Biosorption of Copper by Paenibacillus polymyxa Cells and their Exopolysaccharide

Summary Biosorption of heavy metals by gram-positive, non-pathogenic and non-toxicogenic Paenibacillus polymyxa P13 was evaluated. Copper was chosen as a model element because it is a pollutant originated from several industries. An EPS (exopolysaccharide)-producing phenotype exhibited significant Cu(II) biosorption capacity. Under optimal assay conditions (pH 6 and 25 °C), the adsorption isotherm for Cu(II) in aqueous solutions obeyed the Langmuir model. A high q value (biosorption capacity) was observed with whole cells (q_max=112 mgCu g⁻¹). EPS production was associated with hyperosmotic stress by high salt (1 M NaCl), which led to a significant increase in the biosorption capacity of whole cells (q_max=150 mgCu g⁻¹). Biosorption capacity for Cu(II) of the purified EPS was investigated. The maximum biosorption value (q) of 1602 mg g⁻¹ observed with purified EPS at 0.1 mg ml⁻¹ was particularly promising for use in field applications.     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.     

Medium optimization and proteome analysis of (R,R)-2,3-butanediol production by Paenibacillus polymyxa ATCC 12321

Paenibacillus polymyxa can produce the (R,R)-stereoisomer of 2,3-butanediol (2,3-BDL) which is industrially very useful. Two important factors affecting (R,R)-BDL production by P. polymyxa ATCC 12321, medium composition, and addition of acetic acid to the culture were investigated in this study with accompanying comparative proteomic analysis. For this purpose, a simple control strategy of O₂ supply was applied on the basis of an optimized basal medium: after a short period of batch cultivation with relatively high O₂ supply, the culture is switched into strong O₂ limitation, thereby promoting BDL formation. Three parallel fed-batch cultures starting from the same batch culture in an early stationary phase were then comparatively studied: the first one was running as control with the only change of O₂ supply; the second was, in addition, supplemented with 0.5 g/L yeast extract; and the third one was further added with 6 g/L acetate. Proteomic analyses of the three fed-batch cultures identified more than 86 proteins involved primarily in the central carbon metabolism, amino acid biosynthesis, energy metabolism, and stress responses. The examination of expression patterns of selected proteins, especially combined with fermentation data, gave valuable insights into the metabolic regulation of P. polymyxa under the different given conditions. Based on the proteomic analysis and further medium optimization studies, methionine was identified as one major growth-limiting factor in the basal medium and explains well the effect of yeast extract. Acetic acid was found to trigger the so far less studied acetone biosynthesis pathway in this organism. The latter is suggested in turn to enhance the switch from acidogenesis to solventogenesis. Thus, these findings extended our knowledge about BDL formation in P. polymyxa and provided useful information for further strain and process optimization.     

葡萄糖和木糖双底物生物转化生产2,3-丁二醇和氢气的代谢计量分析

以Klebsiella pneumoniae利用葡萄糖和木糖双底物生物转化生产2,3-丁二醇和氢气过程为研究对象,对其进行代谢计量分析。分析结果显示:2,3-丁二醇和氢气相对于底物葡萄糖和木糖的质量收率依赖于还原能力NADH2氧化磷酸化的分率(δ)。当δ=27时,即在总还原能力NADH2中有27mol NADH2被氧化磷酸化,剩余部分用来产生氢气,呼吸商为14时,2,3-丁二醇和氢气的最优质量收率分别为50%和0.8%;而当δ=1,即还原能力NADH2全部被氧化磷酸化、不产生氢气,呼吸商为4时,2,3-丁二醇的质量收率为37.5%;2,3-丁二醇和氢气的质量收率与底物中葡萄糖和木糖的比值无关。而氢气的摩尔收率与底物中葡萄糖和木糖的比值相关,当底物全部是葡萄糖或木糖时,其最优摩尔收率分别为71%和60%。该分析结果为葡萄糖和木糖双底物生物转化生产2,3-丁二醇过程的实验研究奠定了理论基础。     

Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.     

