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Applied Microbiology and Biotechnology (v.88, #5)

Escherichia coli as a bioreporter in ecotoxicology by Johan Robbens; Freddy Dardenne; Lisa Devriese; Wim De Coen; Ronny Blust (pp. 1007-1025).

Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.

Keywords: E. coli ; Whole-cell biosensor; Ecotoxicology; Bioreporter; Risk assessment

Sulfur-driven autotrophic denitrification: diversity, biochemistry, and engineering applications by Ming-Fei Shao; Tong Zhang; Herbert Han-Ping Fang (pp. 1027-1042).

Sulfur-driven autotrophic denitrification refers to the chemolithotrophic process coupling denitrification with the oxidation of reduced inorganic sulfur compounds. Ever since 1904, when Thiobacillus denitrificans was isolated, autotrophic denitrifiers and their uncultured close relatives have been continuously identified from highly diverse ecosystems including hydrothermal vents, deep sea redox transition zones, sediments, soils, inland soda lakes, etc. Currently, 14 valid described species within α-, β-, γ-, and ε-Proteobacteria have been identified as capable of autotrophic denitrification. Autotrophic denitrification is also widely applied in environmental engineering for the removal of sulfide and nitrate from different water environments. This review summarizes recent researches on autotrophic denitrification, highlighting its diversity, metabolic traits, and engineering applications.

Keywords: Autotrophic denitrification; Sulfide; Nitrate; Chemolithotroph; Proteobacteria; Anoxic

Microbial 2,4,6-trinitrotoluene degradation: could we learn from (bio)chemistry for bioremediation and vice versa? by Ben A. Stenuit; Spiros N. Agathos (pp. 1043-1064).

2,4,6-Trinitrotoluene (TNT) is released in nature from manufacturing or demilitarization facilities but also after munitions firing/detonation or leakage from explosive remnants of war. Due to its toxicity and recalcitrance, life cycle of TNT-containing products and bioremediation are critical issues. As TNT is a strongly electron-deficient aromatic with a positive molecular quadrupole moment and three electrophilic nitro groups, its environmental fate is contingent upon specific sorptive electron donor–acceptor interactions and nucleophilic, reductive (bio)transformations. The microbial degradation of TNT is governed by cometabolism and therefore depends on the growth substrate(s) available in contaminated environments. Long considered an ecotoxicological safety endpoint, the immobilization of TNT metabolites derived from nitro moiety reduction in soil is controversial because they preferentially bind to the dissolved soil organic matter which can be released into surface and groundwaters. The ever-growing biochemical knowledge of TNT degradation has made bioaugmentation and phytoremediation attractive alternatives. While the discovery and engineering of microorganisms with novel/improved degradative abilities are very challenging, the deciphering of the physiological roles of promiscuous enzymes involved in TNT biodegradation, such as type II hydride transferases of the Old Yellow Enzyme family, opens new perspectives for bioremediation. Finally, transgenic plants have enabled effective phytoremediation at the field scale, which is emerging as the preferable in situ option to rehabilitate TNT-contaminated sites.

Keywords: TNT; Self-cleaning explosive formulations; OYE flavoproteins; Redox-active secondary metabolites; Bioaugmentation; Microbially assisted phytoremediation

Metabolic fluxes and beyond—systems biology understanding and engineering of microbial metabolism by Michael Kohlstedt; Judith Becker; Christoph Wittmann (pp. 1065-1075).

The recent years have seen tremendous progress towards the understanding of microbial metabolism on a higher level of the entire functional system. Hereby, huge achievements including the sequencing of complete genomes and efficient post-genomic approaches provide the basis for a new, fascinating era of research—analysis of metabolic and regulatory properties on a global scale. Metabolic flux (fluxome) analysis displays the first systems oriented approach to unravel the physiology of microorganisms since it combines experimental data with metabolic network models and allows determining absolute fluxes through larger networks of central carbon metabolism. Hereby, fluxes are of central importance for systems level understanding because they fundamentally represent the cellular phenotype as integrated output of the cellular components, i.e. genes, transcripts, proteins, and metabolites. A currently emerging and promising area of research in systems biology and systems metabolic engineering is therefore the integration of fluxome data in multi-omics studies to unravel the multiple layers of control that superimpose the flux network and enable its optimal operation under different environmental conditions.

