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Applied Microbiology and Biotechnology (v.95, #5)

Magnetotactic bacteria: promising biosorbents for heavy metals by Wei Zhou; Yanzong Zhang; Xiaohui Ding; Yan Liu; Fei Shen; Xiaohong Zhang; Shihuai Deng; Hong Xiao; Gang Yang; Hong Peng (pp. 1097-1104).

Magnetotactic bacteria (MTB), which can orient and migrate along a magnetic line of force due to intracellular nanosized magnetosomes, have been a subject of research in the medical field, in dating environmental changes, and in environmental remediation. This paper reviews the recent development of MTB as biosorbents for heavy metals. Ultrastructures and taxis of MTB are investigated. Adsorptions in systems of unitary and binary ions are highlighted, as well as adsorption conditions (temperature, pH value, biomass concentration, and pretreatments). The separation and desorption of MTB in magnetic separators are also discussed. A green method to produce metal nanoparticles is provided, and an energy-efficient way to recover precious metals is put forward during biosorption.

Keywords: Magnetotactic bacteria; Magnetosome; Heavy metal; Adsorption; Separation; Nanoparticles

Rapeseed and sunflower meal: a review on biotechnology status and challenges by Anne Lomascolo; Eva Uzan-Boukhris; Jean-Claude Sigoillot; Frédéric Fine (pp. 1105-1114).

Rapeseed and sunflower are two of the world’s major oilseeds. Rapeseed and sunflower meal (RSM and SFM), the by-products of oil extraction, are produced in large quantities. They are mainly composed of proteins, lignocellulosic fibres and minerals. They were initially used as a protein complement in animal feed rations and sometimes as fertilizer or as combustible source. More recently, new alternatives to these traditional uses have been developed that draw on the structure and physicochemical properties of RSM and SFM, which are plentiful sources of nitrogen and carbon nutrients. This feature, together with their cheapness and ready availability, supports the cultivation of various microorganisms in both submerged cultures and solid-state fermentation. Recent studies have thus emphasized the potential utilisation of RSM and SFM in fermentative processes, including saccharification and production of enzymes, antibiotics, antioxidants and other bio-products, opening new challenging perspectives in white biotechnology applications.

Keywords: Biotechnological application; Oil cake; Oil meal; Rapeseed; Sunflower

Multi-catalysis reactions: new prospects and challenges of biotechnology to valorize lignin by Christoph A. Gasser; Gregor Hommes; Andreas Schäffer; Philippe F.-X. Corvini (pp. 1115-1134).

Considerable effort has been dedicated to the chemical depolymerization of lignin, a biopolymer constituting a possible renewable source for aromatic value-added chemicals. However, these efforts yielded limited success up until now. Efficient lignin conversion might necessitate novel catalysts enabling new types of reactions. The use of multiple catalysts, including a combination of biocatalysts, might be necessary. New perspectives for the combination of bio- and inorganic catalysts in one-pot reactions are emerging, thanks to green chemistry-driven advances in enzyme engineering and immobilization and new chemical catalyst design. Such combinations could offer several advantages, especially by reducing time and yield losses associated with the isolation and purification of the reaction products, but also represent a big challenge since the optimal reaction conditions of bio- and chemical catalysis reactions are often different. This mini-review gives an overview of bio- and inorganic catalysts having the potential to be used in combination for lignin depolymerization. We also discuss key aspects to consider when combining these catalysts in one-pot reactions.

Keywords: Lignin; Homogeneous catalysis; Heterogeneous catalysis; Biocatalysis; Chemoenzymatic catalysis

Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem by Sunil Kumar Sirohi; Nasib Singh; Sumit Singh Dagar; Anil Kumar Puniya (pp. 1135-1154).

Rumen microbial community comprising of bacteria, archaea, fungi, and protozoa is characterized not only by the high population density but also by the remarkable diversity and the most complex microecological interactions existing in the biological world. This unprecedented biodiversity is quite far from full elucidation as only about 15–20 % of the rumen microbes are identified and characterized till date using conventional culturing and microscopy. However, the last two decades have witnessed a paradigm shift from cumbersome and time-consuming classical methods to nucleic acid-based molecular approaches for deciphering the rumen microbial community. These techniques are rapid, reproducible and allow both the qualitative and quantitative assessment of microbial diversity. This review describes the different molecular methods and their applications in elucidating the rumen microbial community.

