Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Arsenic-resistant <Emphasis Type="BoldItalic">Pseudomonas</Emphasis> spp. and <Emphasis Type="BoldItalic">Bacillus</Emphasis> sp. bacterial strains reducing As(V) to As(III), isolated from Alps soils,Italy

Five arsenic-resistant bacterial strains (designated MP1400, MP1400a, MP1400d, APSLA3, and BPSLA3) were isolated from soils collected at the Alps region (Italy), which showed no contamination by arsenic. Phylogenetic analysis of the 16S rRNA gene sequences assigned them to the genera Pseudomonas and Bacillus. Bacillus sp. strain 1400d and Pseudomonas spp. strains APSLA3 and MP1400 showed higher tolerance to As(III), as indicated by minimum inhibitory concentrations of 10 mmol/L. Pseudomonas sp. strain MP1400 exhibited higher tolerance to As(V) (minimum inhibitory concentration of 135 mmol/L). The isolated arsenic-resistant strains were able to reduce As(V) to As(III), especially Pseudomonas sp. strain MP1400 reducing 2 mmol/L of As(V) to As(III) within 24 h. The results suggest that the isolated bacterial strains play a role in the arsenic biogeochemical cycle of arsenic-poor soils in the Alps mount area.     

Biodegrading of pyrene by a newly isolated Pseudomonas putida PL2

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic compounds derived from natural sources and anthropogenic processes, which have been recommended as priority pollutants. Degradation of PAHs in the environment is becoming more necessary and urgent. In the current study, strain PL2, which is capable of growing aerobically on pyrene (PYR) as the sole carbon source, was isolated from hydrocarbons-contaminated soil and then identified as Pseudomonas putida by morphological and physiological characteristics as well as 16S rDNA sequence. The strain PL2 was able to degrade 50.0% of the pyrene at 28°C within 6 days in the presence of 50 mg/L pyrene, while the strain PL2 degraded 50.0% of the pyrene within 2 days when a solution of 50 mg/L pyrene and 50 mg/L phenanthrene was used. In addition, phenanthrene was shown to increase the biodegradation efficiency of pyrene by the strain PL2. The order of degradation by the strain PL2 was pH 6.0 > pH 7.0 > pH 5.0 > pH 8.0. The degradation rate of PYR in the soil by the strain PL2 reached 70.0% at the 10^th day. The dynamics of PYR degradation in soil by PL2 was fit to the first order model and the strain PL2 was shown to efficiently degrade PYR in soil. The current study showed that P. putida PL2 was a novel bacterium that could degrade pyrene and holds great promise for use in PAHs bioremediation in soil.     

Biodegradation of methyl parathion and p-nitrophenol by a newly isolated Agrobacterium sp. strain Yw12

Strain Yw12, isolated from activated sludge, could completely degrade and utilize methyl parathion as the sole carbon, phosphorus and energy sources for growth in the basic salt media. It could also completely degrade and utilize p-nitrophenol as the sole carbon and energy sources for growth in the minimal salt media. Phenotypic features, physiological and biochemical characteristics, and phylogenetic analysis of 16S rRNA sequence showed that this strain belongs to the genus of Agrobacterium sp. Response surface methodology was used to optimize degradation conditions. Under its optimal degradation conditions, 50 mg l⁻¹ MP was completely degraded within 2 h by strain Yw12 and the degradation product PNP was also completely degraded within 6 h. Furthermore, strain Yw12 could also degrade phoxim, methamidophos, chlorpyrifos, carbofuran, deltamethrin and atrazine when provided as the sole carbon and energy sources. Enzymatic analysis revealed that the MP degrading enzyme of strain Yw12 is an intracellular enzyme and is expressed constitutively. These results indicated that strain Yw12 might be used as a potential and effective organophosphate pesticides degrader for bioremediation of contaminated sites.     

Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μ_max of 0.094 h⁻¹ and S _m of 1,435 mg l⁻¹. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> LD-8547

A fibrinolytic enzyme producing strain Bacillus subtilis LD-8 was isolated from douchi, a traditional Chinese soybean-fermented food. After mutagenesis treatments by UV, NTG (N-methyl-N′-nitroso-N-nitroso-guanidine) and γ-radiation, a high fibrinolytic enzyme producing strain B. subtilis LD-8547 was obtained. Under optimum condition, LD-8547 was able to yield the average fibrinolytic activity of 4220 U/mL in 15 L fermenter. The strong fibrin-specific enzyme was purified from supernatant of B. subtilis LD-8547 culture broth using the combination of various steps. The optimal temperature and pH value of this fibrinolytic enzyme were 50 °C and 8.0, respectively. The molecular weight was about 30 kDa measured by SDS-PAGE. The amidolytic activity of this fibrinolytic enzyme was inhibited completely by 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), but EDTA and EGTA did not affect the enzyme activity. The apparent K _m and V _max values were 0.521 mmol/L and 0.049 mmol/min, respectively. In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent.     