Keywords: Fluxome; Metabolic flux; Systems biology; Systems metabolic engineering; Multi omics; Metabolic network

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass by Jae-Han Kim; David E. Block; David A. Mills (pp. 1077-1085).

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.

Keywords: Lactobacillus brevis ; Carbon catabolite repression; Simultaneous carbohydrate utilization; Lignocellulosic biomass; SSMSF

Scale-up of Baeyer–Villiger monooxygenase-catalyzed synthesis of enantiopure compounds by Kristian Geitner; Jessica Rehdorf; Radka Snajdrova; Uwe T. Bornscheuer (pp. 1087-1093).

Several Baeyer–Villiger monooxygenases converting a wide spectrum of substrates have been discovered, cloned, and characterized throughout the last few years. Still, only a few of them are applicable for large-scale conversion predominantly due to their sensitivity towards high substrate and/or product concentrations. The recently cloned and characterized 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1 shows excellent enantioselectivity towards 3-phenyl-2-butanone with E > 100 but is inhibited by concentrations >10 mM of both substrate and product. This obstacle could be circumvented by in situ substrate feed and product removal using a hydrophobic Lewatit® adsorbent resin. Thus, the concentration of 3-phenyl-2-butanone could be increased from 1.4 to >26 mM without significant reduction in conversion.

Keywords: Baeyer–Villiger monooxygenase; Reaction scale-up; SFPR; Aryl ketones; Adsorbent resin

Use of a new gelling agent (Eladium©) as an alternative to agar-agar and its adaptation to screen biofilm-forming yeasts by Sabine Gognies; Abdel Belarbi (pp. 1095-1102).

The incidence of yeast-induced infections has increased in the last decade, mainly because of the increasing number of immunodeficient patients. Since biofilm production is believed to be responsible for fungal virulence, we propose screening yeasts of various genera in order to determine their ability to form biofilms. This is an important issue because yeast cells that form biofilms are particularly resistant to anti-fungal agents used in human patients. For screening, we used Eladium©, a new polysaccharide produced by a Rhizobium sp., as an alternative gelling agent to agar. We also established the conditions necessary to detect biofilm formation. The adapted medium provides the missing link between liquid and solid media. Its advantages include enhancement of growth of microorganisms and facilitation of quick and easy monitoring of biofilm formation.

Keywords: Biofilm; Eladium©; Gelling agent; Dynamic growth; Yeast

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose by Tony Sanny; Marina Arnaldos; Stephanie A. Kunkel; Krishna R. Pagilla; Benjamin C. Stark (pp. 1103-1112).

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.

Keywords: Bioethanol; Glucose; Lignocellulose; Vitreoscilla hemoglobin; Xylose

Characterization of endo-1,3–1,4-β-glucanases in GH family 12 from Magnaporthe oryzae by Takumi Takeda; Machiko Takahashi; Tsugumi Nakanishi-Masuno; Yuki Nakano; Hiromasa Saitoh; Akiko Hirabuchi; Shizuko Fujisawa; Ryohei Terauchi (pp. 1113-1123).

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze β-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3–1,4-β-glucans, such as barley β-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3–1,4-β-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3–1,4-β-glucan during infection and spore formation.

Keywords: Endo-1,3–1,4-β-glucanase; β-glucan hydrolysis; GH family 12; Magnaporthe oryzae ; Overexpression; Spore formation

A new esterase EstD2 isolated from plant rhizosphere soil metagenome by Myung Hwan Lee; Kyung Sik Hong; Shweta Malhotra; Ji-Hye Park; Eul Chul Hwang; Hong Kyu Choi; Young Sup Kim; Weixin Tao; Seon-Woo Lee (pp. 1125-1134).

Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K _m and K _cat values were determined to be 79.4 μM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.

