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Applied Microbiology and Biotechnology (v.85, #1)

Plant–microbes interactions in enhanced fertilizer-use efficiency by Anthony O. Adesemoye; Joseph W. Kloepper (pp. 1-12).

The continued use of chemical fertilizers and manures for enhanced soil fertility and crop productivity often results in unexpected harmful environmental effects, including leaching of nitrate into ground water, surface run-off of phosphorus and nitrogen run-off, and eutrophication of aquatic ecosystems. Integrated nutrient management systems are needed to maintain agricultural productivity and protect the environment. Microbial inoculants are promising components of such management systems. This review is a critical summary of the efforts in using microbial inoculants, including plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungi for increasing the use efficiency of fertilizers. Studies with microbial inoculants and nutrients have demonstrated that some inoculants can improve plant uptake of nutrients and thereby increase the use efficiency of applied chemical fertilizers and manures. These proofs of concept studies will serve as the basis for vigorous future research into integrated nutrient management in agriculture.

Keywords: Plant-microbe interaction; Plant growth-promoting rhizobacteria; Arbuscular mycorrhizal fungi; Integrated nutrient management; Fertilizers

Microbial formation of esters by Yong Cheol Park; Catherine Emily Horton Shaffer; George N. Bennett (pp. 13-25).

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.

Keywords: Ester; Aroma; Microorganism; Yeast; Alcohol; Monooxygenase

Molecular basis of and interference into degenerative processes in fungi: potential relevance for improving biotechnological performance of microorganisms by Christian Q. Scheckhuber; Rudolf Mitterbauer; Heinz D. Osiewacz (pp. 27-35).

Biological systems, from simple microorganisms to humans, are characterized by time-dependent degenerative processes which lead to reduced fitness, disabilities, severe diseases, and, finally, death. These processes are under genetic control but also influenced by environmental conditions and by stochastic processes. Studying the mechanistic basis of degenerative processes in the filamentous ascomycete Podospora anserina and in other systems demonstrated that mitochondria play a key role in the expression of degenerative phenotypes and unraveled a number of underlying molecular pathways. Reactive oxygen species (ROS) which are mainly, but not exclusively, formed at the mitochondrial respiratory chain are crucial players in this network. While being essential for signaling processes and development, ROS are, at the same time, a potential danger because they lead to molecular damage and degeneration. Fortunately, a number of interacting pathways including ROS scavenging, DNA and protein repair, protein degradation, and mitochondrial fission and fusion are involved in keeping cellular damage low. If these pathways are overwhelmed by extensive damage, programmed cell death is induced. The current knowledge of this hierarchical system of mitochondrial quality control, although still incomplete, appears now to be ready for the development of strategies effective in interventions into those pathways leading to degeneration and loss of performance also in microorganisms used in biotechnology. Very promising interdisciplinary interactions and collaborations involving academic and industrial research teams can be envisioned to arise which bear a great potential, in particular, when system biology approaches are used to understand relevant networks of pathways in a holistic way.

Keywords: Podospora anserina ; Aging; Mitochondria; Quality control; Health span

Comparison and critical evaluation of PCR-mediated methods to walk along the sequence of genomic DNA by Yuki Tonooka; Masahiro Fujishima (pp. 37-43).

Although researchers can access information on the entire genomic DNA sequence of typical research organisms, convenient genome walking methods in the laboratory are still needed. For the analysis of microorganisms, these methods are especially useful because the available genetic information is often scarce or limited. Many genomic walking methods are based on the polymerase chain reaction (PCR), and useful methods have been developed. This report reviews the methodologies of PCR-mediated genomic walking methods and evaluates their efficiency and usefulness to help microbiologists to select the appropriate method for each target microorganism. The concept and specific features, such as advantages and disadvantages, of five major PCR-mediated genomic walking methods (random PCR, inverse PCR, panhandle PCR, cassette PCR, and rapid amplification of genomic ends) are briefly described. The improved methods and their characteristics are listed, and a report of experimental comparison of such methods is also introduced briefly. Each of these methods has both advantages and disadvantages, and there is a trade-off between the specificity of target amplification and the ease of the method. The cassette PCR seems to be a standard method, but suitable method should be selected in consideration of the characteristics of the material.

Keywords: Genomic walking; Method; PCR

Applications of thiol-disulfide oxidoreductases for optimized in vivo production of functionally active proteins in Bacillus by Thijs R. H. M. Kouwen; Jan Maarten van Dijl (pp. 45-52).

