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Applied Microbiology and Biotechnology (v.92, #5)

Soil microbes and plant fertilization by Mohammad Miransari (pp. 875-885).

With respect to the adverse effects of chemical fertilization on the environment and their related expenses, especially when overused, alternative methods of fertilization have been suggested and tested. For example, the combined use of chemical fertilization with organic fertilization and/or biological fertilization is among such methods. It has been indicated that the use of organic fertilization with chemical fertilization is a suitable method of providing crop plants with adequate amount of nutrients, while environmentally and economically appropriate. In this article, the importance of soil microbes to the ecosystem is reviewed, with particular emphasis on the role of plant growth-promoting rhizobacteria, arbuscular mycorrhizal fungi, and endophytic bacteria in providing necessary nutrients for plant growth and yield production. Such microbes are beneficial to plant growth through colonizing plant roots and inducing mechanisms by which plant growth increases. Although there has been extensive research work regarding the use of microbes as a method of fertilizing plants, it is yet a question how the efficiency of such microbial fertilization to the plant can be determined and increased. In other words, how the right combination of chemical and biological fertilization can be determined. In this article, the most recent advances regarding the effects of microbial fertilization on plant growth and yield production in their combined use with chemical fertilization are reviewed. There are also some details related to the molecular mechanisms affecting the microbial performance and how the use of biological techniques may affect the efficiency of biological fertilization.

Keywords: Arbuscular mycorrhizal (AM) fungi; Biological fertilization; Endopytic bacteria; Plant growth-promoting rhizobateria (PGPR)

Molecular approaches to pathogenesis study of Burkholderia cenocepacia, an important cystic fibrosis opportunistic bacterium by Silvia Bazzini; Claudia Udine; Giovanna Riccardi (pp. 887-895).

Burkholderia cenocepacia is a Gram-negative opportunistic pathogen belonging to the Burkholderia cepacia complex (Bcc). It is spread in a wide range of ecological niches, and in cystic fibrosis patients, it is responsible for serious infections. Its eradication is very difficult due to the high level of intrinsic resistance to clinically relevant antibiotics. One of the main resistance mechanisms in clinical isolates is represented by efflux systems that are able to extrude a variety of molecules, such as antibiotics, out of the cell. Resistance–Nodulation–Cell Division (RND) efflux pumps are known to be mediators of multidrug resistance in Gram-negative bacteria. Since now, the significance of the RND efflux systems in B. cenocepacia has been partially determined. However, the analysis of the completely sequenced genome of B. cenocepacia J2315 allowed the identification of 16 operons coding for these transporters. We focused our attention on the role of these pumps through the construction of several deletion mutants. Since manipulating B. cenocepacia J2315 genome is difficult, we used a peculiar inactivation system, which enables different deletions in the same strain. The characterization of our mutants through transcriptome and phenotype microarray analysis suggested that RND efflux pumps can be involved not only in drug resistance but also in pathways important for the pathogenesis of this microorganism. The aim of this review is an updated overview on host–pathogen interactions and drug resistance, particularly focused on RND-mediated efflux mechanisms, highlighting the importance of molecular techniques in the study of B. cenocepacia.

Keywords: Burkholderia ; RND; Drug efflux transporters; Pathogenesis

The medicinal Agaricus mushroom cultivated in Brazil: biology, cultivation and non-medicinal valorisation by Michèle L. Largeteau; Régulo Carlos Llarena-Hernández; Catherine Regnault-Roger; Jean-Michel Savoie (pp. 897-907).

Sun mushroom is a cultivated mushroom extensively studied for its medicinal properties for several years and literature abounds on the topic. Besides, agronomical aspects were investigated in Brazil, the country the mushroom comes from, and some studies focus on the biology of the fungus. This review aimed to present an overview of the non-medicinal knowledge on the mushroom. Areas of commercial production and marketing trends are presented. Its specific fragrance, taste, nutritional value and potential use of extracts as food additives are compared to those of the most cultivated fungi and laboratory models. The interest of the mushroom for lignocellulosic enzyme production and source of biomolecules for the control of plant pathogens are shown. Investigation of genetic variability among cultivars is reported. Growing and storage of mycelium, as well as cultivation conditions (substrate and casing generally based on local products; indoor and outdoor cultivation; diseases and disorders) are described and compared to knowledge on Agaricus bisporus.