Keywords: Rumen bacteria; Archaea; Anaerobic fungi; Ribosomal RNA; mcrA; DNA fingerprinting; Microbial diversity

Recent research on 3-phenyllactic acid, a broad-spectrum antimicrobial compound by Wanmeng Mu; Shuhuai Yu; Lanjun Zhu; Tao Zhang; Bo Jiang (pp. 1155-1163).

3-Phenyllactic acid (PLA), which is an organic acid widely existing in honey and lactic acid bacteria fermented food, can be produced by many microorganisms, especially lactic acid bacteria. It was proved as an ideal antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. In addition, it could be used as feed additives to replace antibiotics in livestock feeds. This article presented a review of recent studies on the existing resource, antimicrobial activity, and measurement of PLA. In addition, microorganism strains and dehydrogenases producing PLA were reviewed in detail, the metabolic pathway and regulation of PLA synthesis in LAB strains were discussed, and high-level bioproduction of PLA by microorganism fermentation was also summarized.

Keywords: 3-Phenyllactic acid; Antimicrobial compound; Dehydrogenase; Metabolic pathway; Fermentation

Biosilica structures obtained from Nitzschia, Ditylum, Skeletonema, and Coscinodiscus diatom by a filtration-aided acid cleaning method by Yu Wang; Deyuan Zhang; Jun Cai; Junfeng Pan; Mingli Chen; Aobo Li; Yonggang Jiang (pp. 1165-1178).

A filtration-aided acid cleaning method was used to collect biosilica structures from a diatom culture medium, natural seawater, or water bloom. Cell extraction, acid cleaning, and acid removal were all performed on a polytetrafluoroethylene (PTFE) filter cloth, significantly improving the treatment capacity and efficiency of the traditional acid wash method. Five typical diatoms were cultivated in the laboratory for acid cleaning. Different growth speeds were introduced, and different process parameters for acid cleaning were utilized. After the acid cleaning, biosilica structures were collected from the frustules of diatoms using different methods. Girdle bands and valves of Coscinodiscus sp. were separated by floating of the valves. Central spines of Ditylum brightwellii and valves of Skeletonema costatum were separately collected by settling or filtration. Rod-like frustules, such as those of Bacillaris paradoxa, are not suitable for large quantities of acid wash. The silica structures were observed and tested using an AFM-calibrated glass needle to determine their elasticity. Elasticity tests showed that ringent girdle bands are more flexible than complete ones (Coscinodiscus sp.) and that both long-chain clusters of Nitzschia palea and central spines of D. brightwellii have certain elasticities. The required pressure for deforming or breaking the biosilica structures of diatoms was also determined.

Keywords: Frustules; Microstructure; Mechanical characterization; Elastic structure; Microdevice; Nanotechnology

Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli by Nagesh K. Tripathi; Ambuj Shrivastava; Karttik C. Biswal; P. V. Lakshmana Rao (pp. 1179-1189).

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8 M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124 mg of highly purified and biologically active EDIII protein was obtained from 100 g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.

Keywords: Scale-up; Pilot-scale fermentation; Purification; Japanese encephalitis vaccine; Immunogenicity

Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33 by Xingjun Feng; Chunlong Liu; Jiayin Guo; Xueying Song; Jing Li; Wenshan Xu; Zhongqiu Li (pp. 1191-1198).

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5 mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1 l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195 Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC₅₀ = 16–64 μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.

Keywords: Antimicrobial peptides; Hybrid peptides; LfcinB; Thanatin; Prokaryotic expression

Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases by Anaïs A. E. Chavaroche; Lambertus A. M. van den Broek; Carmen Boeriu; Gerrit Eggink (pp. 1199-1210).

Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA⁺) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc⁺) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA⁺ and PmHS2-GlcNAc⁺ is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.

Keywords: Glycosaminoglycan; Heparosan; Oligosaccharide; Heparosan synthase; Single-action transferase; Polymerization

Characterization of a thermostable β-glucuronidase from Thermotoga maritima expressed in Arabidopsis thaliana by Jing Xu; Yong-Sheng Tian; Ri-He Peng; Bo Zhu; Jian-Jie Gao; Quan-Hong Yao (pp. 1211-1219).