Bacillus subtilis is a well-established cellular factory for proteins and fine chemicals. In particular, the direct secretion of proteinaceous products into the growth medium greatly facilitates their downstream processing, which is an important advantage of B. subtilis over other biotechnological production hosts, such as Escherichia coli. The application spectrum of B. subtilis is, however, often confined to proteins from Bacillus or closely related species. One of the major reasons for this (current) limitation is the inefficient formation of disulfide bonds, which are found in many, especially eukaryotic, proteins. Future exploitation of B. subtilis to fulfill the ever-growing demand for pharmaceutical and other high-value proteins will therefore depend on overcoming this particular hurdle. Recently, promising advances in this area have been achieved, which focus attention on the need to modulate the cellular levels and activity of thiol-disulfide oxidoreductases (TDORs). These TDORs are enzymes that control the cleavage or formation of disulfide bonds. This review will discuss readily applicable approaches for TDOR modulation and aims to provide leads for further improvement of the Bacillus cell factory for production of disulfide bond-containing proteins.

Keywords: Bacillus; Bdb; DsbA; PhoA; TDOR; Disulfide bond

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor by Samia Rourou; Arno van der Ark; Samy Majoul; Khaled Trabelsi; Tiny van der Velden; Héla Kallel (pp. 53-63).

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate µ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.

Keywords: Vero cells; Bioreactor; Rabies virus; Serum-free medium; Microcarriers

Effects of pulse feeding of beet molasses on recombinant benzaldehyde lyase production by Escherichia coli BL21(DE3) by Pınar Çalık; Hande Levent (pp. 65-73).

The effect of fed-batch operation (FBO) strategy was investigated using pretreated-beet molasses, containing galactose that induces the lac promoter, on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21(DE3)pLySs. After batch cultivation with 30 g l⁻¹ pretreated-beet molasses consisting of 7.5 g l⁻¹ glucose and 7.5 g l⁻¹ fructose, three FBO strategies were applied at dissolved oxygen (=40%) cascade to air-flow rate. In FBO1 when air-flow rate decreased considerably, feed was given to the system in pulses in such a way that pretreated-beet molasses concentration increased by 10 kg m⁻³ (containing 2.5 g l⁻¹ glucose+2.5 g l⁻¹ fructose); however, decrease in air-flow rate demonstrated only the absence of glucose but not fructose. Thus, in FBO2 when fructose and glucose were completely utilized, pretreated-beet molasses was pulse-fed and its concentration increased by 10 g l⁻¹. In FBO3 with the decreased amount of pretreated-beet molasses (6 g l⁻¹), shift response time from glucose to fructose consumption was avoided, and glucose and fructose consumptions were well correlated with air-flow rate, and the highest C _X (8.04 g l⁻¹) and BAL (2,315 U ml⁻¹) production were obtained (t = 24 h) with the highest substrate yield on cell and product formation.

Keywords: E. coli ; Molasses; Benzaldehyde lyase; Fed-batch; Pulse; Production; Oxygen transfer; Acetate; Yield and maintenance coefficients

Substrate specificity of Myriococcum thermophilum cellobiose dehydrogenase on mono-, oligo-, and polysaccharides related to in situ production of H₂O₂ by S. Pricelius; R. Ludwig; N. Lant; D. Haltrich; G. M. Guebitz (pp. 75-83).

Cellobiose dehydrogenase from the ascomycete fungus Myriococcum thermophilum (MtCDH) was tested for the ability to generate bleaching species at a pH suitable for liquid detergents. The catalytic properties of MtCDH were investigated for a large variety of carbohydrate substrates using oxygen as an electron receptor. MtCDH produces H₂O₂ with all substrates tested (except fructose) but only in the presence of a chelant. Insoluble substrates like cellulose and cotton could as well be oxidized by MtCDH. To enhance the amount of cello-oligosaccharides in solution, different cellulases on cotton were used and in combination with MtCDH an increased H₂O₂ concentration could be measured. Additionally, the degradation of pure anthocyanins in solution (as model substrates for bleaching) was investigated in the absence and presence of a horseradish peroxidase. MtCDH was able to produce a sufficient amount of H₂O₂ to decolorize the anthocyanins within 2 h.

Keywords: Carbohydrate oxidation; Cellobiose dehydrogenase; Destaining; Hydrogen peroxide; Myriococcum thermophilum

Purification and molecular characterization of exo-β-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2 by Ying Peng; Zhen-Ming Chi; Xiang-Hong Wang; Jing Li (pp. 85-94).