一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点.[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性.[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观...     

Degradation of chlorobenzene by strain <Emphasis Type="Italic">Ralstonia pickettii</Emphasis> L2 isolated from a biotrickling filter treating a chlorobenzene-contaminated gas stream

A Ralstonia pickettii species able to degrade chlorobenzene (CB) as the sole source of carbon and energy was isolated from a biotrickling filter used for the removal of CB from waste gases. This organism, strain L2, could degrade CB as high as 220 mg/L completely. Following CB consumption, stoichiometric amounts of chloride were released, and CO₂ production rate up to 80.2% proved that the loss of CB was mainly via mineralization and incorporation into cell material. The Haldane modification of the Monod equation adequately described the relationship between the specific growth rate and substrate concentration. The maximum specific growth rate and yield coefficient were 0.26 h⁻¹ and 0.26 mg of biomass produced/mg of CB consumed, respectively. The pathways for CB degradation were proposed by the identification of metabolites and assay of ring cleavage enzymes in cell extracts. CB was degraded predominantly via 2-chlorophenol to 3-chlorocatechol and also partially via phenol to catechol with subsequent ortho ring cleavage, suggesting partially new pathways for CB-utilizing bacteria.     

Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969

Laccase is a widespread group of multi-copper enzymes which can catalyze the oxidation of a variety of organic compounds, with concomitant reduction of molecular oxygen to water. It has a wide application in industrial processes, particularly in renewable bio-energy industry. In this study, Pleurotus ostreatus strain 10969 with high yield of laccase, previously isolated from edible fungus growing on Juncao, was applied for optimization of fermentation media and growth parameters for the maximal enzyme production through response surface methodology and further characterization of the laccase activity. The results show that glucose and Mg²⁺ are the key ingredients for laccase production with the optimum concentration of 0.0988 g/mL and 7.3 mmol/L respectively. Compared to the initial medium, the highest laccase yield observed is approximately increased by 2.5 times under the optimized conditions. Extracellular laccase was then purified and its characters were analyzed. The molecular weight of the laccase is about 40 kDa, and the optimum pH and temperature for its activity is 4.0 and 50 °C with the corresponding Km and Vmax of 0.31 mmol/L and 303.25 mmol/min respectively. DTT, β-mercaptoethanol and NaN₃ nearly inhibit all activity of the laccase, as well as the metal ions especially Ag⁺. In summary, our results will facilitate the utilization of plant lignin in biomass energy industry.     

一株红球菌属细菌LV4在高盐条件下的吡啶降解特性

由微生物介导的吡啶降解技术是解决高盐吡啶环境污染的经济有效方法之一,开发具有吡啶降解性能且能够耐受高盐分的微生物是该类研究的重要前提。本研究从山西太原钢铁公司焦化废水处理厂活性污泥中分离培养了一株耐盐吡啶降解菌,通过菌落形态和16S rDNA基因系统发育分析,鉴定其为红球菌属(Rhodococcus sp.)的细菌。耐盐性实验结果表明,菌株LV4能够在0%–6%盐度范围内生长,并完全降解初始浓度为500 mg/L的吡啶;但当盐度高于4%时,菌株LV4因其生长变缓而导致吡啶完全降解时间明显延长。扫描电镜结果显示,高盐环境会使菌株LV4的菌体细胞分裂变慢,诱导细胞表面分泌更多的颗粒状胞外聚合物(extracellular polymeric substance, EPS)。当盐度不高于4%时菌株LV4主要依靠EPS中蛋白含量的增加来响应高盐环境的冲击。单因素实验优化发现,菌株LV4在盐度为4%的高盐环境中降解吡啶的最佳条件为温度30℃、pH 7.0、转速为120 r/min (DO 10.30 mg/L)。最优条件下菌株LV4对于初始浓度为500 mg/L的吡啶,在经过12 h的适应期后,...     