Keywords: Agaricus blazei ; Agaricus brasiliensis ; Agaricus subrufescens ; Biology; Cultivation; Genetic variability; Marketing; Non-medicinal use; Pathology; Production areas

From photons to biomass and biofuels: evaluation of different strategies for the improvement of algal biotechnology based on comparative energy balances by Christian Wilhelm; Torsten Jakob (pp. 909-919).

Microalgal based biofuels are discussed as future sustainable energy source because of their higher photosynthetic and water use efficiency to produce biomass. In the context of climate CO₂ mitigation strategies, algal mass production is discussed as a potential CO₂ sequestration technology which uses CO₂ emissions to produce biomass with high-oil content independent on arable land. In this short review, it is presented how complete energy balances from photon to harvestable biomass can help to identify the limiting processes on the cellular level. The results show that high productivity is always correlated with high metabolic costs. The overall efficiency of biomass formation can be improved by a photobioreactor design which is kinetically adapted to the rate-limiting steps in cell physiology. However, taking into account the real photon demand per assimilated carbon and the energy input for biorefinement, it becomes obvious that alternative strategies must be developed to reach the goal of a real CO₂ sequestration.

Keywords: Biofuel; Energy balance; Microalgae; Metabolic costs; Photosynthesis

Murein and pseudomurein cell wall binding domains of bacteria and archaea—a comparative view by Ganesh Ram R. Visweswaran; Bauke W. Dijkstra; Jan Kok (pp. 921-928).

The cell wall, a major barrier protecting cells from their environment, is an essential compartment of both bacteria and archaea. It protects the organism from internal turgor pressure and gives a defined shape to the cell. The cell wall serves also as an anchoring surface for various proteins and acts as an adhesion platform for bacteriophages. The walls of bacteria and archaea are mostly composed of murein and pseudomurein, respectively. Cell wall binding domains play a crucial role in the non-covalent attachment of proteins to cell walls. Here, we give an overview of the similarities and differences in the biochemical and functional properties of the two major murein and pseudomurein cell wall binding domains, i.e., the Lysin Motif (LysM) domain (Pfam PF01476) and the pseudomurein binding (PMB) domain (Pfam PF09373) of bacteria and archaea, respectively.

Keywords: Murein; Pseudomurein; Motifs and domains

Increased NADPH availability in Escherichia coli: improvement of the product per glucose ratio in reductive whole-cell biotransformation by Solvej Siedler; Stephanie Bringer; Michael Bott (pp. 929-937).

A basic requirement for the efficiency of reductive whole-cell biotransformations is the reducing capacity of the host. Here, the pentose phosphate pathway (PPP) was applied for NADPH regeneration with glucose as the electron-donating co-substrate using Escherichia coli as host. Reduction of the prochiral β-keto ester methyl acetoacetate to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) served as a model reaction, catalyzed by an R-specific alcohol dehydrogenase. The main focus was maximization of the reduced product per glucose yield of this pathway-coupled cofactor regeneration with resting cells. With a strain lacking the phosphoglucose isomerase, the yield of the reference strain was increased from 2.44 to 3.78 mol MHB/mol glucose. Even higher yields were obtained with strains lacking either phosphofructokinase I (4.79 mol MHB/mol glucose) or phosphofructokinase I and II (5.46 mol MHB/mol glucose). These results persuasively demonstrate the potential of NADPH generation by the PPP in whole-cell biotransformations.

Keywords: Escherichia coli ; Pathway engineering; NADPH yield; Resting cells; Reductive whole-cell biotransformation; pfkA ; pfkB ; pgi

The viability to a wall shear stress and propagation of Bifidobacterium longum in the intensive membrane bioreactor by Ilseon S. Jung; Min Kyo Oh; Young Chai Cho; In Soo Kong (pp. 939-949).

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.

Keywords: Bifidobacterium longum ; Membrane bioreactor; Viability; Wall shear stress

Cloning and expression of a tyrosinase from Aspergillus oryzae in Yarrowia lipolytica: application in l-DOPA biotransformation by Ashit Rao; Priya Pimprikar; Chandrika Bendigiri; Ameeta Ravi Kumar; Smita Zinjarde (pp. 951-959).

Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po1g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po1g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with l-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed l-tyrosine to l-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60°C and with 3.5 mg of biomass), 0.4 mg/ml of l-DOPA was obtained.

Keywords: Yarrowia lipolytica ; Tyrosinase; Aspergillus oryzae ; l-DOPA biotransformation

Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of Aspergillus oryzae by Hiroki Sato; Yoshiyuki Toyoshima; Takahiro Shintani; Katsuya Gomi (pp. 961-969).

We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.