The extracellular β-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific β-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40°C, respectively. The enzyme was significantly stimulated by Li⁺, Ni²⁺, and Ba²⁺. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the β-1,3-glucanases from other yeasts.

Keywords: Molecular characterization; Marine yeast; β-1,3-Glucanases; Williopsis saturnus ; Laminarin

Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library by Danya Isogawa; Takeshi Fukuda; Kouichi Kuroda; Hideo Kusaoke; Hisashi Kimoto; Shin-ichiro Suye; Mitsuyoshi Ueda (pp. 95-104).

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and β-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and β-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose.

Keywords: Chitosanase; Paenibacillus fukuinensis ; Family 8 glycoside hydrolase; Proton acceptor; Yeast cell surface engineering

Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars by Miho Sasaki; Toru Jojima; Hideo Kawaguchi; Masayuki Inui; Hideaki Yukawa (pp. 105-115).

Corynebacterium glutamicum strains CRA1 and CRX2 are able to grow on l-arabinose and d-xylose, respectively, as sole carbon sources. Nevertheless, they exhibit the major shortcoming that their sugar consumption appreciably declines at lower concentrations of these substrates. To address this, the C. glutamicum ATCC31831 l-arabinose transporter gene, araE, was independently integrated into both strains. Unlike its parental strain, resultant CRA1-araE was able to aerobically grow at low (3.6 g·l⁻¹) l-arabinose concentrations. Interestingly, strain CRX2-araE grew 2.9-fold faster than parental CRX2 at low (3.6 g·l⁻¹) d-xylose concentrations. The corresponding substrate consumption rates of CRA1-araE and CRX2-araE under oxygen-deprived conditions were 2.8- and 2.7-fold, respectively, higher than those of their respective parental strains. Moreover, CRA1-araE and CRX2-araE utilized their respective substrates simultaneously with d-glucose under both aerobic and oxygen-deprived conditions. Based on these observations, a platform strain, ACX-araE, for C. glutamicum-based mixed sugar utilization was designed. It harbored araBAD for l-arabinose metabolism, xylAB for d-xylose metabolism, d-cellobiose permease-encoding bglF 317A, β-glucosidase-encoding bglA and araE in its chromosomal DNA. In mineral medium containing a sugar mixture of d-glucose, d-xylose, l-arabinose, and d-cellobiose under oxygen-deprived conditions, strain ACX-araE simultaneously and completely consumed all sugars.

Keywords: Corynebacterium glutamicum ; Simultaneous utilization; d-xylose; l-arabinose; l-arabinose transporter

Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface by Zhen-lin Han; Shuang-yan Han; Sui-ping Zheng; Ying Lin (pp. 117-126).

To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60°C for 5 h and increased synthetic activity in organic solvent at 60°C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.

Keywords: Disulfide bond; Rhizomucor miehei ; Thermostability; Lipase; Yeast surface display

Global transcriptome analysis of the Mycobacterium bovis BCG response to sodium hypochlorite by Hyeung-Jin Jang; Chantal Nde; Freshteh Toghrol; William E. Bentley (pp. 127-140).

Tuberculosis is a common and often deadly infectious disease caused by mycobacteria, mainly Mycobacterium tuberculosis and infrequently by other subspecies of the M. tuberculosis complex, such as M. bovis. Sodium hypochlorite (bleach) is routinely used in hospitals and health care facilities for surface sterilization; however, the modes of action of bleach on M. bovis BCG and how this organism develops resistance to sodium hypochlorite have not been elucidated. In this study, we performed a global toxicogenomic analysis of the M. bovis response to 2.5 mM sodium hypochlorite after 10 and 20 min. M. bovis BCG growth was monitored by measuring the quantity of ATP in picomoles produced over a short exposure time (10–60 min) to sodium hypochlorite. This study revealed significant regulation of oxidative stress response genes of M. bovis BCG, such as oxidoreductase, peroxidase, heat shock proteins and lipid transport, and metabolism genes. We interpreted this response as a potentially more lethal interplay between fatty acid metabolism, sulfur metabolism, and oxidative stress. Our results also suggest that sodium hypochlorite repressed transcription of genes involved in cell wall synthesis of M. bovis. This study shows that the treatment of M. bovis BCG with bleach inhibits the biosynthesis of outer cell wall mycolic acids and also induces oxidative damage.