Effects of 6-methoxy-2-benzoxazolinone on the germination and α-amylase activity in lettuce seeds

Germination of lettuce seeds was inhibited by 6-methoxy-2-benzoxazolinone (MBOA) at concentrations greater than 0.03 mmol/L. MBOA also inhibited the induction of α-amylase activity in the lettuce seeds at concentrations greater than 0.03 mmol/L. These two concentration–response curves for the germination and α-amylase indicate that the percentage of the germination was positively correlated with the activity of α-amylase in the seeds. Lettuce seeds germinated around 18 h after incubation and inhibition of α-amylase by MBOA occurred within 6 h after seed incubation. These results show that MBOA may inhibit the germination of lettuce seeds by inhibiting the induction of α-amylase activity.     

Isolation and complete genome sequence of Halorientalis hydrocarbonoclasticus sp. nov., a hydrocarbon-degrading haloarchaeon

Bioremediation in hypersaline environments is particularly challenging since the microbes that tolerate such harsh environments and degrade pollutants are quite scarce. Haloarchaea, however, due to their inherent ability to grow at high salt concentrations, hold great promise for remediating the contaminated hypersaline sites. This study aimed to isolate and characterize novel haloarchaeal strains with potentials in hydrocarbon degradation. A haloarchaeal strain IM1011 was isolated from Changlu Tanggu saltern near Da Gang Oilfield in Tianjin (China) by enrichment culture in hypersaline medium containing hexadecane. It could degrade 57 ± 5.2% hexadecane (5 g/L) in the presence of 3.6 M NaCl at 37 °C within 24 days. To get further insights into the mechanisms of petroleum hydrocarbon degradation in haloarchaea, complete genome (3,778,989 bp) of IM1011 was sequenced. Phylogenetic analysis of 16S rRNA gene, RNA polymerase beta-subunit (rpoB’) gene and of the complete genome suggested IM1011 to be a new species in Halorientalis genus, and the name Halorientalis hydrocarbonoclasticus sp. nov., is proposed. Notably, with insights from the IM1011 genome sequence, the involvement of diverse alkane hydroxylase enzymes and an intact β-oxidation pathway in hexadecane biodegradation was predicted. This is the first hexadecane-degrading strain from Halorientalis genus, of which the genome sequence information would be helpful for further dissecting the hydrocarbon degradation by haloarchaea and for their application in bioremediation of oil-polluted hypersaline environments.

     

Application of polyethylene glycol immobilized Clostridium sp. LS2 for continuous hydrogen production from palm oil mill effluent in upflow anaerobic sludge blanket reactor

A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium sp. LS2 for continuous hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. Palm oil mill effluent (POME) was used as the substrate carbon source. The optimal amount of PEG-immobilized cells for anaerobic hydrogen production was 12% (w/v) in the UASB reactor. The UASB reactor containing immobilized cells was operated at varying hydraulic retention times (HRT) that ranged from 24 to 6 h at 3.3 g chemical oxygen demand (COD)/L/h organic loading rate (OLR), or at OLRs that ranged from 1.6 to 6.6 at 12 h HRT. The best volumetric hydrogen production rate of 336 mL H₂/L/h (or 15.0 mmol/L/h) with a hydrogen yield of 0.35 L H₂/g COD_removed was obtained at a HRT of 12 h and an OLR of 5.0 g COD/L/h. The average hydrogen content of biogas and COD reduction were 52% and 62%, respectively. The major soluble metabolites during hydrogen fermentation were butyric acid followed by acetic acid. It is concluded that the PEG-immobilized cell system developed in this work has great potential for continuous hydrogen production from real wastewater (POME) using the UASB reactor.     

<Emphasis Type="Italic">MLXIPL</Emphasis> variant in individuals with low and high triglyceridemia in white population in Central Europe

We tested the hypothesis that the MLXIPL rs3812316 variant predicts plasma triglyceride (TG) levels. We compared three groups of adult individuals: 162 persons with TG > 10 mmol/L, 266 persons with TG < 0.65 mmol/L, and 2,043 population-based controls (range of TG concentrations 0.7–8.7 mmol/L). We found a small difference in the frequency of the Gln allele carriers between population controls (20.4%) and persons with low TG (26.3%, P = 0.033). We found no difference between individuals with high TG and population controls, and there was no association between the MLXIPL variant and plasma TG levels among the population controls.     