Novel (2R,3R)-2,3-butanediol dehydrogenase from potential industrial strain Paenibacillus polymyxa ATCC 12321

A (2R,3R)-2,3-butanediol dehydrogenase (BDH99::67) from Paenibacillus polymyxa ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of meso- and (2S,3S)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which meso- and (2S,3S)-2,3-butanediol dehydrogenases belong.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

An integrated high throughput strategy to screen mutants of Paenibacillus polymyxa with high polymyxin E-productivity

An integrated high-throughput screening (HTS) strategy was developed to screen large numbers of polymyxin E-producing mutants of Paenibacillus polymyxa. Various types of mutants were transferred onto the surfaces of solidified agar in 96-well microtiter plates, and then inoculated to 96-deep-well microtiter plates for micro-cultivation. The culture conditions were optimized for the production of polymyxin E. The supernatants from the micro-culture plates were transferred to 96-well bioassay microtiter plates containing Escherichia coli JM109 for high-throughput bioassay. By using this high-throughput screening (HTS) procedure, one best producer P. polymyxa PE 5.808 was identified from a large NTG mutated library with about 5,000 isolates. The volumetric productivity of polymyxin E of P. polymyxa PE 5.808 was 1,200 μg/ml in shake flasks, about 140% improvement compared with that of the wild type strain.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

Optimization, purification, characterization and antioxidant activity of an extracellular polysaccharide produced by Paenibacillus polymyxa SQR-21

The optimization, purification and characterization of an extracellular polysaccharide (EPS) from a bacterium Paenibacillus polymyxa SQR-21 (SQR-21) were investigated. The results showed that SQR-21 produced one kind of EPS having molecular weight of 8.96 × 10⁵ Da. The EPS was comprised of mannose, galactose and glucose in a ratio of 1.23:1.14:1. The ratio of monosaccharides and glucuronic acid was 7.5:1. The preferable culture conditions for EPS production were pH 6.5, temperature 30 °C for 96 h with yeast extract and galactose as best N and C sources, respectively. The maximum EPS production (3.44 g L⁻¹) was achieved with galactose 48.5 g L⁻¹, Fe³⁺ 242 μM and Ca²⁺ 441 μM. In addition, the EPS showed good superoxide scavenging, flocculating and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities were determined. These results showed the great potential of EPS produced by SQR-21 to be used in industry in place of synthetic compounds.     

生物法生产2,3-丁二醇研究进展

2,3-丁二醇是一种重要的化工原料,可广泛应用于多个领域。二战期间由于合成橡胶需要大量1,3-丁二烯,2,3-丁二醇生产空前发展。近年来,由于聚对苯二甲酸丁烯树脂、γ-丁内酯,Spandex弹性纤维及其前体的需求增长,2,3-丁二醇的需求和产量也稳步增长。多年来,生物法生产2,3-丁二醇虽然得到了广泛的研究,但一直没有实现工业化。本文从产生2,3-丁二醇的菌种及2,3-丁二醇的生理意义、代谢途径、旋光异构体的形成机理、影响发酵的因素与产物的提纯等方面对生物法生产2,3-丁二醇进行了综述并提出了生物法生产2,3-丁二醇要解决的几个问题。     

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C₅-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M _r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M _r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - M _r relative molecular mass - MTPA-Cl -methoxy--trifluoromethylphenyl acetic acid chloride - PEIC 1-phenylethylisocyanate     

Auxin production and detection of the gene coding for the Auxin Efflux Carrier (AEC) protein in <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis>