Keywords: Toxicogenomics; Microarray; Mycobacterium bovis BCG; Sodium hypochlorite; Bleach

Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae by Yuji Noguchi; Motoaki Sano; Kyoko Kanamaru; Taro Ko; Michio Takeuchi; Masashi Kato; Tetsuo Kobayashi (pp. 141-154).

XlnR is a Zn(II)₂Cys₆ transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus. Overexpression of the aoxlnR gene in Aspergillus oryzae (A. oryzae xlnR gene) resulted in elevated xylanolytic and cellulolytic activities in the culture supernatant, in which nearly 40 secreted proteins were detected by two-dimensional electrophoresis. DNA microarray analysis to identify the transcriptional targets of AoXlnR led to the identification of 75 genes that showed more than fivefold increase in their expression in the AoXlnR overproducer than in the disruptant. Of these, 32 genes were predicted to encode a glycoside hydrolase, highlighting the biotechnological importance of AoXlnR in biomass degradation. The 75 genes included the genes previously identified as AoXlnR targets (xynF1, xynF3, xynG2, xylA, celA, celB, celC, and celD). Thirty-six genes were predicted to be extracellular, which was consistent with the number of proteins secreted, and 61 genes possessed putative XlnR-binding sites (5′-GGCTAA-3′, 5′-GGCTAG-3′, and 5′-GGCTGA-3′) in their promoter regions. Functional annotation of the genes revealed that AoXlnR regulated the expression of hydrolytic genes for degradation of β-1,4-xylan, arabinoxylan, cellulose, and xyloglucan and of catabolic genes for the conversion of d-xylose to xylulose-5-phosphate. In addition, genes encoding glucose-6-phosphate 1-dehydrogenase and l-arabinitol-4-dehydrogenase involved in d-glucose and l-arabinose catabolism also appeared to be targets of AoXlnR.

Keywords: Aspergillus ; Regulation; XlnR ; Xylanase; Cellulase; Xylose catabolism

Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe by Hiroyuki Mukaiyama; Yuko Giga-Hama; Hideki Tohda; Kaoru Takegawa (pp. 155-164).

The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

Keywords: Heterologous protein production; Schizosaccharomyces pombe ; Human transferrin; Secretion

Pressure treatment of Saccharomyces cerevisiae in low-moisture environments by Marwen Moussa; Vincent Espinasse; Jean-Marie Perrier-Cornet; Patrick Gervais (pp. 165-174).

We investigated the influence of cell hydration on the ability of Saccharomyces cerevisiae CBS 1171 to withstand extreme hydrostatic pressure in order to determine the mechanisms involved in cell resistance. Hydration conditions were modified in two different ways. We first modulated the chemical potential of water by adding glycerol in cell suspensions. Another procedure consisted in dehydrating cells aerobically and immersing them in perfluorooctane, an innocuous hydrophobic liquid used as a pressure-transmitting medium, prior to pressure treatments. This original method made it possible to transmit isostatic pressure to yeast powders without changing the initial water activity (a _w) level at which cells had been equilibrated. The a _w ranged between 0.11 and 0.99. Pressure treatments were applied at levels of up to 600 MPa for 10 min, 24 h, and 6 days. The dehydration of cells was found to strongly limit, or even prevent, cell inactivation under pressure. Notably, cells suspended in a water–glycerol mixture with a _w levels of 0.71 or below were completely protected against all pressure treatments. Moreover, cells dehydrated aerobically survived for 6 days at 600 MPa even when a _w levels were relatively high (up to 0.94). We highlighted the crucial role of water content in determining cellular damage under pressure. When water is available in a sufficient amount, high pressure induces membrane permeabilization, causing uncontrolled mass transfers that could lead to death during a prolonged holding under pressure. Possible mechanisms of membrane permeabilization are discussed.

Keywords: Cell inactivation; High pressure; Hydration; Membrane damage; Yeast cells

Microbial fuel cell with an azo-dye-feeding cathode by Liang Liu; Fang-bai Li; Chun-hua Feng; Xiang-zhong Li (pp. 175-183).