Leucine improves the component of isovalerylspiramycins for the production of bitespiramycin

Bitespiramycin, a group of 4″-O-acylated spiramycins with 4″-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4″-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 ± 0.33% total isovalerylspiramycins was recorded compared to 40.9 ± 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4″ side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.     

Nicotine degradation enhancement by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZCJ during aging process of tobacco leaves

Nicotine is a key harmful component of tobacco and cigarettes, and the development of low-nicotine cigarettes is of increasing importance in the market. The objectives of this study are to isolate native nicotine-degrading strains and evaluate their feasibility for nicotine reduction during the aging (or fermentation) of tobacco leaves. A novel nicotine-degrading strain was isolated and identified as Pseudomonas stutzeri ZCJ based on its 16S rDNA sequence and morphological-biochemical characteristics. In submerged cultures, P. stutzeri ZCJ could tolerate 4.5 g/L nicotine and completely degrade 1.5 g/L nicotine within 24 h at 37°C and pH 7.4. The addition of glucose (1 g/L) could improve nicotine degradation by P. stutzeri ZCJ in submerged cultures. After submerged culturing, the cell suspension of P. stutzeri ZCJ could be utilized to improve nicotine reduction in tobacco leaves during solid-state fermentation. The nicotine content of tobacco leaves decreased by as much as 32.24% after 7 days of solid-state fermentation by P. stutzeri ZCJ, suggesting the industrial application potential of the native strain to enhance nicotine degradation during the aging of tobacco leaves.     

Optimization of phenazine-1-carboxylic acid production by a gacA/qscR-inactivated Pseudomonas sp. M18GQ harboring pME6032Phz using response surface methodology

Phenazine-1-carboxylic acid (PCA) production was enhanced in Pseudomonas sp. M18 wild strain and its mutants carrying recombinant pME6032Phz for phz gene cluster overexpression, among which Pseudomonas sp. strain M18GQ/pME6032Phz, a gacA and qscR double gene chromosomally inactivated mutant harboring pME6032Phz, showed the highest PCA yield. The conditions for fermentation and isopropyl-β-d-1-thiogalactopyranoside (IPTG) induction were optimized for strain M18GQ/pME6032Phz in shake flask experiments. A one-factor-at-a-time approach, followed by a fractional factorial design identified soybean meal, corn steep liquor, and ethanol as statistically significant factors. Optimal concentrations and mutual interactions of the factors were then determined by the method of steepest ascent and by response surface methodology based on the center composite design. The predicted PCA production was 6,335.2 mg/l after 60 h fermentation in the optimal medium of 65.02 g soybean meal, 15.36 g corn steep liquor, 12 g glucose, 21.70 ml ethanol, and 1 g MgSO₄ per liter in the flask fermentations, with induction of 1.0 mmol/l IPTG 24 h after inoculation. In an experimental validation under these conditions, the maximum PCA production was 6,365.0 mg/l. This represents a ∼60% increase over production by strain M18GQ in optimal conditions. The negative effect of plasmid pME6032 on the expression of chromosomally located phz gene cluster was found in Pseudomonas sp. M18GQ, and the possible reason was discussed in the text.     

A new fermentation strategy for S-adenosylmethionine production in recombinant Pichia pastoris

A recombinant Pichia pastoris MutS expressing SAM2 gene of Saccharomyces cerevisiae was cultured for S-adenosylmethionine (SAM) accumulation. Effect of the amount of methanol added (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 6.0%, 10.0%, and 12.0%) and cell densities (9.57, 13.47, 21.74, 30.90, and 41.24 g/L dry cell weight (DCW)) on yield of SAM was found in flask cultivations. In flask experiments, maximal yield of SAM (1.29 g/L) was obtained at 2.0% methanol added and 30.90 g/L DCW which gave the maximal methanol consumption rate. Conjunct effect of amount of methanol added and cell density was found through Origin 7.0 (7.0 Microcal, USA). Scale up in 3.7 L bioreactor, 51% specific yield of SAM was enhanced at 0.6% methanol compared to that of 0.1% methanol. In fed-batches of different cell densities at 0.6% methanol, maximal yield of SAM was 8.66 g/L at 100 g/L DCW with 64% yield of SAM enhanced again. Methanol consumption rate at 100 g/L DCW was 4.81 mL/L h. Maintenance coefficient of 100 g/L DCW was lower than that of others significantly, although methanol consumption rate of 90 g/L DCW was higher (5.07 mL/L h) than that of 100 g/L DCW.