Keywords: Aspergillus oryzae ; α-Amylase; Cell wall; Chitin; Submerged culture; Protein production

Characterisation of a γ-butyrolactone receptor of Streptomyces tacrolimicus: effect on sporulation and tacrolimus biosynthesis by Zahra Salehi-Najafabadi; Carlos Barreiro; Miriam Martínez-Castro; Elena Solera; Juan F. Martín (pp. 971-984).

Streptomyces tacrolimicus (ATCC 55098) was reported to produce the immunosuppressant tacrolimus. The wild-type strain sporulates sparsely and produces very low levels of this immunosuppressant. The lack of genetic knowledge of this strain has hampered strain improvement. In this work, we have cloned the gene encoding a γ-butyrolactone receptor protein (Gbr). The gbr gene is linked to two genes encoding two subunits of the dihydroxyacetone kinase, putatively involved in the biosynthesis of the dihydroxyacetone phosphate precursor of γ-butyrolactone but is not flanked by γ-butyrolactone synthetase genes. The Gbr protein was overexpressed in Escherichia coli and purified. Electrophoretic mobility shift assays showed that Gbr binds to a specific autoregulatory element sequence located 338 bp upstream of the gbr gene, indicating that its expression is self-regulated. The deletion mutant Δgbr showed a very early and intense sporulation in two different media. A phenotype similar to that of the wild-type strain was restored by complementation of the Δgbr mutant with a wild-type gbr allele. Duplication of the gbr gene resulted in a slower sporulation. The Δgbr mutant produced much lower amount (32%) of tacrolimus quantified by high performance liquid chromatography. This analysis, using an optimised system, allowed the resolution of tacrolimus from ascomycin and other contaminant metabolites. Our results indicate that the Gbr protein regulates negatively the sporulation and positively the production of tacrolimus.

Keywords: Tacrolimus; FK506; Streptomyces ; Secondary metabolite; Sporulation

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum by Vipin Gopinath; Tobias M. Meiswinkel; Volker F. Wendisch; K. Madhavan Nampoothiri (pp. 985-996).

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.

Keywords: Corynebacterium glutamicum ; Amino acid production; Lignocellulosic hydrolysates; Pentose utilization; Renewables; Metabolic engineering

Comparative structural bioinformatics analysis of Bacillus amyloliquefaciens chemotaxis proteins within Bacillus subtilis group by Anna Yssel; Oleg Reva; Ozlem Tastan Bishop (pp. 997-1008).

Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites.

Keywords: Homology modeling; Chemotaxis receptor; Positive selection; Purifying selection

The ECF sigma factor SigT regulates actinorhodin production in response to nitrogen stress in Streptomyces coelicolor by Wei-Hong Feng; Xu-Ming Mao; Zhen-Hua Liu; Yong-Quan Li (pp. 1009-1021).

Sigma factors of the extracytoplasmic function (ECF) subfamily are important regulators of stress responses in bacteria. This work described the characterization of ECF sigma factor SigT in Streptomyces coelicolor. We found the absence of sigT almost abolished the production of the antibiotics actinorhodin (Act) under nitrogen stress. Under nitrogen-limited conditions, significantly reduced Act production and linked actII-ORF4 transcription with respect to wild type were observed in the sigT-null mutant. Using reporter (xylE) fusion to sigT promoter, we demonstrated that sigT was induced by nitrogen limitation in a SigT-dependent manner. Transcriptional analyses showed that SigT controlled the expression of relA, the ppGpp synthetase gene, and consequently affected the Act production upon nitrogen starvation. Co-transcription analysis revealed that sigT was co-transcribed with rstB (gene upstream of sigT) but not with rstA (gene downstream of sigT). Phenotypic and transcriptional results suggested RstA may modulate the activity of SigT positively.

Keywords: Sigma factor; Nitrogen stress; Secondary metabolism; Streptomyces coelicolor

A sirA-like gene, sirA2, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading Pseudomonas sp. HZN6 strain by Jiguo Qiu; Yun Ma; Liansheng Chen; Lifei Wu; Yuezhong Wen; Weiping Liu (pp. 1023-1032).

A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain’s main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.

Keywords: Nicotine; 3-Succinoyl-pyridine; Degradation; sirA2 ; Gene deletion; Pseudomonas sp. HZN6

Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNA sequence analysis by Can Su; Wen Gu; Wei Zhe; Ke-Qin Zhang; Yanqing Duan; Jinkui Yang (pp. 1033-1044).

