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Applied Microbiology and Biotechnology (v.71, #5)
Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino by Shantanu Karkare; Deepak Bhatnagar (pp. 575-586).
Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone. Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability. They have become a general and versatile tool for DNA and RNA recognition. LNA is a general and versatile tool for specific, high-affinity recognition of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). LNA can be used for designing LNA oligoes for hybridization studies or as real time polymerase chain reaction probes in the form of Taqman probes. LNA also has therapeutic and diagnostic applications. PNA is another type of DNA analog with neutral charge. The extreme stability of PNA makes it an ideal candidate for the antisense and antigene application. PNA is used as probe for gene cloning, mutation detection, and in homologous recombination studies. It was also used to design transcription factor decoy molecules for target gene induction. Morpholino, another structural type, was devised to circumvent cost problems associated with DNA analogs. It has become the premier knockdown tool in developmental biology due to its cytosolic delivery in the embryos by microinjection. Thus, the nucleic acid analogs provide an advantage to design and implementation, therapies, and research assays, which were not implemented due to limitations associated with standard nucleic acids chemistry.
Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery by V. V. Zverlov; O. Berezina; G. A. Velikodvorskaya; W. H. Schwarz (pp. 587-597).
Clostridial acetone–butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis.
Genetically modified crops: success, safety assessment, and public concern by Om V. Singh; Shivani Ghai; Debarati Paul; Rakesh K. Jain (pp. 598-607).
With the emergence of transgenic technologies, new ways to improve the agronomic performance of crops for food, feed, and processing applications have been devised. In addition, ability to express foreign genes using transgenic technologies has opened up options for producing large quantities of commercially important industrial or pharmaceutical products in plants. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of genetically modified (GM) crops on the environment. Potential contamination of the environment and food chains has prompted detailed consideration of how such crops and the molecules that they produce can be effectively isolated and contained. One of the reasonable steps after creating a transgenic plant is to evaluate its potential benefits and risks to the environment and these should be compared to those generated by traditional agricultural practices. The precautionary approach in risk management of GM plants may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Effective risk assessment and monitoring mechanisms are the basic prerequisites of any legal framework to adequately address the risks and watch out for new risks. Several agencies in different countries monitor the release of GM organisms or frame guidelines for the appropriate application of recombinant organisms in agro-industries so as to assure the safe use of recombinant organisms and to achieve sound overall development. We feel that it is important to establish an internationally harmonized framework for the safe handling of recombinant DNA organisms within a few years.
Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate by microbial function by Yuichiro Otsuka; Masaya Nakamura; Kiyotaka Shigehara; Kosuke Sugimura; Eiji Masai; Seiji Ohara; Yoshihiro Katayama (pp. 608-614).
Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria–Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more.
Biochemical retrosynthesis of 2′-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase by Nobuyuki Horinouchi; Jun Ogawa; Takako Kawano; Takafumi Sakai; Kyota Saito; Seiichiro Matsumoto; Mie Sasaki; Yoichi Mikami; Sakayu Shimizu (pp. 615-621).
2′-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2′-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2′-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2′-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker’s yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5′-triphosphate generated from adenosine 5′-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via d-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2′-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2′-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2′-deoxyribonucleoside synthesis. 2′-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.
Keywords: Deoxyribonucleoside; Deoxyriboaldolase; Glycolysis; Phosphopentomutase; Nucleoside phosphorylase; nucleoside
Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan by C. B. Faulds; G. Mandalari; R. B. Lo Curto; G. Bisignano; P. Christakopoulos; K. W. Waldron (pp. 622-629).
The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.
Molecular cloning of the gene encoding β-1,3(4)-glucanase A from a marine bacterium, Pseudomonas sp. PE2, an essential enzyme for the degradation of Pythium porphyrae cell walls by Etsushi Kitamura; Yuto Kamei (pp. 630-637).
