在PJ3生长的最适pH值进行优化的基础上,利用分光光度法测定了该菌株在不同浓度咔唑和联苯的矿物培养基中的生长速率以及对咔唑和联苯的降解性能。结果表明,在pH为7、8、9时,PJ3菌株的生长速率一致,而且高于其他pH值的生长速率。在pH为7、咔唑浓度为0.1mg.ml-1的CNFMM培养基中,PJ3菌株生长速度较快,而且15d咔唑的降解率可达到73%。在pH为7、联苯浓度为0.5mg.ml-1的MSB培养基中PJ3菌株生长最好。在0.1和0.5mg.ml-1联苯浓度下,15d对联苯的降解率可达80%～85%。综合评价,PJ3菌株降解咔唑比较适宜的浓度范围应该为0.1～0.2mg.ml-1,降解联苯较适宜的浓度范围为0.1～0.5mg.ml-1。     

Production of murine collagen-induced arthritis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant.

Collagen-induced arthritis (CIA) was produced in mice with non H-2q and H-2r haplotypes by repeated immunization of porcine type-II collagen (CII) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes that appeared in joints of repeatedly immunized mice were characterized by destruction of normal joint structure, synovial hyperplasia with proliferation of synovial cells, and infiltration of inflammatory cells. No such lesions were produced in mice receiving repeated injections of CII alone or KO3 LPS alone. Development of the humoral antibody and the delayed-type hypersensitivity to CII was exclusively found in mice immunized with the mixture of CII and KO3 LPS. It was therefore suggested that arthritis lesions induced by repeated immunization with the mixture of CII and KO3 LPS might be caused by an autoimmune mechanism, and that the experimental model might be useful for characterization of human rheumatoid arthritis (RA).     

Biodegradation of pyrene by Candida sp. S1 under high salinity conditions

Polycyclic aromatic hydrocarbon is a toxic recalcitrant environmental pollutant and its removal from the environment is very essential. In this study, a novel S1 strain isolated from the tropical rain forest was identified as Candida species based on 18S rRNA. The pyrene biodegradation was performed by Candida sp. S1. Pyrene was 35% degraded in 15 days. The percentage of pyrene biodegradation increased up to 75% with 24 g L⁻¹ of sodium chloride and decreased along with increasing salinity. Under the acidic condition, the biodegradation was increased up to 60% at pH 5. It was also found that the increasing glucose concentration of more than 10 g L⁻¹ had no significant effect on pyrene biodegradation, while agitation proved to have greater influence. There was a positive relationship between biomass growth and biodegradation rate of pyrene. One pyrene metabolite was identified from the extract solution and analyzed by a thin-layer chromatography, UV–visible absorption and gas chromatography–mass spectrometry. The metabolite found in the pyrene degradation was benzoic acid. Suitable conditions must be found to promote a successful microbial augmentation in liquid culture.

     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.     

Whole-genome analysis of Klebsiella pneumoniae from bovine mastitis milk in the U.S.

Dairy cattle mastitis has long been one of the most common and costly diseases in the dairy industry worldwide, due to its significant impact on milk production and animal welfare. Among all mastitis causing bacterial pathogens, Klebsiella pneumoniae causes the largest milk loss. To better understand the genomic features of this population, 180 K. pneumoniae strains isolated from dairy cattle mastitis milk in 11 U.S. states were sequenced. The phylogenetic analysis classified all mastitis-causing K. pneumoniae into two major phylogroups, with exclusive predominance in phylogroup KpI. Analysis of more than 61 sequence types, 51 capsular types and 12 lipopolysaccharide O-antigen types revealed great genomic diversity of this K. pneumoniae population. Approximately 100 gene units in accessory genomes were detected with significantly higher prevalence in bovine mastitis strains, compared to human-sourced or dairy environmental strains. The most notable genes were identified associated with ferric citrate uptake, lactose fermentation and resistance to heavy metals. The acquired antimicrobial resistance genes were identified in sporadic mastitis strains. This comprehensive genomic epidemiological study provides insights for a better understanding of the virulence of mastitis-causing K. pneumoniae strains and may lead to the development of novel diagnostic tools and preventive strategies.     

Nitrogenase of Klebsiella pneumoniae nifV mutants.

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.     

Contemplating 3-Hydroxypropionic Acid Biosynthesis in Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP) is a commercially valuable platform chemical from which an array of C₃ compounds can be generated. Klebsiella pneumoniae has been considered a promising species for biological production of 3-HP. Despite a wealth of reports related to 3-HP biosynthesis in K. pneumoniae, its commercialization is still in infancy. The major hurdle hindering 3-HP overproduction lies in the poor understanding of glycerol dissimilation in K. pneumoniae. To surmount this problem, this review aims to portray a picture of 3-HP biosynthesis, involving 3-HP-synthesizing strains, biochemical attributes, metabolic pathways and key enzymes. Inspired by the state-of-the-art advances in metabolic engineering and synthetic biology, here we advocate protocols for overproducing 3-HP in K. pneumoniae. These protocols range from cofactor regeneration, alleviation of metabolite toxicity, genome editing, remodeling of transport system, to carbon flux partition via logic gate. The feasibility for these protocols was also discussed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0513-0) contains supplementary material, which is available to authorized users.     

The capsular network of Klebsiella pneumoniae.

Attempts at improving chemical fixation for electron-microscopic observation of the capsule of Klebsiella pneumoniae were made. The capsule was preserved by using alcian blue - lanthanum and tris-(1-aziridinyl) phosphine oxide (TAPO) - aldehyde - osmium procedures. Despite the different retention of the overall capsular material and minor variations in morphological details, in both cases the interpretation of ultrastructural patterns suggested that the capsule be composed of a meshed network of thin polysaccharide fibrils radiating from the cell wall. This organization is in keeping with all recognized chemical properties of bacterial polysaccharide capsules or, at least, does not contradict them. Moreover, an effective preservation of bacterial structures other than capsule has been obtained, mostly in specimens fixed by the TAPO-aldehyde-osmium method, a fact which gives further reliability to the technical approach used for capsule visualization.     

5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae. 1. Purification and properties

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62-1 (= Klebsiella pneumoniae ATCC 25306). A 3300-fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE-cellulose, Sephadex G-75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49%. An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of Mr = 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme-catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3-phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 60 degrees C, the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations. Non-linear Lineweaver-Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme-substrate complexes: forward reaction: 43 microM (PEP) and 22 microM (S3P); reverse reaction: 1.3 microM (EPSP) and 2.6 mM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction.