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Applied Microbiology and Biotechnology (v.80, #2)


Phthalates biodegradation in the environment by Da-Wei Liang; Tong Zhang; Herbert H. P. Fang; Jianzhong He (pp. 183-198).
Phthalates are synthesized in massive amounts to produce various plastics and have become widespread in environments following their release as a result of extensive usage and production. This has been of an environmental concern because phthalates are hepatotoxic, teratogenic, and carcinogenic by nature. Numerous studies indicated that phthalates can be degraded by bacteria and fungi under aerobic, anoxic, and anaerobic conditions. This paper gives a review on the biodegradation of phthalates and includes the following aspects: (1) the relationship between the chemical structure of phthalates and their biodegradability, (2) the biodegradation of phthalates by pure/mixed cultures, (3) the biodegradation of phthalates under various environments, and (4) the biodegradation pathways of phthalates.

Keywords: Phthalates; Biodegradation; Environment; Pathway


Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges by Rumpa Biswas Bhattacharjee; Aqbal Singh; S. N. Mukhopadhyay (pp. 199-209).
The potential of nitrogen-fixing (NF) bacteria to form a symbiotic relationship with leguminous plants and fix atmospheric nitrogen has been exploited in the field to meet the nitrogen requirement of the latter. This phenomenon provides an alternative to the use of the nitrogenous fertiliser whose excessive and imbalanced use over the decades has contributed to green house emission (N2O) and underground water leaching. Recently, it was observed that non-leguminous plants like rice, sugarcane, wheat and maize form an extended niche for various species of NF bacteria. These bacteria thrive within the plant, successfully colonizing roots, stems and leaves. During the association, the invading bacteria benefit the acquired host with a marked increase in plant growth, vigor and yield. With increasing population, the demand of non-leguminous plant products is growing. In this regard, the richness of NF flora within non-leguminous plants and extent of their interaction with the host definitely shows a ray of hope in developing an ecofriendly alternative to the nitrogenous fertilisers. In this review, we have discussed the association of NF bacteria with various non-leguminous plants emphasizing on their potential to promote host plant growth and yield. In addition, plant growth-promoting traits observed in these NF bacteria and their mode of interaction with the host plant have been described briefly.

Keywords: Biological nitrogen fixation (BNF); Nitrogen-fixing (NF) bacteria; Endophyte; Rhizobium ; Non-legume; Growth promotion


Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system by Setefilla Molina; Jorge Rencoret; José C. del Río; Anne Lomascolo; Eric Record; Angel T. Martínez; Ana Gutiérrez (pp. 211-222).
Different model lipids—alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides—were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase–mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase–mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase–mediator treatment of different pulp types.

Keywords: Laccase–mediator system; Fungal enzymes; Model lipids; Reaction mechanisms; Paper pulps; Pitch deposits


Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus by Shin Kanamasa; Lies Dwiarti; Mitsuyasu Okabe; Enoch Y. Park (pp. 223-229).
A filamentous fungus Aspergillus terreus produces itaconic acid, which is predicted to be derived from cis-aconitic acid via catalysis by cis-aconitic acid decarboxylase (CAD) in the carbon metabolism of the fungus. To clarify the enzyme’s function and a pathway for itaconic acid biosynthesis, we cloned a novel gene encoding the enzyme. The open reading frame of this gene (CAD1) consists of 1,529 bp encoding 490 amino acids and is interrupted by a single intron. Among the identified proteins in the database, the primary structure of the protein encoded by CAD1 shared high identity with the MmgE/PrpD family of proteins, including a number of 2-methylcitrate dehydratases of bacteria. The cloned gene excluding an intron was introduced into the expression plasmid pAUR-CAD1 controlled by the ADH1 promoter. The CAD activity in Saccharomyces cerevisiae was confirmed by directly detecting itaconic acid as a product from cis-aconitic acid as a substrate. This result reveals for the first time that this gene encodes CAD, which is essential for itaconic acid production in A. terreus.

Keywords: cis-Aconitic acid decarboxylase; Aspergillus terreus ; Itaconic acid; Gene cloning; Gene expression


Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9 by Ning Li; Kun Meng; Yaru Wang; Pengjun Shi; Huiying Luo; Yingguo Bai; Peilong Yang; Bin Yao (pp. 231-240).
A new xylanase gene, xynAS9, was cloned from Streptomyces sp. S9, which was isolated from Turpan Basin, China. The full-length gene consists of 1,395 bp and encodes 465 amino acids including 38 residues of a putative signal peptide. The overall amino acid sequence shares the highest identity (50.8%) with a putative endo-1,4-β-xylanase from Streptomyces avermitilis of the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.5 and 60°C, respectively. The enzyme showed broad temperature adaptability, retaining more than 65% of the maximum activity when assayed at 50–80°C. The enzyme also had good thermal and pH stability. The K m values for oat spelt xylan and birchwood xylan substrates were 2.85 and 2.43 mg ml−1, with the V max values of 772.20 and 490.87 μmol min−1 mg−1, respectively. The hydrolysis products of xylan were mainly xylose and xylobiose. These favorable properties should make XynAS9 a good candidate in various industrial applications.

Keywords: Streptomyces sp. S9; Turpan Basin; Xylanase; Xylose


Cloning and characterization of a novel chitinase gene (chi46) from Chaetomium globosum and identification of its biological activity by Z.H. Liu; Q. Yang; S. Hu; J.D. Zhang; J. Ma (pp. 241-252).
Chitinases play a major role in the defensive strategies of plants against fungal pathogens. In the current study, the gene for a 46-kDa endochitinase (chi46) was cloned from Chaetomium globosum, an important biocontrol fungus. The corresponding complementary deoxyribonucleic acid sequence was 1,350 bp in length, encoding 449 amino acid residues. The temporal expression of chi46, in response to the treatments of cell walls of six pathogens and confrontation against two fungal pathogens, was measured in C. globosum using real-time reverse transcription polymerase chain reaction. The expression of chi46 can be highly induced by exposure to the cell walls of plant pathogens and living pathogens, suggesting a role in plant disease resistance. The chi46 gene was inserted into the pPIC9 vector and transferred into the cells of Pichia pastoris GS115 for heterologous expression. The optimal reaction conditions for chitinase CHI46 activity were: 45°C, pH of 5.0, and 5 mmol l−1 of Cu2+. The maximum enzyme activity was 1.42 U ml−1 following exposure to the cell wall chitin of Septoria tritici. The CHI46 enzyme can efficiently degrade cell walls of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Valsa sordida, S. tritici, and Phytophthora sojae, demonstrating that it may be involved in the biocontrol mechanism of C. globosum.

Keywords: Chaetomium globosum ; Chitinase; Gene cloning; Yeast expression; Biological control


Regioselective synthesis of plant (iso)flavone glycosides in Escherichia coli by Xian-Zhi He; Wen-Sheng Li; Jack W. Blount; Richard A. Dixon (pp. 253-260).
The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4’-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.

Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling by Bo Xu; Zhihua Jin; Hengzheng Wang; Qingchao Jin; Xin Jin; Peilin Cen (pp. 261-267).
Improvement of pristinamycin production by Streptomyces pristinaespiralis was performed by using recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-μg/ml pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/l, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable. It is concluded that genome shuffling improves the production of pristinamycin by enhancing product-resistance in a stepwise manner. Pristinamycin fermentation experiments by recombinant G 4-17 were carried out in a 5-l fermentor, and its production of pristinamycin reached 0.90 g/l after 60 h of fermentation.

Keywords: Genome shuffling; Protoplast fusion; Pristinamycin; Streptomyces pristinaespiralis ; Pristinamycin resistant recombinant


Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion by Kwanghyun Hwang; Seung G. Shin; Jaai Kim; Seokhwan Hwang (pp. 269-276).
In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.

Keywords: Anaerobic batch system; Archaea; Denaturing gradient gel electrophoresis (DGGE); Methanogens; Methanosarcinales ; Methanomicrobiales ; Methanobacteriales ; 16S rRNA gene primer


Characterization of a negative regulator AveI for avermectin biosynthesis in Streptomyces avermitilis NRRL8165 by Lei Chen; Yinhua Lu; Jun Chen; Weiwen Zhang; Dan Shu; Zhongjun Qin; Sheng Yang; Weihong Jiang (pp. 277-286).
A transcriptional activator for actinorhodin biosynthesis, AtrA, was previously characterized in Streptomyces coelicolor A3(2), and an orthologue of atrA, named aveI, is identified in the Streptomyces avermitilis NRRL8165 genome (Uguru et al., Mol Microbiol, 58:131–150, 2005). In this study, genetic and functional characterization of aveI gene was reported. Deletion of aveI gene led to increased biosynthesis of avermectin B1a by about 16-fold. The increased synthesis of avermectin B1a was suppressed by complementation with either aveI gene or its orthologue gene atrA from S. coelicolor, suggesting AveI and AtrA shared the similar functionality and were negative regulators for avermectin biosynthesis in S. avermitilis. However, when aveI was introduced into S. coelicolor on a multi-copy plasmid, the production of actinorhodin was significantly increased, indicating that aveI had a positive effect on actinorhodin biosynthesis in S. coelicolor, the same as its orthologue atrA. Electrophoretic mobility shift assays revealed AveI can bind specifically to the promoter region of actII-ORF4 in vitro but not that of aveR. Although its mechanism still needs to be defined, the species-differential regulation by the same regulator may represent an example of the evolutional strategy that enables bacteria to adapt the existing molecular machinery to a variety of functionalities for growth and survival.

Keywords: Avermectin biosynthesis; Negative regulator; Streptomyces avermitilis ; Streptomyces coelicolor


Taxonomic distribution of Streptomyces species capable of producing bioactive compounds among strains preserved at NITE/NBRC by Kozue Anzai; Michiyo Ohno; Takuji Nakashima; Natsumi Kuwahara; Rieko Suzuki; Tomohiko Tamura; Hisayuki Komaki; Shinji Miyadoh; Shigeaki Harayama; Katsuhiko Ando (pp. 287-295).
The taxonomic distribution of Streptomyces species capable of producing bioactive compounds was investigated. Nine hundred and six strains were tested for the following four biological activities: antimicrobial, anti-tyrosinase, antioxidant, and hemolytic. Approximately 30% of strains tested showed antimicrobial activities, except for anti-Escherichia coli activity, which was present in only a few strains, while the rates of positivity for the anti-tyrosinase, antioxidant, and hemolytic activities were much lower. The distribution of Streptomyces strains capable of producing bioactive compounds was analyzed by the taxonomy based on 16S rRNA gene sequences. Moreover, the strains of Streptomyces hygroscopicus tested were divided into two clades in the phylogenetic tree, and all of the strains belonging to one clade showed antibacterial and antifungal activities. For detection of polyenes, the UV–visible spectra of metabolic extracts in the strains showing antifungal activities were measured. It was suggested that Streptomyces strains produce universal active compounds under different growth conditions. Further information on the relationship between the microbial taxonomy and the bioactive compounds produced would be useful for the utilization of industrial microorganisms.

Keywords: Streptomyces ; Taxonomy; 16S rRNA; Bioactivity


Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation by Anita N. Jakobsen; Inga M. Aasen; Kjell D. Josefsen; Arne R. Strøm (pp. 297-306).
Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate- and glycerol-containing growth medium. Exponentially growing cells had a lipid content of 13% (w/w) of dry weight. A fattening of cells fed excess glycerol occurred in the post-exponential growth phase, after the medium was depleted of N or P. Lipid accumulation was also initiated by O2 limitation (below 1% of saturation). N starvation per se, or in combination with O2 limitation, gave the highest lipid content, i.e., 54% to 63% (w/w) of dry weight. The corresponding maximum culture density was 90 to 100 g/l dry biomass. The content of docosahexaenoic acid (22:6n-3) in N starved, well-oxygenated cells reached 29% (w/w) of total fatty acids but increased to 36% to 52% in O2-limited cells, depending on the time span of the limitation. O2-limited cells did not accumulate the monounsaturated fatty acids that were normally present. We inferred that the biological explanation is that O2 limitation hindered the O2-dependent desaturase(s) and favored the O2-independent polyunsaturated fatty acid synthase. The highest overall volumetric productivity of docosahexaenoic acid observed was 93 mg/l/h. Additionally, we present a protocol for quantitative lipid extraction, involving heat and protease treatment of freeze-dried thraustochytrids.

Keywords: Thraustochytrid; Aurantiochytrium ; Docosahexaenoic acid; Lipid accumulation; Lipid extraction; Nutrient starvation


Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa by Walid Hammami; Caroline Labbé; Florian Chain; Benjamin Mimee; Richard R. Bélanger (pp. 307-315).
This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.

Keywords: Pseudozyma; Flocculosa; Cellobiose lipid; Ustilagic acid


Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria by Ingrid Sumeri; Liisa Arike; Kaarel Adamberg; Toomas Paalme (pp. 317-324).
The aim of the present study was to design an in vitro model system to evaluate the probiotic potential of food. A single bioreactor system—gastrointestinal tract simulator (GITS) was chosen for process simulation on account of its considerable simplicity compared to multi-vessel systems used in previous studies. The bioreactor was evaluated by studying the viability of four known probiotic bacteria (Lactobacillus acidophilus La-5, Lactobacillus johnsonii NCC 533, Lactobacillus casei strain Shirota, and Lactobacillus rhamnosus GG) as a function of their physiological state. L. acidophilus and L. johnsonii survived in GITS better when introduced at an early stationary or exponential phase compared to being previously stored for 2 weeks at 4 °C. These two species were more resistant to bile salts and survived better than L. casei and L. rhamnosus GG. The latter two species gave large losses (up to 6 log) in plate counts independent of growth state due to the bile. However, experiments with some commercial probiotic products containing Lb. GG bacteria showed much better survival compared with model food (modified deMan–Rogosa–Sharpe growth medium), thus demonstrating the influence of the food matrix on the viability of bacteria. The study demonstrated that GITS can be successfully used for evaluation of viability of probiotic bacteria and functionality of probiotic food.

Keywords: Probiotics; GIT model; Bile salts; Survival


Electricity generation from model organic wastewater in a cassette-electrode microbial fuel cell by Takefumi Shimoyama; Shoko Komukai; Akira Yamazawa; Yoshiyuki Ueno; Bruce E. Logan; Kazuya Watanabe (pp. 325-330).
A new highly scalable microbial fuel cell (MFC) design, consisting of a series of cassette electrodes (CE), was examined for increasing power production from organic matter in wastewater. Each CE chamber was composed of a box-shaped flat cathode (two air cathodes on both sides) sandwiched in between two proton-exchange membranes and two graphite-felt anodes. Due to the simple design of the CE-MFC, multiple cassettes can be combined to form a single unit and inserted into a tank to treat wastewater. A 12-chamber CE-MFC was tested using a synthetic wastewater containing starch, peptone, and fish extract. Stable performance was obtained after 15 days of operation in fed-batch mode, with an organic removal efficiency of 95% at an organic loading rate of 2.9 kg chemical oxygen demand (COD) per cubic meter per day and an efficiency of 93% at 5.8 kg COD per cubic meter per day. Power production was stable during this period, reaching maximum power densities of 129 W m−3 (anode volume) and 899 mW m−2 (anode projected area). The internal resistance of CE-MFC decreased from 2.9 (day 4) to 0.64 Ω (day 25). These results demonstrate the usefulness of the CE-MFC design for energy production and organic wastewater treatment.

