Isolation and application of a styrene-degrading strain of Pseudomonas putida to biofiltration

A bacterial strain ST201 capable of degrading styrene was isolated from soil and identified as Pseudomonas putida. This strain had high tolerance to styrene and could degrade it completely in 48 h at concentrations up to 600 mg/l. P. putida ST201 was also demonstrated to degrade a mixture of benzene, toluene, ethylbenzene and p-xylene. A packed tower biofilter inoculated with P. putida ST201 was constructed which removed styrene vapor with a styrene elimination capacity of 90 g/m3.h.     

Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate)

The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyal-kanoate (PHA) is described here. PET was pyrolised at 450 degrees C resulting in the production of a solid, liquid, and gaseous fraction. The liquid and gaseous fractions were burnt for energy recovery, whereas the solid fraction terephthalic acid (TA) was used as the feedstock for bacterial production of PHA. Strains previously reported to grow on TA were unable to accumulate PHA. We therefore isolated bacteria from soil exposed to PET granules at a PET bottle processing plant From the 32 strains isolated, three strains capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 accumulates 3-hydroxydecanoic acid as the predominant monomer with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium. Strains GO16 and GO19 accumulate PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h before the rate of PHA accumulation decreased dramatically. Strain GO23 accumulates PHA at a lower maximal rate of 4.4 mg PHA/l/h but there was no slow down in the rate of PHA accumulation over time. Each of the PHA polymers is a thermoplastic with the onset of thermal degradation occurring around 308 degrees C with the complete degradation occurring by 370 degrees C. The molecular weight ranged from 74 to 123 kDa. X-ray diffraction indicated crystallinity of the order of 18-31%. Thermal analysis shows a low glass transition (-53 degrees C) with a broad melting endotherm between 0 and 45 degrees C.     

Taste recognition threshold concentrations of styrene in oil-in-water emulsions and yoghurts

Taste recognition threshold concentrations (TRTC) of styrene were determined in samples of oil-in-water emulsions (30–300 g kg^?1 oil) and yoghurts (1–30 g kg^?1 fat), spiked with styrene. The observed TRTC increased linearly with increasing fat content and ranged from 0–3 to 2–1 mg kg^?1 for the emulsions and from 36 to 171 g kg^?1 for the yoghurts. Styrene equilibrium partition coefficients between emulsions and their respective vapour phases were determined. The concentrations of styrene in the continuous aqueous phase of the emulsions and yoghurts were calculated at the TRTC. The styrene concentrations in the continuous phase had constant values of about 15 g kg^?1 indicating that perception of styrene for oil-in–water emulsions is determined by the aqueous phase of the emulsion. The concentrations of styrene in the vapor phases above the emulsions and yoghurts were also calculated and were found to be constant at the TRTC. This relationship probably resulted from the equilibrium of distribution of styrene between the respective phases. Commercial yoghurt packed in polystyrene beakers contained styrene levels in the range 2–11 g kg^?1, much lower then the TRTC reported.     

Green technology for conversion of food scraps to biodegradable thermoplastic polyhydroxyalkanoates

A new technology is developed and demonstrated that couples anaerobic digestion of food scraps with production of biodegradable thermoplastics, polyhydroxyalkanoates (PHAs). The food wastes were digested in an anaerobic reactor producing four major organic acids. The concentrations of acetic, propionic, butyric, and lactic acids reached 5.5, 1.8, 27.4, and 32.7 g/L, respectively. The fermentative acids were transferred through membranes via molecule diffusion into an air-bubbling reactor where the acids were utilized to produce PHAs in an enriched culture of Ralstonia eutropha. With a silicone rubber membrane, butyric acid and small amounts of acetic and propionic acids were transferred and used, producing a homopolymer PHA, poly(3-hydroxybutyrate). The dry cell weight and PHA content reached 11.3 g/L and 60.2% (w/w), respectively. With a dialysis membrane, the mass transfer rates of fermentative acids were enhanced, and the PHA production was significantly improved. The dry cell weight and its PHA content reached 22.7 g/L and 72.6% (w/w), respectively. The formed PHA was a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (HV) with 2.8 mol % HV monomer unit. The polymer content (72.6% of dry cell mass) reported in this study is the highest one obtained from organic wastes and is comparable with the PHA content from pure glucose fermentation.     