Keywords: Esterase; Plant rhizosphere; Soil metagenome

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca by Hanna M. Dudek; Daniel E. Torres Pazmiño; Cristina Rodríguez; Gonzalo de Gonzalo; Vicente Gotor; Marco W. Fraaije (pp. 1135-1143).

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.

Keywords: Phenylacetone monooxygenase; Baeyer–Villiger oxidation; Coenzyme specificity; Enantioselectivity

Investigations on the microbial catabolism of the organic sulfur compounds TDP and DTDP in Ralstonia eutropha H16 employing DNA microarrays by Katja Peplinski; Armin Ehrenreich; Christina Döring; Mechthild Bömeke; Alexander Steinbüchel (pp. 1145-1159).

In this study, we have investigated the transcriptome of Ralstonia eutropha H16 during cultivation with gluconate in presence of 3,3′-thiodipropionic acid (TDP) or 3,3′-dithiodipropionic acid (DTDP) during biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptopropionate). Genome-wide transcriptome analyses revealed several genes which were upregulated during cultivation in presence of the above-mentioned compounds. Obtained data strongly suggest that two ABC-type transport system and three probable extracytoplasmic solute receptors mediate the uptake of TDP and DTDP, respectively. In addition, genes encoding the hydrolase S-adenosylhomocysteinase AhcY and the thiol-disulfide interchange proteins DsbA, DsbD, and FrnE were upregulated during cultivation on DTDP and, in case of AhcY and FrnE, on TDP as well. It is assumed that the corresponding enzymes are involved in the cleavage of TDP and DTDP. Several genes of the fatty acid metabolism exhibited increased expression levels: genes encoding two acetyltransferases, a predicted acyltransferase, the acetoacetyl-CoA reductase phaB3, an enoyl-CoA hydratase as well as an acyl dehydratase, an acetyl-CoA synthetase, two acyl-CoA dehydrogenases, the methylmalonyl-CoA mutase encoded by sbm1 and sbm2 and phaY1 were detected. Furthermore, ORF H16_A0217 encoding a hypothetical protein and exhibiting 54% amino acids identical to an acyl-CoA thioesterase from Saccharomonospora viridis was found to be highly upregulated. As the 2-methylcitrate synthase PrpC exhibited a three- to fourfold increased activity in cells grown in presence of TDP or DTDP as compared to gluconate, metabolization of the cleavage products 3MP and 3-hydroxypropionate to propionyl-CoA is proposed.

Keywords: 3,3′-dithiodipropionic acid; 2-methylcitric acid cycle; Poly(3-hydroxybutyrate); Polythioester; Ralstonia eutropha H16; 3,3′-thiodipropionic acid

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1 by Byoung Seung Jeon; Byung-Chun Kim; Youngsoon Um; Byoung-In Sang (pp. 1161-1167).

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.

Keywords: Clostridium sp. BS-1; Sludge; Hexanoic acid; d-galactitol; Coculture

Anti-cancer effect of rubropunctatin against human gastric carcinoma cells BGC-823 by Yunquan Zheng; Yanwen Xin; Xianai Shi; Yanghao Guo (pp. 1169-1177).

The Monascus pigment, rubropunctatin, was extracted and purified from red mold rice (RMR) and its cytotoxic activities against human gastric adenocarcinoma BGC-823 cells were studied both in vitro and in vivo. Rubropunctatin inhibited the proliferation of BGC-823 cells with an inhibitory concentration (IC₅₀) of 12.57 μM, while it exhibited no significant toxicity to normal gastric epithelial cell GES-1 at the same concentration. Treatment of BGC-823 cells with rubropunctatin resulted in a dose- and time-dependent apoptosis, as validated by the increase in the percentage of cells in sub-G1 phase and phosphotidylserine externalization. The in vivo experimental data demonstrated that rubropunctatin could offer similar therapeutic benefits in comparison with the same dose of taxol. After five times of intravenous injection, tumor weight in BGC-823-bearing nude mice reduced 23.5% at the dose of 8 mg/kg and 37.7% at the dose of 32 mg/kg, respectively. The expressions of 30 genes related to induction of apoptosis were found up-regulated significantly. The two most expressed genes were tumor necrosis factor (TNF) and DNA-damage inducible transcript 3. TNF was considered as a major mediator of apoptosis induced by rubropunctatin. This is the first report describing the anti-proliferative effect of rubropunctatin and its apoptosis mechanism on BGC-823 cells. Rubropunctatin has potential to be developed as a new natural anti-cancer agent.