Keywords: Microbial fuel cell; Energy recovery; Organic waste; Biomass; Wastewater


Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus by Takashi Watanabe; Noriatsu Ozaki; Kazuhiro Iwashita; Tsutomu Fujii; Haruyuki Iefuji (pp. 331-338).
Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.

Keywords: Phosphorus; Wastewater treatment; Yeast; Polyphosphate accumulation; PHO pathway


Assessment of the positive effect of salinity on the nitrogen removal performance and microbial composition during the start-up of CANON process by Sitong Liu; Fenglin Yang; Zheng Gong; Zhencheng Su (pp. 339-348).
In this study, a non-woven rotating biological contactor reactor was operated for the start-up of completely autotrophic nitrogen removal over nitrite (CANON) process. In this perfectly attached growth system, nitrite oxidizing was identified, which interfered with the nitrogen removal performance. Batch tests indicated that 10 g NaCl per liter salinity was a preferable definite level to stand out ammonium-oxidizing activity and anammox activity, and selectively suppress nitrite-oxidizing activity under oxygen-limited conditions. Reactor operation showed that the maximum TN removal rate was increased from 425 mg N l−1 day−1 to 637 mg N l−1 day−1 after the addition of 10 g NaCl per liter salinity on analogous technological parameters. Microbiological community analysis revealed that bacteria strains similar to the genus Nitrospira sp. were specialized nitrite oxidizers existing in CANON reactor, which were then eliminated under salinity exposure for their no salinity-tolerant relative. However, anammox bacteria belonging to Planctomycetes and some aerobic ammonium oxidizers belonging to Nitrosomonas could be highly enriched under this oxygen-limited salinity conditions. Salinity-contained high ammonium wastewater will be so considered as suitable influent for CANON process in further industrial application.

Keywords: CANON; Anammox; Salinity; Oxygen-limited; Nitrospira


Electricity generation and treatment of paper recycling wastewater using a microbial fuel cell by Liping Huang; Bruce E. Logan (pp. 349-355).
Increased interest in sustainable agriculture and bio-based industries requires that we find more energy-efficient methods for treating cellulose-containing wastewaters. We examined the effectiveness of simultaneous electricity production and treatment of a paper recycling plant wastewater using microbial fuel cells. Treatment efficiency was limited by wastewater conductivity. When a 50 mM phosphate buffer solution (PBS, 5.9 mS/cm) was added to the wastewater, power densities reached 501 ± 20 mW/m2, with a coulombic efficiency of 16 ± 2%. There was efficient removal of soluble organic matter, with 73 ± 1% removed based on soluble chemical oxygen demand (SCOD) and only slightly greater total removal (76 ± 4%) based on total COD (TCOD) over a 500-h batch cycle. Cellulose was nearly completely removed (96 ± 1%) during treatment. Further increasing the conductivity (100 mM PBS) increased power to 672 ± 27 mW/m2. In contrast, only 144 ± 7 mW/m2 was produced using an unamended wastewater (0.8 mS/cm) with TCOD, SCOD, and cellulose removals of 29 ± 1%, 51 ± 2%, and 16 ± 1% (350-h batch cycle). These results demonstrate limitations to treatment efficiencies with actual wastewaters caused by solution conductivity compared to laboratory experiments under more optimal conditions.

Keywords: Microbial fuel cell; Paper recycling wastewater; Cellulose; Solution conductivity; Power production


Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-α) gene by Qiang Chen; Yang Zhao; Zhuo Cheng; Yixiang Xu; Chundong Yu (pp. 357-363).
Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-α is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-α in macrophages, we attempt to establish a reporter system to assess TNF-α gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-α promoter and 3′-untranslated region (3′-UTR). The TNF-α-luciferase reporter cell line is used for assessing the expression of TNF-α gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-α such as dexamethasone and emodin, and also for measuring change of expression of TNF-α gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-α. The luciferase activity correlated well with the ELISA results for TNF-α production, therefore, the TNF-α-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-α gene.

Keywords: TNF-α; Luciferase activity; Macrophage; LPS
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