Microorganisms play important roles in the tobacco aging process. However, microbial communities on flue-cured tobacco leaves (FCTL) remain largely unknown. In this study, the total microbial genomic DNA of unaged and aging FCTL from Zimbabwe were isolated using a culture-independent method, and the bacterial communities were investigated through analyzing two 16S rRNA gene libraries. Eighty-four and 65 operational taxonomic units were obtained from the libraries of the unaged and aging FCTL, respectively. The following genera were represented more than 4% in both libraries (aging and unaged library): Sphingomonas (4.84%, 4.18%), Stenotrophomonas (4.84%, 5.23%), Erwinia (5.81%, 4.88%), Pantoea (19.35%, 18.47%), and Pseudomonas (21.29%, 24.04%). The dominant species varied between the two libraries. Specifically, several dominant species in unaged FCTL including Pseudomonas fulva, Pseudomonas sp. (AM909658), Klebsiella sp. (HM584796), and Pantoea sp. (AY501386) were not identified in aging FCTL, while several dominant species in aging FCTL such as Pantoea sp. (GU566350), Pseudomonas sp. (EF157292), and Buttiauxella izardii were not found in unaged FCTL. The phylogenetic analysis showed that bacteria from unaged and aging FCTL were divided into two clades, and two unique subclades were identified in aging FCTL. Our results revealed for the first time the bacterial diversities on Zimbabwe tobacco, and provided a basis for clarifying the roles of bacteria in aging process of FCTL.

Keywords: Zimbabwe tobacco; 16S rRNA clone library; Culture-independent method; Bacterial diversity; Phylogenetic analysis

Assay-dependent effect of silver nanoparticles to Escherichia coli and Bacillus subtilis by Shin Woong Kim; Yong-Wook Baek; Youn-Joo An (pp. 1045-1052).

We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of dissolved silver ions were influenced profoundly by the microbial test method employed.

Keywords: Silver nanoparticle; Microbial assay; Toxicity; Escherichia coli ; Bacillus subtilis

Rapid detection of food- and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates by Cui Fan; Zhiqiang Hu; Azlin Mustapha; Mengshi Lin (pp. 1053-1061).

Development of rapid and sensitive methods to detect pathogens is important to food and water safety. This study aimed to detect and discriminate important food- and waterborne bacteria (i.e., Escherichia coli O157:H7, Staphylococcus epidermidis, Listeria monocytogenes, and Enterococcus faecelis) by surface-enhanced Raman spectroscopy (SERS) coupled with intracellular nanosilver as SERS substrates. An in vivo molecular probing using intracellular nanosilver for the preparation of bacterial samples was established and assessed. Satisfactory SERS performance and characteristic SERS spectra were obtained from different bacterial samples. Distinctive differences were observed in SERS spectral data, specifically in the Raman shift region of 500–1,800 cm⁻¹, and between bacterial samples at the species and strain levels. The detection limit of SERS coupled with in vivo molecular probing using silver nanosubstrates could reach the level of single cells. Experiments with a mixture of E. coli O157:H7 and S. epidermidis for SERS measurement demonstrate that SERS could be used for classification of mixed bacterial samples. Transmission electron microscopy was used to characterize changes of morphology and cellular composition of bacterial cells after treatment of intracellular nanosilver. The results indicate that SERS coupled with intracellular silver nanosubstrates is a promising method for detection and characterization of food- and waterborne pathogenic and non-pathogenic bacterial samples.

Keywords: SERS; Bacteria; Silver nanosubstrates; Detection

Development and characterization of DehaloR^2, a novel anaerobic microbial consortium performing rapid dechlorination of TCE to ethene by Michal Ziv-El; Anca G. Delgado; Ying Yao; Dae-Wook Kang; Katherine G. Nelson; Rolf U. Halden; Rosa Krajmalnik-Brown (pp. 1063-1071).

A novel anaerobic consortium, named DehaloR^2, that performs rapid and complete reductive dechlorination of trichloroethene (TCE) to ethene is described. DehaloR^2 was developed from estuarine sediment from the Back River of the Chesapeake Bay and has been stably maintained in the laboratory for over 2 years. Initial sediment microcosms showed incomplete reduction of TCE to DCE with a ratio of trans- to cis- isomers of 1.67. However, complete reduction to ethene was achieved within 10 days after transfer of the consortium to sediment-free media and was accompanied by a shift to cis-DCE as the prevailing intermediate metabolite. The microbial community shifted from dominance of the Proteobacterial phylum in the sediment to Firmicutes and Chloroflexi in DehaloR^2, containing the genera Acetobacterium, Clostridium, and the dechlorinators Dehalococcoides. Also present were Spirochaetes, possible acetogens, and Geobacter which encompass previously described dechlorinators. Rates of TCE to ethene reductive dechlorination reached 2.83 mM Cl⁻ d⁻¹ in batch bottles with a Dehalococcoides sp. density of 1.54E+11 gene copies per liter, comparing favorably to other enrichment cultures described in the literature and identifying DehaloR^2 as a promising consortium for use in bioremediation of chlorinated ethene-impacted environments.