TmGUSI, a gene identical to that encoding a thermostable β-glucuronidase in the hyperthermophilic anaerobe Thermotoga maritima, has been synthesized using a PCR-based two-step DNA synthesis and codon optimization for plants, and expressed in both Escherichia coli and Arabidopsis thaliana. TmGUSI expressed in transformed E. coli cells exhibited maximum hydrolytic activity at 65 °C and pH 6.5 and retained more than 80% activity after incubation at 85 °C for 30 min. TmGUSI activity in transgenic A. thaliana plants containing TmGUSI was also stable over the temperature range 65–80 °C. Our data suggest that β-glucuronidase from T. maritima can serve as a useful thermostable marker in higher plants.

Keywords: Arabidopsis thaliana ; β-Glucuronidase; Thermostable enzyme; Thermotoga maritima

In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum by Erica Elisa Ferrandi; Giulia Maria Bertolesi; Fausto Polentini; Armando Negri; Sergio Riva; Daniela Monti (pp. 1221-1233).

Nicotinamide adenine dinucleotide phosphate-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenases (7β-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7α-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman sequencing. After PCR amplifications of a gene fragment with degenerate primers, cloning of the complete gene (786 nt) has been achieved by sequencing of C. absonum genomic DNA. The sequence coding for the 7β-HSDH (783 nt) has been obtained by sequencing of the genomic DNA region flanking the 5′ termini of 7α-HSDH gene, the two genes being contiguous and presumably part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in Escherichia coli, purified by affinity chromatography and submitted to kinetic analysis for determination of Michaelis constants (K _m) and specificity constants (k _cat/K _m) in the presence of various bile acids derivatives. Both enzymes showed a very strong substrate inhibition with all the tested substrates. The lowest K _S values were observed with chenodeoxycholic acid and 12-ketochenodeoxycholic acid as substrates in the case of 7α-HSDH, whereas ursocholic acid was the most effective inhibitor of 7β-HSDH activity.

Keywords: Hydroxysteroid dehydrogenases; Clostridium absonum ; Bile acids; Cloning; Biochemical characterization; Substrate inhibition

Stability, oviposition attraction, and larvicidal activity of binary toxin from Bacillus sphaericus expressed in Escherichia coli by Luciano da Silva Pinto; Relber Aguiar Gonçales; Fabricio Rochedo Conceição; Paula Ferreira Knabah; Sibele Borsuk; Vinicius Farias Campos; Francisco Vassiliepe Arruda; Fabio Pereira Leivas Leite (pp. 1235-1241).

Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P < 0.05). These data suggest that biologically active recombinant BinA and BinB toxins might be useful in mosquito control programs, delivered by inactivated bacterial cells or in traps.

Keywords: Biological control; Culex quinquefasciatus ; Oviposition attraction; Binary toxin BinAB

Determining novel molecular markers in the Chinese caterpillar fungus Ophiocordyceps sinensis by screening a shotgun genomic library by Yong-Jie Zhang; Fei-Rong Bai; Shu Zhang; Xing-Zhong Liu (pp. 1243-1251).

The Chinese caterpillar fungus Ophiocordyceps sinensis, endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. Genetic differentiation within this fungus was observed; however, due to lack of highly efficient molecular markers, the overall genetic structure of this fungus has not been clarified. In this study, a shotgun genomic library of O. sinensis was constructed, and >181,848 nt were analyzed from >250 random clones. Primers from 33 sequenced fragments were then designed to amplify O. sinensis samples collected from widely separated regions on the Tibetan Plateau. Ten of the 33 fragments had no amplification or poor sequencing quality from all or certain samples. Sequence variations of the remaining 23 fragments among different samples were investigated in detail. Three fragments (OSRC14, OSRC19, and OSRC32) were the most variable with 7–43 single-nucleotide polymorphism (SNP) sites, representing the SNP frequency of 1.2–6.7 % per nucleotide site. These three fragments have the potential to be useful molecular markers for studying the population genetics of O. sinensis. These results also showed that constructing and screening a shotgun genomic library was an efficient approach to identify novel molecular markers from non-model organisms.

Keywords: Ophiocordyceps sinensis ; Shotgun genomic library; Molecular marker; Single-nucleotide polymorphism; Population genetics

Upconversion nanoparticles modified with aminosilanes as carriers of DNA vaccine for foot-and-mouth disease by Huichen Guo; Rongzeng Hao; Haisheng Qian; Shiqi Sun; Dehui Sun; Hong Yin; Zaixin Liu; Xiangtao Liu (pp. 1253-1263).