Different species of Paenibacillus are considered to be plant growth-promoting rhizobacteria (PGPR) due to their ability to repress soil borne pathogens, fix atmospheric nitrogen, induce plant resistance to diseases and/or produce plant growth-regulating substances such as auxins. Although it is known that indole-3-acetic acid (IAA) is the primary naturally occurring auxin excreted by Paenibacillus species, its transport mechanisms (auxin efflux carriers) have not yet been characterized. In this study, the auxin production of P. polymyxa and P. graminis, which are prevalent in the rhizospheres of maize and sorghum sown in Brazil, was evaluated. In addition, the gene encoding the Auxin Efflux Carrier (AEC) protein from P. polymyxa DSM36(T) was sequenced and used to determine if various strains of P. polymyxa and P. graminis possessed this gene. Each of the 68 P. polymyxa strains evaluated in this study was able to produce IAA, which was produced at concentrations varying from 1 to 17 microg/ml. However, auxin production was not detected in any of the 13 P. graminis strains tested in this study. Different primers were designed for the PCR amplification of the gene coding for the AEC in P. polymyxa, and the predicted protein of 319 aa was homologous to AEC from Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis. However, no product was observed when these primers were used to amplify the genomic DNA of seven strains of P. graminis, which suggests that this gene is not present in this species. Moreover, none of the P. graminis genomes tested were homologous to the gene coding for AEC, whereas all of the P. polymyxa genomes evaluated were. This is the first study to demonstrate that the AEC protein is present in P. polymyxa genome.     

Identification of LI-F type antibiotics and di-n-butyl phthalate produced by Paenibacillus polymyxa

Paenibacillus polymyxa strain JSa-9, a soil isolate that displayed antibacterial and antifungal activities in vitro, had been found to produce two types of antimicrobial substances. The two compounds were extracted from the fermentation broth of JSa-9 using ethyl acetate and subsequently purified by high performance liquid chromatography. By means of liquid chromatography-mass spectrometry and tandem mass spectrometry analysis, one of two antagonistic compounds was determined as di-n-butyl phthalate. And another was characterized as a mixture of related peptides of molecular masses of 883, 897, 911, 947, and 961 Da, with the most likely structure of them determined to be a cyclic depsipeptide with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety bound to a free amino group. These peptides were therefore members of the LI-F group of cyclic depsipeptides.     

Production of 2,3-butanediol from glucose byBacillus licheniformis

Bacillus licheniformis produced 2,3-butanediol from glucose with an optimum yield of 47 g/100 g glucose after 72 h of growth on a peptone/beef extract medium containing 2% (w/v) glucose at pH 6.0 and 37°C. This yield of 2,3-butanediol was higher than those previously reported forKlebsiella oxytoca (37 g/100 g glucose) andBacillus polymyxa (24 g/100 glucose).     

不同有机酸对粘质沙雷氏菌合成2,3-丁二醇的影响

考察了不同的短链有机酸对粘质沙雷氏菌合成2,3－丁二醇的影响,结果表明乙酸、乳酸、丙酮酸、琥珀酸、延胡索酸和柠檬酸均能在一定程度上提高2,3－丁二醇的产量,其中乙酸的效果最为明显,在基础培养基中添加6g／L乙酸,与对照相比,2,3．丁二醇的产量提高了91．06％,此外菌体干重也提高了58．28％。为了揭示其中的调控机制,构建了启动子：lacZ融合报告载体,fncz活性测定显示六种有机酸均可提高报告基因B．半乳糖苷酶的表达,其中乙酸可提高B．半乳糖苷酶活性近4倍,暗示六种有机酸促进2,3－丁二醇的合成可能与诱导该合成途径相关基因的表达有关。     

粘质沙雷氏菌产2,3-丁二醇培养基的优化

研究了各种碳源、氮源、柠檬酸及无机盐对细胞生长与产物形成的影响,通过单因子、正交及中心组合设计响应面分析优化发酵培养基。结果表明在培养基中添加柠檬酸不但可以促进细胞生长与糖耗速度,还可以缩短发酵周期,提高2,3-丁二醇的产量。采用优化后的培养基,2,3-丁二醇的产量由14.03g/L增加到39.27g/L,提高了近3倍。     

Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture

The optimum dilution rate for 2, 3-butanediol (BDL) production by Paenibacillus polymyxa was 0.2 h1 and the optical purity of BDL remained above 98 % at all dilution rates. With decreasing culture pH, ethanol and BDL production increased, whereas the optical purity of BDL decreased to 94 % at pH 5.7. In the chemostat culture at pH 6.3 and a 0.1 h1 dilution rate, the optimum air supply for BDL production was 200 ml min–1 in which the O2 uptake rate was 6.7 mmol l–1 h–1. Under this condition, the optical purity of BDL decreased to 93 %. © Rapid Science Ltd. 1998