Keywords: Dehalococcoides ; Chlorinated ethenes; Sediment microorganisms; Reductive dechlorination

Response of methanotrophs and methane oxidation on ammonium application in landfill soils by Na Yang; Fan Lü; Pinjing He; Liming Shao (pp. 1073-1082).

To test the dose effect of ammonium (NH₄ ⁺) fertilization on soil methane (CH₄) oxidation by methanotrophic communities, batch incubations were conducted at a wide scale of NH₄ ⁺ amendments: 0, 100, 250, 500, and 1,000 mg N kg_dry soil ⁻¹. Denaturing gradient gel electrophoresis and real-time quantitative PCR analysis were conducted to investigate the correlation between the CH₄ oxidation capacity and methanotrophic communities. Immediately after the addition of NH₄ ⁺, temporal inhibition of CH₄ oxidation occurred, and this might have been due to the non-specific salt effect (osmotic stress). After a lag phase, the CH₄ oxidation rates of the soils with NH₄ ⁺ fertilization were promoted to levels higher than those of the controls. More than 100 mg N kg_dry soil ⁻¹ of NH₄ ⁺ addition resulted in the reduction of type II/type I MOB ratios and an obvious evolution of type II MOB communities, while less than 100 mg N kg_dry soil ⁻¹ of NH₄ ⁺ addition induced nearly no change of methanotrophic community compositions. The NH₄ ⁺-derived stimulation after the lag phase was attributed to the improvement of N availability for type I MOB. Compared with the controls, 100 mg N kg_dry soil ⁻¹ of NH₄ ⁺ addition doubled the CH₄ oxidation peak value to more than 20 mg CH₄ kg_dry soil ⁻¹ h⁻¹. Therefore, an appropriate amount of leachate irrigation on the landfill cover layer might efficiently mitigate the CH₄ emissions.

Keywords: Methane oxidation; Ammonium stimulation; Nitrogen limitation; Landfill cover soil; Denaturing gradient gel electrophoresis (DGGE); Real-time quantitative polymerase chain reaction (qPCR)

Analysis of the microbial community of the biocathode of a hydrogen-producing microbial electrolysis cell by Elsemiek Croese; Maria Alcina Pereira; Gert-Jan W. Euverink; Alfons J. M. Stams; Jeanine S. Geelhoed (pp. 1083-1093).

The microbial electrolysis cell (MEC) is a promising system for hydrogen production. Still, expensive catalysts such as platinum are needed for efficient hydrogen evolution at the cathode. Recently, the possibility to use a biocathode as an alternative for platinum was shown. The microorganisms involved in hydrogen evolution in such systems are not yet identified. We analyzed the microbial community of a mixed culture biocathode that was enriched in an MEC bioanode. This biocathode produced 1.1 A m⁻² and 0.63 m³ H₂ m⁻³ cathode liquid volume per day. The bacterial population consisted of 46% Proteobacteria, 25% Firmicutes, 17% Bacteroidetes, and 12% related to other phyla. The dominant ribotype belonged to the species Desulfovibrio vulgaris. The second major ribotype cluster constituted a novel taxonomic group at the genus level, clustering within uncultured Firmicutes. The third cluster belonged to uncultured Bacteroidetes and grouped in a taxonomic group from which only clones were described before; most of these clones originated from soil samples. The identified novel taxonomic groups developed under environmentally unusual conditions, and this may point to properties that have not been considered before. A pure culture of Desulfovibrio strain G11 inoculated in a cathode of an MEC led to a current development from 0.17 to 0.76 A m⁻² in 9 days, and hydrogen gas formation was observed. On the basis of the known characteristics of Desulfovibrio spp., including its ability to produce hydrogen, we propose a mechanism for hydrogen evolution through Desulfovibrio spp. in a biocathode system.

Keywords: Desulfovibrio G11; MEC; Hydrogen; Exocellular electron transfer; Sulfate-reducing bacteria

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Applied Microbiology and Biotechnology (v.92, #5)