The potential of the upconversion nanoparticles NaYF₄:Yb/Er@silica(UCPs)/plasmid DNA (pcDNA3.1/VP1-GFP) complex in inducing immune responses was evaluated using the UCPs as carriers of the foot-and-mouth disease virus (FMDV AsiaI/Jiangsu2005) DNA vaccine. The UCPs protection against DNaseI degradation was measured using an in vitro inhibition assay. The expression of the plasmid in vivo was determined via confocal microscopy. Its biocompatibility was evaluated through cytotoxicity assay. Based on the results, the aminosilane-modified UCPs can electrostatically bind, condense, and protect plasmid DNA. Cell viability assays demonstrated that the cytotoxicity of the UCPs/plasmid DNA complex is lower than that of the cationic lipid/plasmid DNA complex, and that the transfection efficiency of UCPs is the same as that of the cationic lipid. Furthermore, the UCP/plasmid DNA complex was intramuscularly administered to guinea pigs. Humoral and cellular immune responses were detected using indirect enzyme-linked immunosorbent assay (ELISA), micro-neutralization assay, and T-lymphocyte proliferation assay. Anti-FMDV specific antibodies, neutralizing antibodies, and T-lymphocyte proliferation responses were induced after vaccination. In the challenge test, all the guinea pigs vaccinated with the UCPs/plasmid DNA complex were fully protected from the FMDV challenge. The current study encourages the use of UCPs as an effective nanosystem for gene delivery to cells for in vitro and in vivo vaccination, and other therapeutic applications.

Keywords: Upconversion nanoparticles; Gene delivery; DNA carrier; Foot-and-mouth disease virus; DNA vaccine

The effects of pH oscillation on Lactobacillus rhamnosus batch cultivation by Janne Wallenius; Dorothee Barth; Tero Eerikäinen (pp. 1265-1273).

Inhomogeneous mixing in industrial-sized fermentation processes causes oscillations in process parameters such as temperature or pH value in the cultivation medium, which causes stress to the bacteria being cultivated. In this work, the impact of extracellular pH oscillations on the production of Lactobacillus rhamnosus, a well-studied probiotic bacteria, were investigated by means of a scale-down batch process, simulating inhomogeneous pH values by controlling the pH value of the medium on sinusoidal trajectories. Effects of pH stimulation on the bacteria were assessed by testing storage and freeze-drying stability of harvested cells, two factors relevant for the industrial process. Furthermore, gene expressions of six selected genes, i.e. atpA, fat, cfa, groEL, hrcA, and pstS, known to be related to stress response were monitored. Although storage stability is only slightly negatively affected by pH stimulation of the bacteria, gene expression of four of the studied genes, i.e. fat, hrcA, groEL, and pstS show to correlate with amplitude and frequency of the oscillation.

Keywords: Gene expression; Oscillatory pH stimuli; Freezing; Freeze drying; Lactobacillus rhamnosus ; qPCR; atpA ; fat ; cfa ; groEL ; hrcA ; pstS ; ldhD

Isolation and characterization of Staphylococcus sp. strain NBRIEAG-8 from arsenic contaminated site of West Bengal by Shubhi Srivastava; Praveen C. Verma; Ankit Singh; Manisha Mishra; Namrata Singh; Neeta Sharma; Nandita Singh (pp. 1275-1291).

Arsenic contaminated rhizospheric soils of West Bengal, India were sampled for arsenic resistant bacteria that could transform different arsenic forms. Staphylococcus sp. NBRIEAG-8 was identified by16S rDNA ribotyping, which was capable of growing at 30,000 mg l⁻¹ arsenate [As(V)] and 1,500 mg l⁻¹ arsenite [As(III)]. This bacterial strain was also characterized for arsenical resistance (ars) genes which may be associated with the high-level resistance in the ecosystems of As-contaminated areas. A comparative proteome analysis was conducted with this strain treated with 1,000 mg l⁻¹ As(V) to identify changes in their protein expression profiles. A 2D gel analysis showed a significant difference in the proteome of arsenic treated and untreated bacterial culture. The change in pH of cultivating growth medium, bacterial growth pattern (kinetics), and uptake of arsenic were also evaluated. After 72 h of incubation, the strain was capable of removing arsenic from the culture medium amended with arsenate and arsenite [12% from As(V) and 9% from As(III)]. The rate of biovolatilization of As(V) was 23% while As(III) was 26%, which was determined indirectly by estimating the sum of arsenic content in bacterial biomass and medium. This study demonstrates that the isolated strain, Staphylococcus sp., is capable for uptake and volatilization of arsenic by expressing ars genes and 8 new upregulated proteins which may have played an important role in reducing arsenic toxicity in bacterial cells and can be used in arsenic bioremediation.