Microbial fuel cells (MFCs) were constructed using azo dyes as the cathode oxidants to accept the electrons produced from the respiration of Klebsiella pneumoniae strain L17 in the anode. Experimental results showed that a methyl orange (MO)-feeding MFC produced a comparable performance against that of an air-based one at pH 3.0 and that azo dyes including MO, Orange I, and Orange II could be successfully degraded in such cathodes. The reaction rate constant (k) of azo dye reduction was positively correlated with the power output which was highly dependent on the catholyte pH and the dye molecular structure. When pH was varied from 3.0 to 9.0, the k value in relation to MO degradation decreased from 0.298 to 0.016 μmol min⁻¹, and the maximum power density decreased from 34.77 to 1.51 mW m⁻². The performances of the MFC fed with different azo dyes can be ranked from good to poor as MO > Orange I > Orange II. Furthermore, the cyclic voltammograms of azo dyes disclosed that the pH and the dye structure determined their redox potentials. A higher redox potential corresponded to a higher reaction rate.

Keywords: Microbial fuel cell; Azo dyes; K. pneumoniae ; Orange II; Degradation

Influence of trace erythromycin and erythromycin-H₂O on carbon and nutrients removal and on resistance selection in sequencing batch reactors (SBRs) by Caian Fan; Patrick K. H. Lee; Wun Jern Ng; Lisa Alvarez-Cohen; Eoin L. Brodie; Gary L. Andersen; Jianzhong He (pp. 185-195).

Three sequencing batch reactors (SBRs) were operated in parallel to study the effects of trace erythromycin (ERY) and ERY-H₂O on the treatment of a synthetic wastewater. Through monitoring (1) daily effluents and (2) concentrations of nitrogen (N) and phosphorous (P) in certain batch cycles of the three reactors operated from transient to steady states, the removal of carbon, N, and P was affected negligibly by ERY (100 µg/L) or ERY-H₂O (50 µg/L) when compared with the control reactor. However, through analyzing microbial communities of the three steady state SBRs on high-density microarrays (PhyloChip), ERY, and ERY-H₂O had pronounced effects on the community composition of bacteria related to N and P removal, leading to diversity loss and abundance change. The above observations indicated that resistant bacteria were selected upon exposure to ERY or ERY-H₂O. Short-term batch experiments further proved the resistance and demonstrated that ammonium oxidation (56–95%) was inhibited more significantly than nitrite oxidation (18–61%) in the presence of ERY (100, 400, or 800 µg/L). Therefore, the presence of ERY or ERY-H₂O (at µg/L levels) shifted the microbial community and selected resistant bacteria, which may account for the negligible influence of the antibiotic ERY or its derivative ERY-H₂O (at µg/L levels) on carbon, N, and P removal in the SBRs.

Keywords: Antibiotics; Erythromycin (ERY); Dehydrated erythromycin (ERY-H₂O); Sequencing batch reactors (SBRs); Nutrients removal

The concentration of polysaccharides and proteins in EPS of Pseudomonas putida and Aureobasidum pullulans as revealed by ¹³C CPMAS NMR spectroscopy by Ulrich Metzger; Ulrich Lankes; Kai Fischpera; Fritz H. Frimmel (pp. 197-206).

Extracellular polymeric substances were extracted from the bacterial strain Pseudomonas putida and the fungal species Aureobasidium pullulans using three different methods (formaldehyde–NaOH, ethylenediaminetetraacetic acid (EDTA) and cation-exchange-resin). The composition of the extracellular polymeric substances (EPS) was analysed by biochemical and high-resolution solid state ¹³C nuclear magnetic resonance (NMR) spectroscopic methods. The EPS yield was strongly dependent on the extraction method, with the formaldehyde–NaOH method showing the best extraction efficiency. The NMR method revealed that when using the EDTA extraction method, about 40% of the EDTA accumulated in the EPS and that was responsible for the apparent high extraction yields. EPS protein content determined by the NMR method was up to 30% higher than the protein content determined using the biochemical (Lowry) method for P. putida and for A. pullulans. The average protein carbon content determined by the NMR method was approximately 70% of the total carbon content. NMR results could be supported by elemental analysis, which showed a high nitrogen content (~10%) in the EPS. The carbohydrate carbon content detected with both methods in the cell aggregates and the EPS was approximately 20% in each. In this study, quantitative ¹³C cross-polarisation magic angle spinning NMR spectroscopy was conducted on unlabeled cell strains, and EPS and could be used to quantify protein and carbohydrate of different samples.

Keywords: EPS; ¹³C CPMAS NMR; Extraction; Protein; Carbohydrate

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Applied Microbiology and Biotechnology (v.85, #1)

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Applied Microbiology and Biotechnology (v.85, #1)