The β-1,3(4)-glucanase A (GluA)-encoding gene named gluA was cloned from the genomic library of a marine bacterium Pseudomonas sp. PE2 by expression in Escherichia coli, and the complete DNA sequence was determined. The recombinant enzyme from Pseudomonas sp. PE2 was examined to determine the essential enzymes for degrading Pythium porphyrae cell walls, comparatively using other two recombinant enzymes, chitinase A and β-1,3-glucanase B from the same bacterial strain. GluA most degraded the cell walls among these three enzymes, suggesting that GluA seems to be most important to P. porphyrae cell-wall-degrading activity. The deduced GluA is a modular enzyme composed of an N-terminal signal peptide, the tandem-duplicated carbohydrate-binding module family 6 (CBMGluA-1 and CBMGluA-2), and a glycoside hydrolase family 16 catalytic domain. Deletion analysis clearly indicated that GluA lacking CBMGluA-1 and CBMGluA-2 does not bind to Avicel and xylan. These results suggest that the tandem-repeated CBM of GluA may play a key role in the binding of Avicel and xylan as well as β-1,3- and β-1,3;1,4-glucans and is very important to bind to insoluble polysaccharides.
Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Stenotrophomonas maltrophilia CCTCC M 204024 by Jian-Fen Zhang; Yu-Guo Zheng; Ya-Ping Xue; Yin-Chu Shen (pp. 638-645).
A soluble glucoside 3-dehydrogenase (G3DH) from Stenotrophomonas maltrophilia CCTCC M 204024, recently isolated from wheat soil in our laboratory, was purified to 37.4-fold with a yield of 24.7% and was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 66 kDa. 2,6-Dichlorophenolindophenol (DCPIP) and ferricyanide were able to act as artificial electron acceptors for the enzyme. The optimal pH of G3DH was in the range of 6.0–7.0 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4–10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. They produced a characteristic spectrum by alkaline treatment with a peak at 340 nm. The apparent K m values for validoxylamine A and d-glucose were 8.3 and 1.1 mM, respectively. Cu2+, Ag2+, and Hg2Cl2 inhibited the activity of G3DH.
Production and characterization of laccase from Cyathus bulleri and its use in decolourization of recalcitrant textile dyes by Salony; S. Mishra; V. S. Bisaria (pp. 646-653).
Many fungi (particularly the white rot) are well suited for treatment of a broad range of textile dye effluents due to the versatility of the lignin-degrading enzymes produced by them. We have investigated decolourization of a number of recalcitrant reactive azo and acid dyes using the culture filtrate and purified laccase from the fungus Cyathus bulleri. For this, the enzyme was purified from the culture filtrate to a high specific activity of 4,022 IU mg−1 protein, produced under optimized carbon, nitrogen and C/N ratio with induction by 2,6-dimethylaniline. The protein was characterized as a monomer of 58±5.0 kDa with carbohydrate content of 16% and was found to contain all three Cu(II) centres. The three internal peptide sequences showed sequence identity (80–92%) with laccases of a number of white rot fungi. Substrate specificity indicated highest catalytic efficiency (k cat/K M) on guaiacol followed by 2,2′-azino-bis(3-ethylthiazoline-6-sulfonic acid) (ABTS). Decolourization of a number of reactive azo and acid dyes was seen with the culture filtrate of the fungus containing predominantly laccase. In spite of no observable effect of purified laccase on other dyes, the ability to decolourize these was achieved in the presence of the redox mediator ABTS, with 50% decolourization in 0.5–5.4 days.
Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans by Takamitsu Arai; Akihiko Kosugi; Helen Chan; Roger Koukiekolo; Hideaki Yukawa; Masayuki Inui; Roy H. Doi (pp. 654-660).
The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHΔCBM and EngYΔCBM devoid of the CBM lost activity toward all substrates including CMC. EngKΔCBM and EngMΔCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.
Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli by Zhixia Zhong; Zhinan Xu; Li Peng; Lei Huang; Xiangming Fang; Peilin Cen (pp. 661-667).
Human beta-defensin-2 (hBD2) is a cysteine-rich cationic antimicrobial peptide with low molecular weight that exhibits a broad range of antimicrobial activity. To improve the expression level of hBD2 in Escherichia coli, tandem repeats of mature hBD2 gene were constructed and expressed as fusion proteins (TrxA-nmhBD2, n=1, 2, 4, 8) by constructing the vectors of pET32-nsmhBD2 (n=1, 2, 4, 8). The results showed that the tandem repeats of mhBD2 gene were highly expressed in our constructed system. Comparing the expression levels of soluble mhBD2, BL21(DE3)/pET32-2smhBD2 was selected as an ideal recombinant strain for mature hBD2 production. Under the optimized conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the maximum expression level of soluble mature hBD2 (0.76 g/l) with the highest percentage of fusion protein in soluble proteins (62.2%) was obtained in the present work, which was the highest yield of hBD2 reported so far.
Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha by Soazig C. Delamarre; Carl A. Batt (pp. 668-679).
Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (Pphb), acetoin (PacoE, PacoD, and PacoX) or pyruvate (PpdhE) metabolism were constructed and tested. The promoters were representative either of the enterobacterial σ70 (Pphb, PacoE, and PpdhE)- or σ54 (PacoD and PacoX)-dependent promoters. To obtain polymers consisting of C4–C12 monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 Ps) tandemly linked to the W. eutropha genes encoding β-ketothiolase (phbA We) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB We) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the PacoD and PacoX promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters—independent of the addition of acetoin—resulted in the production of PHA polymers with high 3HB fractions (90–100 mol%) and with high 3HO contents (70–86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the PacoE promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.
Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing by Dmitriy V. Volokhov; Joseph George; Sue X. Liu; Pranvera Ikonomi; Christine Anderson; Vladimir Chizhikov (pp. 680-698).
We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction–microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.
Analysis of the genomic response of a wine yeast to rehydration and inoculation by Tristan Rossignol; Olivier Postaire; Julien Storaï; Bruno Blondin (pp. 699-712).
We used DNA microarrays to study the transcriptome of a wine yeast before and after rehydration and during the first hours following inoculation of a synthetic must. There was a substantial transcriptional remodeling during this period, including 1,874 genes regulated more than threefold. Dried yeasts displayed an expression profile typical of respiratory-grown cells starved for nitrogen and carbon and which had been highly stressed. During rehydration, many genes involved in biosynthetic pathways, in transcription or in protein synthesis were coordinately induced while genes subject to glucose repression were down-regulated. The transcriptional response was very rapid indicating that yeast quickly recovered the capacity to sense environmental signals and to respond appropriately. Our data show that genes involved in the general stress response were repressed during rehydration while acid stress specific genes were induced probably in response to organic acid accumulation. The glycolytic genes and acid stress-responsive genes were simultaneously and transiently repressed after inoculation into the fermentation medium suggesting that regulation of glycolytic genes may correspond to an adjustment to the energetic needs of the cells. Surprisingly, inoculation into the must did not trigger a stress response despite the high concentrations of sugars.
Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains by H. D. Mortensen; K. Gori; H. Siegumfeldt; P. Nissen; L. Jespersen; N. Arneborg (pp. 713-719).
The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30–35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pKa,i) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pKa,i close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pKa,i to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.
Co-production of caffeic acid and p-hydroxybenzoic acid from p-coumaric acid by Streptomyces caeruleus MTCC 6638 by Ashish Sachan; Shashwati Ghosh; Sukanta Kumar Sen; Adinpunya Mitra (pp. 720-727).
In a culture medium of Streptomyces caeruleus MTCC 6638 grown with p-coumaric acid (5 mM) as the sole source of carbon, co-production of caffeic acid and p-hydroxybenzoic acid was observed. Both caffeic acid and p-hydroxybenzoic acid are important phenolic compounds with pharmaceutical importance. These biotransformed products were identified by high-performance liquid chromatography and electrospray ionization mass spectrometry. Obtained data suggest that p-coumaric acid was possibly utilized by two different routes, resulting in the formation of a hydroxycinnamate and a hydroxybenzoate compound. However, higher concentration of p-coumaric acid (10 mM) favoured caffeic acid formation. Addition of 5 mM p-coumaric acid into S. caeruleus cultures pre-grown on minimal medium with 1.0 g/l glucose resulted in the production of 65 mg/l caffeic acid. Furthermore, S. caeruleus cells were able to produce the maximum amount of caffeic acid when pre-grown on nutrient broth for 16 h. Under this condition, the addition of 5 mM p-coumaric acid was sufficient for the S. caeruleus culture to produce 150 mg/l caffeic acid, with a molar yield of 16.6% after 96 h of incubation.
Isolation and characterization of a phenol-degrading bacterium from an industrial activated sludge by Anli Geng; Alwyn En Wei Soh; Ci Ji Lim; Leslie Ching Thin Loke (pp. 728-735).