Keywords: Arsenic hypertolerance; Arsenic; Staphylococcus sp. ; Protein; Ars operon; Biovolatilization

Aoyap1 regulates OTA synthesis by controlling cell redox balance in Aspergillus ochraceus by Massimo Reverberi; Katia Gazzetti; Federico Punelli; Marzia Scarpari; Slaven Zjalic; Alessandra Ricelli; Anna A. Fabbri; Corrado Fanelli (pp. 1293-1304).

Among the various factors correlated with toxin production in fungi, oxidative stress is a crucial one. In relation to this, an important role is played by oxidative stress-related receptors. These receptors can transduce the “oxidative message” to the nucleus and promote a transcriptional change targeted at restoring the correct redox balance in the cell. In Aspergillus parasiticus, the knockout of the ApyapA gene, a homologue of the yeast Yap-1, disables the fungus’s capacity to restore the correct redox balance in the cell. As a consequence, the onset of secondary metabolism and aflatoxins synthesis is triggered. Some clues as to the involvement of oxidative stress in the regulation of ochratoxin A (OTA) synthesis in Aspergillus ochraceus have already been provided by the disruption of the oxylipin-producer AoloxA gene. In this paper, we add further evidence that oxidative stress is also involved in the regulation of OTA biosynthesis in A. ochraceus. In fact, the use of certain oxidants and, especially, the deletion of the yap1-homologue Aoyap1 further emphasize the role played by this stress in controlling metabolic and morphological changes in A. ochraceus.

Keywords: Oxidative stress; Ochratoxin A; Aspergillus ochraceus ; Aoyap1

Two-step denaturing gradient gel electrophoresis (2S-DGGE), a gel-based strategy to capture full-length 16S rRNA gene sequences by Shanquan Wang; Jianzhong He (pp. 1305-1312).

Obtaining full-length 16S rRNA gene sequences is important for generating accurate taxonomy assignments of bacteria, which normally is realized via clone library construction. However, the application of clone library has been hindered due to its limitations in sample throughput and in capturing minor populations (<1 % of total microorganisms). To overcome these limitations, a new strategy, two-step denaturing gradient gel electrophoresis (2S-DGGE), is developed to obtain full-length 16S rRNA gene sequences. 2S-DGGE can compare microbial communities based on its first-round DGGE profiles and generate partial 16S rRNA gene sequences (8–534 bp, Escherichia coli numbering). Then, strain-specific primers can be designed based on sequence information of bacteria of interest to PCR amplify their remaining 16S rRNA gene sequences (515–1541 bps, E. coli numbering). The second-round DGGE can confirm DNA sequence purity of these PCR products. Finally, the full-length 16S rRNA gene sequences can be obtained through combining the two partial DNA sequences. By employing 2S-DGGE, taxonomies of a group of dehalogenating bacteria have been assigned based on their full-length 16S rRNA gene sequences, several of which existed in dehalogenating microcosms as minor populations. In all, 2S-DGGE can be utilized as a medium throughput method for taxonomic identification of interested/minor populations from single or multiple microbial consortia.

Keywords: 2S-DGGE; 16S rRNA genes; Taxonomic identification; Microbial community

SMP production by activated sludge in the presence of a metabolic uncoupler, 3,3′,4′,5-tetrachlorosalicylanilide (TCS) by Yan Li; Ai-Min Li; Juan Xu; Bing Liu; Li-Chun Fu; Wen-Wei Li; Han-Qing Yu (pp. 1313-1321).