Influence of the antibiotic erythromycin on anaerobic treatment of a pharmaceutical wastewater

A laboratory-scale anaerobic sequencing batch reactor was used to treat a model substrate mixture representing pharmaceutical wastewater at an organic loading rate of 2.9 g COD/(L d). After reaching stable operation the reactor was first exposed to low (1 mg/L) and, subsequently, to high (200 mg/L) concentrations of the antibiotic erythromycin. The addition of low levels of erythromycin resulted in a significant but limited reduction of biogas production by 5% and the higher level of erythromycin did not impact biogas production further, suggesting that a substantial fraction of the microbial populations in the ASBR were resistant to the antibiotic. Effluent soluble COD could not be accounted for in measured volatile fatty acids, perhaps suggesting the production of soluble microbial products. In batch tests evaluating the specific methanogenic activity, conversion of the model substrate mixture was only slightly affected by the presence of erythromycin. However, the conversion of butyric acid was inhibited when erythromycin was present. After 47 days of exposure to erythromycin, the conversion of butyric acid was inhibited to a lesser extent, suggesting the development of antibiotic resistance in the biomass. Exposure to antibiotics can affect specific substrate degradation pathways, leading to the accumulation of volatile fatty acids, soluble microbial products, and potentially to overall system instabilities.     

大豆油的热解特性及其产物分析

本实验利用自制固定床反应器对大豆油进行热解,然后利用GC-MS、FT-IR以及元素分析对其进行分析。探讨了不同热解参数(热解温度、氮气流速、进样速率、大豆油和甲醇比例)对大豆油热解特性及其产物生物油组成的影响。结果表明:大豆油热解的最佳条件为热解温度为550 ℃、氮气流速150 mL/min、油脂进样速率为0.3 mL/min、大豆油和甲醇体积比为3:1。大豆油热解产物由脂肪烃和含氧化合物(酯、醇、酸、醚等)组成,其中脂肪烃含量可达到31.37%。     

MIGRATION OF STYRENE MONOMER FROM POLYSTYRENE PACKAGING MATERIALS INTO FOOD SIMULANTS

Migration studies of residual styrene from Impact Polystyrene cups into soybean oil were carried out at several temperatures. Experimental migration curves were compared to theoretical ones calculated from diffusion-type equations; the former showed higher (by approximately 20%) migration rates and equilibrium styrene concentrations in the oil. Even though the residual styrene was in most cases below 0.1% (far below the levels allowed by the FDA), significant migration occurred, and the resulting styrene concentrations in the oil exceeded the previously determined organoleptic threshold by more than an order of magnitude. The diffusion coefficient of styrene in polystyrene was found to decrease and the partition coefficient to increase with the decrease of residual styrene indicating an increase in monomer-polymer interaction.     

Continuous Production of Glucose Syrup in an Ultrafiltration Reactor

An ultrafiltration reactor was developed for the continuous saccharification of liquefied corn starch using glucoamylase. At an enzyme concentration of 1 g/L and a substrate concentration of 300 g/L, maltose and maltotriose were still detected in the reactor permeate after 4 hr of operation. At higher enzyme concentrations (6 and 12 g/L), the reactor achieved steady-state operation within 1–3 hr at all substrate concentrations studied. At an enzyme concentration of 12 g/L, residence time did not affect the final conversion of liquefied starch to glucose. The ultrafiltration reactor produced glucose syrups at residence times of 2–3 hr and substrate concentrations up to 30% w/v.     

A dynamic study and modeling of the formation of polyhydroxyalkanoates combined with treatment of high strength wastewater