This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.0% identity to 16S rRNA gene sequences of Acinetobacter calcoaceticus. Strain EDP3 could aerobically grow on a number of aromatic compounds, such as phenol, sodium benzoate, p-hydroxybenzoate, phenylacetate, benzene, ethylbenzene, benzylalcohol, and so on. In particular, it could mineralize up to 1,000 mg l−1 phenol at room temperature (25°C). The growth kinetics of strain EDP3 on phenol as a sole carbon and energy source at 25°C can be described using the Haldane equation. It has a maximal specific growth rate (μmax) of 0.28 h−1, a half-saturation constant (K S) of 1,167.1 mg l−1, and a substrate inhibition constant (K i) of 58.5 mg l−1. Values of yield coefficient (Y X/S) are between 0.4 and 0.6 mg dry cell (mg phenol)−1. Strain EDP3 has high tolerance to the toxicity of phenol (up to 1,000 mg l−1). It therefore could be an excellent candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated soils.
Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy by A. Bosch; D. Serra; C. Prieto; J. Schmitt; D. Naumann; O. Yantorno (pp. 736-747).
Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200–900 cm−1 region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm−1) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.
Changes in bacterial community structure correlate with initial operating conditions of a field-scale denitrifying fluidized bed reactor by C. Hwang; W.-M. Wu; T. J. Gentry; J. Carley; S. L. Carroll; C. Schadt; D. Watson; P. M. Jardine; J. Zhou; R. F. Hickey; C. S. Criddle; M. W. Fields (pp. 748-760).
High levels of nitrate are present in groundwater migrating from the former waste disposal ponds at the Y-12 National Security Complex in Oak Ridge, TN. A field-scale denitrifying fluidized bed reactor (FBR) was designed, constructed, and operated with ethanol as an electron donor for the removal of nitrate. After inoculation, biofilms developed on the granular activated carbon particles. Changes in the bacterial community of the FBR were evaluated with clone libraries (n=500 partial sequences) of the small-subunit rRNA gene for samples taken over a 4-month start-up period. Early phases of start-up operation were characterized by a period of selection, followed by low diversity and predominance by Azoarcus-like sequences. Possible explanations were high pH and nutrient limitations. After amelioration of these conditions, diversification increased rapidly, with the appearance of Dechloromonas, Pseudomonas, and Hydrogenophaga sequences. Changes in NO3, SO4, and pH also likely contributed to shifts in community composition. The detection of sulfate-reducing-bacteria-like sequences closely related to Desulfovibrio and Desulfuromonas in the FBR have important implications for downstream applications at the field site.
Characteristics of aerobic granular sludge in a sequencing batch reactor with variable aeration by Yong-Qiang Liu; Joo-Hwa Tay; Benjamin Yan-Pui Moy (pp. 761-766).
Aerobic granules can be used for the treatment of industrial or municipal wastewater, but high aeration rate is required for the stable operation of the granular sludge system. Therefore, the aim of this research was to reduce aeration rate greatly to decrease the energy consumption for the technology of aerobic granules. Based on the characteristics of sequencing batch reactor with distinct feast and famine periods, aeration rate was reduced from 1.66 to 0.55 cm s−1 in the famine period after granules were formed. It was found that the settleability of aerobic granules in reactor R1 with reduced aeration was the same as that of aerobic granules in reactor R2 with constant aeration rate of 1.66 cm s−1. However, the outer morphology of aerobic granules gradually changed from round shape to long shape, and minor population showed certain shift after aeration rate was reduced in the famine period. Since good settleability is the most essential feature of aerobic granules, it can be said that reducing aeration rate in famine period did not influence the stable operation of aerobic granular sludge system. Furthermore, the experimental results indicated that aeration rate in feast period was much more important to the stable operation of aerobic granules than that in famine period.
Construction of a large phage display antibody library by in vitro package and in vivo recombination by Xiaodong Cen; Qun Bi; Shenggeng Zhu (pp. 767-772).
Capacity and diversity are extremely important to the quality of various phage display libraries. In this work, λ phage-based in vitro package was applied to construct a filamentous phage display antibody library so as to enlarge its capacity and introduce more sequence diversity in the final library. In vivo recombination via Cre recombinase/lox sites was also exploited to create VH/VL combination diversity based on multivalent package of λ phage packaging extracts on phagemid DNA concatemers. The library constructed with 10 μg concatenated phagemid DNA and ten vials of λ phage packaging extracts was calculated to contain 1.40×1010 independent clones. Higher capacity can be easily achieved when more materials are consumed. This strategy is somewhat more efficient than prior methods.