3,3′,4′,5-Tetrachlorosalicylanilide (TCS) is an effective metabolic uncoupler utilized for microbial yield reduction. However, its potential impact, in particular on the soluble microbial products (SMP) formation, is unknown yet. Herein we study the effect of TCS on SMP production and analyze the related mechanism. The addition of TCS in activated sludge system led to an increased production of SMP, especially proteins. The SMP were produced in proportion to the substrate utilization at a low TCS concentration, while more non-substrate-associated SMP were released at a high TCS concentration. TCS simulated the production of extracellular polymeric substances (EPS) and enhanced cell lysis, which both contributed to SMP production. FTIR and EEM analyses show that the SMP, EPS, and cell lysis products have similar functional groups and fluorescence properties, indicating a similar origin of these substances. In addition, a dose of TCS increased the release of high molecular weight compounds due to cell lysis. This study might benefit for a better understanding of the response of activated sludge to metabolic uncouplers like TCS.

Keywords: 3,3′,4′,5-Tetrachlorosalicylanilide (TCS); Activated sludge; Cell lysis; Extracellular polymeric substances (EPS); Metabolic uncoupler; Soluble microbial products (SMP)

Assessing the impact of alumina nanoparticles in an anaerobic consortium: methanogenic and humus reducing activity by Luis H. Alvarez; Francisco J. Cervantes (pp. 1323-1331).

The impact of γ-Al₂O₃ nanoparticles (NP) on specific methanogenic activity (SMA) and humus reducing activity (HRA) in an anaerobic consortium was evaluated. SMA in sludge incubations without γ-Al₂O₃ was always higher compared with those performed in the presence of 100 g/L of γ-Al₂O₃. Nevertheless, the SMA in incubations with γ-Al₂O₃ was not completely inhibited, indicating that some methanogenic microorganisms were physiologically active even in the presence of γ-Al₂O₃ NP during the incubation period (~400 h). SMA and HRA of the anaerobic consortium were also conducted in the presence of γ-Al₂O₃ NP coated with humic acids (HA). Microbial HA reduction occurred 3.7-fold faster using HA immobilized on γ-Al₂O₃ NP (HA_Imm), compared with the control with suspended HA (HA_Sus). Furthermore, immobilized HA decreased the toxicological effects of γ-Al₂O₃ NP on methanogenesis. Scanning electron microscopy (SEM) images revealed cell membrane damage in those sludge incubations exposed to uncoated γ-Al₂O₃ NP. In contrast, cell damage was not observed in incubations with HA-coated γ-Al₂O₃ NP. Methanogenesis out-competed microbial humus reduction regardless if HA was HA_Imm or HA_Sus. The present study provides a clear demonstration that HA immobilized in γ-Al₂O₃ NP are effective terminal electron acceptor for microbial respiration and suggests that HA could mitigate the toxicological effects of metal oxide NP on anaerobic microorganisms.

Keywords: Anaerobic consortium; Nanoparticles; Humic substances; Humus reduction; Methanogenesis

Effects of dissolved organic matter (DOM) at environmentally relevant carbon concentrations on atrazine degradation by Chelatobacter heintzii SalB by Karlien Cheyns; Jasper Calcoen; Fabrice Martin-Laurent; David Bru; Erik Smolders; Dirk Springael (pp. 1333-1341).

The dissolved organic matter (DOM) is the term used for organic components of natural origin present in the soil solution and is probably the most available C-source that primes microbial activity in subsoils. Contrasting effects of organic C components on pesticide degradation have been reported; however, most studies have used model organic compounds with compositions and concentrations which differ substantially from those found in the environment. Degradation of atrazine (AT) by Chelatobacter heintzii SalB was monitored in liquid batch assays in the absence or presence of well-defined model C compounds (glucose, gluconate and citrate) as model DOM (mDOM) or complex, less-defined, environmental DOM solutions (eDOM: isolated humic substances, soil and plant residue extracts) at environmentally relevant concentrations. Glucose significantly increased AT degradation rate by more than a factor of 8 at and above 2.5 mg C L^− 1. Optical density measurements showed that this stimulation is related to microbial growth. Gluconate and citrate had no effects unless at non-relevant concentrations (1,000 mg DOC L^− 1) at which stimulations (gluconate) or inhibitions (citrate) were found. The effects of eDOM added at 10 mg DOC L^− 1 on AT degradation were generally small. The AT degradation time was reduced by factors 1.4–1.9 in the presence of humic acids and eDOM from soils amended with plant residues; however, no effects were found for fulvic acids or eDOM from a soil leachate solution or extracted from unamended peat or forest soil. In conclusion, DOM supplied as both mDOM and eDOM did not inhibit AT degradation at environmentally relevant concentrations, and stimulation can be found for selected DOM samples and this is partly related to its effect on growth.