Production of biodegradable thermoplastics, polyhydroxyalkanoates (PHAs), from organic wastes may provide multiple benefits to environmental conservation efforts. In this study, microfiltration-coupled reactors were set up to study the dynamic behavior of a typical PHA-producing bacteria, Ralstonia eutropha, fed with a real acidic solution from starch acidogenesis in a continuous flow system. The majorfermentation acids (butyric and acetic acids) were utilized by the PHA producers at a high conversion rate (>95%) when the cells were suspended in a small volume of mineral solution (pH 7), but at a low conversion rate (<10%) when the cells were suspended in an acidic solution (pH 4). The acids were consumed mainly for PHA synthesis and maintenance energy, which resulted in slow growth of PHA-producing cells and a washout of the cells in the continuous flow system. PHAs, however, were continuously synthesized and accumulated during the washout. A simple dynamic model is proposed for estimation of specific growth rates and PHA formation rates during the washout at two hydraulic retention times (HRT) or dilution rates. The net specific growth rate of PHA-producing cells was near zero at a hydraulic retention time (HRT) of around 30 h, but it increased to 0.01 h(-1) when the HRT was reduced to 18 h. The model also reveals that PHA was synthesized faster based on the active biomass (ABM) during the short HRT (10.3 mg PHA/g ABM.h) than during the long HRT (3.4 mg PHA/g ABM.h).     

Recycling of meat and bone meal animal feed by vacuum pyrolysis

Due to the recent bovine spongiform encephalopathy (BSE) crisis in the European beef industry, the use of animal-derived products to feed cattle is now severely restricted. Large quantities of waste animal meat and bone meal (MBM), also known as animal flour, have to be safely disposed of or transformed. One disposal option is pyrolysis. Vacuum pyrolysis of an animal flour sample has been performed in a laboratory reactor. The results obtained revealed that vacuum pyrolysis can be an attractive alternative to incineration and cement kilns. The process generated a combustible gas (15.1 wt %), a high calorific value oil (35.1 wt %), a solid residue rich in minerals (39.1 wt %), and an aqueous phase rich in organics (10.7 wt %). The gas and the aqueous phase can be used to provide heat to the vacuum pyrolysis reactor and the MBM drying unit. The oil can be used alone or mixed with petroleum products as a fuel in boilers or gas turbines. Conversion of animal waste by pyrolysis into fuels can contribute to the reduction of greenhouse gases. It is suggested to use the solid residue for agricultural soil enrichment in minerals and as a soil moisturizer.     

Metabolic engineering study on the direct fermentation of 2-keto-L-gulonic acid, a key intermediate of L-ascorbic acid in Pseudomonas putida IFO3738

We have achieved production of 2-keto-L-gulonic acid (2-KLGA) in recombinant Pseudomonas putida IFO3738. Firstly, the genes for sorbose dehydrogenase (SDH)/sorbosone dehydrogenase (SNDH) were introduced into P. putida. The recombinant P. putida/pBBR-SDH produced 0.7 mg/ml of 2-KLGA in a culture broth containing 5% L-sorbose. Replacement of the native SNDH promoter by the Escherichia coli tufB promoter (pBBR-SDH-tufB) improved the productivity of 2-KLGA up to 11.4 mg/ml. Secondly, the sorbitol dehydrogenase (SLDH) gene was also introduced into P. putida. The recombinant P. putida/pUCP19-3DH carrying the genes for SDH, SNDH and SLDH had the ability to produce 2-KLGA (7.5 mg/ml) in a 5% d-sorbitol broth. The productivity of 2-KLGA was improved up to 9.8 mg/ml by changing to an expression system with two plasmids, pBBR-SDH-tufB (for SDH/SNDH) and pUCP19-SLDH (for SLDH), respectively. Moreover, the replacement of the native SLDH promoter by the E. coli tufB promoter (pUCP19-SLDH-tufB) improved the 2-KLGA productivity up to 11.6 mg/ml. Optimization of cultivation conditions increased the conversion yield of 2-KLGA to 32% and that of l-idonate, a metabolite of 2-KLGA, to 40%. These results indicate P. putida IFO3738 is one of the candidate strains for direct fermentation of 2-KLGA.     

Biokinetic parameter estimation for degradation of 2,4,6-trinitrotoluene (TNT) with Pseudomonas putida KP-T201

The purpose of this study is to determine the biodegradation kinetics of 2,4,6-trinitrotoluene (TNT) by a newly isolated microorganism. Hundreds of microorganisms were isolated from explosives-contaminated soil in Korea. Examination of culture tests revealed that a few species of microorganisms have good ability to degrade TNT. The most efficient one was selected and identified as Pseudomonas putida KP-T201. Biodegradation of TNT was tested in a batch reactor using a pure culture of P. putida KP-T201. The effect of different concentrations of TNT on the rate of bacterial biodegradation was investigated. The Haldane equation seems to be an adequate expression for the cell growth data, and the kinetic constants obtained were mu(m)=0.65 h(-1), K(S)=0.62 mgl(-1), and K(i)=115 mgl(-1). The dependence of the bacterial specific growth rate on the concentration of TNT could be explained as a conventional model of substrate inhibition.     