Keywords: Monascus products; Rubropunctatin; BGC-823; Anti-cancer

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples by Léonid M. Irenge; Jean-François Durant; Herbert Tomaso; Paola Pilo; Jaran S. Olsen; Vincent Ramisse; Jacques Mahillon; Jean-Luc Gala (pp. 1179-1192).

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Keywords: Bacillus anthracis ; LNA probe; PCR; Environmental samples

Microbial biodegradation of a novel fluorotelomer alcohol, 1H,1H,2H,2H,8H,8H-perfluorododecanol, yields short fluorinated acids by Atsushi Arakaki; Yuko Ishii; Takahito Tokuhisa; Seiichiro Murata; Katsuyuki Sato; Takehiro Sonoi; Haruyoshi Tatsu; Tadashi Matsunaga (pp. 1193-1203).

The accumulation of perfluorooctanoic acid (PFOA) has been detected in wildlife, soil, and water. Further, 8:2 fluorotelomer alcohol (8:2 FTOH) is used for the industrial synthesis of other fluorotelomer compounds, surfactants, and polymeric materials; however, it was recently found to be a potential source of PFOA contamination in the environment. 1H,1H,2H,2H,8H,8H-perfluorododecanol (degradable telomer fluoroalcohol (DTFA)), which is a newly developed fluorotelomer, contains the –CH₂– group in the fluorinated carbon backbone, making it potentially degradable through biological reactions. In this study, we investigated the biodegradation of DTFA in a mixed bacterial culture obtained from activated sludge. Optimized quantitative liquid chromatography–mass spectrometry analysis of the predicted metabolites generated in the culture revealed accumulations of the transformation products from DTFA to 2H,2H,8H,8H-PFDoA and 2H,8H,8H-2-PFUDoA via multiple processes. Furthermore, the production of short fluorinated compounds, perfluorobutanoic acid, perfluoropentanoic acid, and perfluoropentanedioic acid, which are believed to have lower accumulation potential and toxicity toward organisms than PFOA, was determined.

Keywords: Fluorotelomer alcohol; Biodegradation; Liquid chromatography–mass spectrometry

Composition of activated sludge settling and planktonic bacterial communities treating industrial effluent and their correlation to settling problems by Nalina Nadarajah; D. Grant Allen; Roberta R. Fulthorpe (pp. 1205-1214).

Problems with deflocculation and solids separation in biological wastewater treatment systems are linked to fluctuations in physicochemical conditions. This study examined the composition of activated sludge bacterial communities in lab-scale sequencing batch reactors treating bleached kraft mill effluent, under transient temperature conditions (30 to 45 °C) and their correlation to sludge settleability problems. The bacterial community composition of settled and planktonic biomass samples in the reactors was monitored via denaturing gradient gel electrophoresis of 16S ribosomal RNA gene fragments. Our analysis showed that settled biomass has a different community composition from the planktonic biomass (49 ± 7% difference based on Jaccard similarity coefficients; p < 0.01). During times of poor sludge compression, the settled and planktonic biomass became more similar. This observation supports the hypothesis that settling problems observed were due to deflocculation of normally settling flocs rather than the outgrowth of non-settling bacterial species.

Keywords: Activated sludge; Deflocculation; DGGE; Population composition; Planktonic community

Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation by Takanori Tanino; Atsushi Hotta; Tomonori Ito; Jun Ishii; Ryosuke Yamada; Tomohisa Hasunuma; Chiaki Ogino; Naoto Ohmura; Takayuki Ohshima; Akihiko Kondo (pp. 1215-1221).

A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.

Keywords: Xylose isomerase; Xylose fermentation; Xylitol; Delta integration

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Applied Microbiology and Biotechnology (v.88, #5)