Keywords: Atrazine; Biodegradation; Dissolved organic matter; Chelatobacter heintzii SalB

Evaluation of the toxic effects of arsenite, chromate, cadmium, and copper using a battery of four bioassays by Kyung-Seok Ko; Pyeong-Koo Lee; In Chul Kong (pp. 1343-1350).

The sensitivities of four different kinds of bioassays to the toxicities of arsenite, chromate, cadmium, and copper were compared. The different bioassays exhibited different sensitivities, i.e., they responded to different levels of toxicity of each of the different metals. However, with the exception of the α-glucosidase enzyme activity, arsenite was the most toxic compound towards all the tested organisms, exhibiting the highest toxic effect on the seeds of Lactuca, with an EC₅₀ value of 0.63 mg/L. The sensitivities of Lactuca and Raphanus were greater than the sensitivities of two other kinds of seeds tested. Therefore, these were the seeds appropriate for use in a seed germination assay. A high revertant mutagenic ratio (5:1) of Salmonella typhimurium was observed with an arsenite concentration of 0.1 μg/plate, indicative of a high possibility of mutagenicity. These different results suggested that a battery of bioassays, rather than one bioassay alone, is needed as a more accurate and better tool for the bioassessment of environmental pollutants.

Keywords: Bioassays; Bioluminescence; Enzyme activity; Metals; Mutation; Seed germination

Quantification and characterization of β-lactam resistance genes in 15 sewage treatment plants from East Asia and North America by Ying Yang; Tong Zhang; Xu-Xiang Zhang; Da-Wei Liang; Ming Zhang; Da-Wen Gao; He-Guang Zhu; Qing-Guo Huang; Herbert H. P. Fang (pp. 1351-1358).

The emerging antibiotic resistance genes in the aquatic environment have aroused public concern. As β-lactam is the most widely used group of antibiotics, β-lactam resistance genes were selected to investigate their distribution and diversity in the activated sludge from 15 geographically different sewage treatment plants (STPs) of China, Singapore, USA, and Canada. Specific PCR and quantitative real-time PCR (q-PCR) were used to investigate the occurrence and abundance of nine β-lactam resistance genes. Five genes (OXA-1, OXA-2, OXA-10, ampC, and TEM-1) were detected in most of the sludge collected, while three genes (mecA, CTX-M-1, and SME) were not found in any sludge sample. The total abundances of the six detected β-lactam resistance genes in the 15 STPs varied from 5.34 × 10¹ copies/ng DNA (ampC) to 5.49 × 10⁴ copies/ng DNA (OXA-1). Overall, OXA-1 had the highest total concentration, followed by IMP and OXA-10. Noticeably, the abundances of TEM-1 in Chinese STPs were generally higher than those in the STPs of other countries, while the abundances of OXA-2 and IMP in the STPs of North America were much greater than those of East Asia. A total of 78 clones carrying β-lactam resistance genes were randomly selected from six clone libraries for phylogenetic diversity analysis; the similarity of these cloned genes to known β-lactam resistance genes with sequence identities ranged from 96% to 100%. Furthermore, OXA-1, ampC, and IMP were found to be more diverse than the other β-lactam resistance genes.

Keywords: Activated sludge; β-lactam resistance genes; Phylogenetic analysis; quantitative real-time PCR (q-PCR)

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing by Nan Hu; Bo Yuan; Juan Sun; Shi-An Wang; Fu-Li Li (pp. 1359-1368).

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40 °C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200 g L⁻¹) at 40 °C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2 g L⁻¹, which corresponded to the theoretical ethanol yield of 90.0 and 79.7 %, respectively. In the range of 30 to 40 °C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.

Keywords: Bioethanol; Consolidated bioprocessing (CBP); Jerusalem artichoke; Kluyveromyces marxianus ; Saccharomyces cerevisiae ; Thermotolerance

Retraction Note to: Antituberculars which target decaprenylphosphoryl-β-D-ribofuranose 2'-oxidase DprE1: state of art by Silvia Buroni; Maria Rosalia Pasca; Ana Luisa de Jesus Lopes Ribeiro; Giulia Degiacomi; Elisabetta Molteni; Giovanna Riccardi (pp. 1369-1369).

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Applied Microbiology and Biotechnology (v.95, #5)

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Applied Microbiology and Biotechnology (v.95, #5)