Extraction of Astaxanthin Plgment from Crawfish Waste Using a Soy Oil Process

The carotenoid astaxanthin has been extracted from processed crawfish waste using a soy oil process. Analyses have included pigment quantitative analysis, effects of protease enzymes on pigment release, use of ethoxyquin as an antioxidant stabilizer agent, and studies of the ratio of added soybean oil to crawfish waste (v/w) for optimal extraction. Hydrolysis of crawfish waste with a commercial protease, Milezyme® 8X resulted in a 58% increase in astaxanthin release. Maximal pigment extraction and oil recovery was obtained from a 1:1 (v/w) ratio of oil to crawfish waste. The astaxanthin-enriched oil (60 mg/100g oil), obtained from a 1:10 (v/w) ratio, or 3:10 (v/w) after the second stage of extraction, has application for a variety of pigmentation purposes, notably in aquatic diets for salmonid fishes.     

Comparative effect of overexpressed phaJ and fabG genes supplementing (R)-3-hydroxyalkanoate monomer units on biosynthesis of mcl-polyhydroxyalkanoate in Pseudomonas putida KCTC1639

phaJ and fabG genes encoding (R)-specific 2-enoyl-CoA hydratase and 3-ketoacyl-acyl carrier protein reductase, respectively, were cloned from Pseudomonas putida KCTC1639 and amplified in the parent strain to achieve oversupplementation of (R)-3-hydroxyalkanoate monomer units for the biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The comparative effect of the overexpressed fabG and phaJ genes in P. putida KCTC1639 on the biosynthesis of mcl-PHA and the cell growth were elucidated. Overexpression of phaJ enhanced the biosynthesis of mcl-PHA, increasing its content and concentration from 18% to 27% and 0.38 to 0.51 g/l, respectively. Conversely, fabG overexpression tended to depress the biosynthesis of mcl-PHA, possibly due to the reversible conversion of (R)-3-hydroxyalkanoate monomer units into 3-ketoacyl-CoA.     

Styrene monomer migration as monitored by purge and trap gas chromatography and sensory analysis for polystyrene containers

A plastic-like off-flavor in food packaged in multilayered polystyrene containers was determined to be predominately caused by residual styrene monomer, in conjunction with ethylbenzene, that had diffused into the food product. Purge and trap gas chromatography was used to measure μg/L (ppb) concentrations of styrene monomer that migrated from containers over time. The flavor panel used in this study was able to detect the presence of styrene in apple juice at a concentration of 50 μg/L. A mixture of styrene and ethylbenzene was found to lower the concentration at which an off-flavor could be detected to 25 μg/L. Deionized water stored in containers at various temperatures was analyzed for styrene and ethylbenzene by purge and trap gas chromatography, and the initial data fitted a simple diffusion model showing that styrene pickup was proportional to the square root of time.     

生物柴油制备中原料麻疯树籽油预酯化条件的研究

生物柴油一般是通过天然油脂经过碱催化酯交换反应制得。以攀枝花地区的麻疯树籽油为原料,研究了KOH催化酯交换过程对原料油酸值的要求,以及麻疯树籽油性质对浓硫酸催化预酯化过程的适应性。研究发现,KOH催化酯交换过程中,原料油的酸值应控制在1.5 mgKOH/g以下以避免引起产品皂化;预酯化过程中,用油重1%的浓硫酸催化,当甲醇用量为油重12%时,原料油的酸值应小于20 mgKOH/g;随着原料中水分含量的增加,预酯化转化率下降;磷脂含量对于预酯化转化率影响不大,但应在1%以下才能便于后续分离。     

Effect of a fullerene water suspension on bacterial phospholipids and membrane phase behavior

Several fullerene-based nanomaterials generate reactive oxygen species that can damage cells. In this study, we investigated the effect of buckminsterfullerene (C60) introduced as colloidal aggregates in water (nC60) on bacterial membrane lipid composition and phase behavior. Pseudomonas putida (Gram-negative) and Bacillus subtilis (Gram-positive) responded to nC60 by altering membrane lipid composition, phase transition temperature, and membrane fluidity. P. putida decreased its levels of unsaturated fatty acids and increased the proportions of cyclopropane fatty acids in the presence of nC60, possibly to protect the bacterial membrane from oxidative stress. Fourier transform infrared spectroscopy measurement of intact bacterial cells showed slightly increased phase transition temperatures (Tm) and increased membrane fluidity for cells grown in the presence of high, growth-inhibiting concentrations (0.5 mg L(-1)) of nC60. B. subtilis responded to a low dose of nC6o (0.01 mg L(-1)) by significantly increasing the levels of iso- and anteiso-branched fatty acids (from 5.8 to 31.5% and 12.9 to 32.3% of total fatty acids, respectively) and to a high, growth-inhibiting concentration of nC60 (0.75 mg L-1) by increasing synthesis of monounsaturated fatty acids. In contrast to P. putida, B. subtilis response was a decrease in Tm and an increase in membrane fluidity. These findings represent the first demonstrated physiological adaptation response of bacteria to a manufactured nanomaterial, and they showthat response inlipid composition and membrane phase behavior depends on both the nC60 concentration and the cell wall morphology.     

木醋杆菌流加静置培养生产细菌纤维素

木醋杆菌在传统的分批静置培养方式下产生的细菌纤维素具有良好的成膜性,但难于实现工业化生产,为了解决这个问题,对传统的静置培养装置进行改造,形成流加静置培养方式,能够将静置培养与动态培养的优点结合起来。对分批静置和流加静置2种培养方式进行比较,结果表明,流加培养时,细菌纤维素产量达到11.7g/L,其产量是分批培养的3.44倍,反应器的效率为0.585 g/(L·d),高于分批培养的0.425 g/(L·d)。流加培养条件下细菌纤维素产量和反应器效率得到提高,主要是由于在整个培养过程中营养供给充足,促进木醋杆菌的生长,使糖转化率及细菌平均比生长速率都达到较高水平,大量转化和合成细菌纤维素。     

A GC–FID Method to Determine Styrene in Polystyrene Glasses

The styrene levels of polystyrene (PS) glasses from the most consumed brands of disposable glasses intended for water and coffee in Brazil were determined. A GC–FID method was developed and validated, showing good precision and accuracy. The method was successfully used to determine styrene in 11 PS glass brands. The styrene levels ranged from 1.68 to 43.69 mg/100 g glass, depending on the kind of polymer, thickness, and glass brand. It could be used to control the content of styrene in the polymer. The migration of styrene from the glasses into water and 20 % ethanol was not detected.     

Continuous production of L-carnitine with NADH regeneration by a nanofiltration membrane reactor with coimmobilized L-carnitine dehydrogenase and glucose dehydrogenase

L-carnitine dehydrogenase (CDH) was partially purified from Pseudomonas putida IAM12014 for the stereospecific reduction of 3-dehydrocarnitine to L-carnitine. CDH and glucose dehydrogenase (GDH) were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of L-carnitine from 3-dehydrocarnitine with NADH regeneration. In the NFMBR, NAD was partially immobilized through rejection by the nanofiltration membrane and effectively regenerated by the conjugation reaction of CDH and GDH. Since 3-dehydrocarnitine was unstable at neutral pH, it was maintained under acidic conditions (pH 0.7) and supplied to the NFMBR separately from the other substrates, glucose and coenzyme NAD. As 50 mM 3-dehydrocarnitine in HCl solution, 0.05 mM NAD, and 100 mM glucose in 0.5 M Tris buffer (pH 8) were continuously supplied to the NFMBR with immobilized CDH (200 U/ml) and GDH (200 U/ml) at the retention time of 80 min and temperature of 25 degrees C, the maximum conversion, reactor productivity, and NAD regeneration number were 78%, 113 g/l/d, and 780, respectively. The half-life of the NFMBR was longer than 500 h.