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Applied Microbiology and Biotechnology (v.87, #1)

Spotlights on advances in mycotoxin research by Markus Bohnert; Barbara Wackler; Dirk Hoffmeister (pp. 1-7).

A remarkable feature of filamentous fungi is their ability to produce small yet structurally complex and often bioactive natural products. In this mini-review, we cover advances in the research on fungal secondary metabolites, particularly mycotoxins, and focus on biosynthetic aspects as well as on the complex regulatory mechanisms which control the expression of biosynthetic genes. We also highlight the increasing impact of genomics and transcriptomics, which help explore the realm of secondary metabolism of fungi.

Keywords: Ascomycetes; Basidiomycetes; Biosynthesis; Genomics; Mycotoxin; Natural products

Biotransformation of ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases by Chang-Su Park; Mi-Hyun Yoo; Kyeong-Hwan Noh; Deok-Kun Oh (pp. 9-19).

Ginsenosides are the principal components responsible for the pharmaceutical activities of ginseng. The minor ginsenosides, which are also pharmaceutically active, can be produced via the hydrolysis of the sugar moieties in the major ginsenosides using acid hydrolytic, heating, microbial, and enzymatic transformation techniques. The enzymatic method has a profound potential for ginsenoside transformation, owing to its high specificity, yield, and productivity, and this method is increasingly being recognized as a useful tool in structural modification and metabolism studies. In this article, the transformation methods of ginsenosides, the characterization of microbial glycosidases with ginsenoside hydrolyzing activities, and the enzymatic production of minor ginsenosides are reviewed. Moreover, the conversions of ginsenosides using cell extracts from food microorganisms and recombinant thermostable β-d-glycosidases are proposed as feasible methods for use in industrial processes.

Keywords: Ginsenosides; Ginsenoside hydrolyzing enzyme; Ginsenoside biotransformation; Glycosidase; Enzyme characterization

Bikaverin production and applications by M. Carmen Limón; Roberto Rodríguez-Ortiz; Javier Avalos (pp. 21-29).

Bikaverin is a reddish pigment produced by different fungal species, most of them from the genus Fusarium, with antibiotic properties against certain protozoa and fungi. Chemically, bikaverin is a polyketide with a tetracyclic benzoxanthone structure, resulting from the activity of a specific class I multifunctional polyketide synthase and subsequent group modifications introduced by a monooxygenase and an O-methyltransferase. In some fungi, bikaverin is found with smaller amounts of a precursor molecule, called norbikaverin. Production of these metabolites by different fungal species depends on culture conditions, but it is mainly affected by nitrogen availability and pH. Regulation of the pathway has been investigated in special detail in the gibberellin-producing fungus Fusarium fujikuroi, whose genes and enzymes responsible for bikaverin production have been recently characterized. In this fungus, the synthesis is induced by nitrogen starvation and acidic pH, and it is favored by other factors, such as aeration, sulfate and phosphate starvation, or sucrose availability. Some of these inducing agents increase mRNA levels of the enzymatic genes, organized in a coregulated cluster. The biological properties of bikaverin include antitumoral activity against different cancer cell lines. The diverse biological activities and the increasing information on the biochemical and genetic basis of its production make bikaverin a metabolite of increasing biotechnological interest.

Keywords: Mycotoxin; Polyketide; PKS; Nitrogen regulation; pH regulation; Antibiotic; Antitumoral activity

Pathogenicity, virulence factors, and strategies to fight against Burkholderia cepacia complex pathogens and related species by Jorge H. Leitão; Sílvia A. Sousa; Ana S. Ferreira; Christian G. Ramos; Inês N. Silva; Leonilde M. Moreira (pp. 31-40).

The Burkholderia cepacia complex (Bcc) is a group of 17 closely related species of the β-proteobacteria subdivision that emerged in the 1980s as important human pathogens, especially to patients suffering from cystic fibrosis. Since then, a remarkable progress has been achieved on the taxonomy and molecular identification of these bacteria. Although some progress have been achieved on the knowledge of the pathogenesis traits and virulence factors used by these bacteria, further work envisaging the identification of potential targets for the scientifically based design of new therapeutic strategies is urgently needed, due to the very difficult eradication of these bacteria with available therapies. An overview of these aspects of Bcc pathogenesis and opportunities for the design of future therapies is presented and discussed in this work.

Keywords: Burkholderia cepacia complex; Pathogenicity; Virulence factors; Antimicrobials

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives by Qun Ren; Katinka Ruth; Linda Thöny-Meyer; Manfred Zinn (pp. 41-52).

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.

Keywords: R-hydroxycarboxylic acids; Polyhydroxyalkanotes (PHAs); Biotechnological processes; Chiral compounds

Engineering of microorganisms towards recovery of rare metal ions by Kouichi Kuroda; Mitsuyoshi Ueda (pp. 53-60).

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.

Keywords: Rare metals; Metal recovery; Bioadsorption; Cell surface engineering; Arming yeast

Removal of the endocrine disrupter butyl benzyl phthalate from the environment by Subhankar Chatterjee; Petr Karlovsky (pp. 61-73).

Butyl benzyl phthalate (BBP), an aryl alkyl ester of 1,2-benzene dicarboxylic acid, is extensively used in vinyl tiles and as a plasticizer in PVC in many commonly used products. BBP, which readily leaches from these products, is one of the most important environmental contaminants, and the increased awareness of its adverse effects on human health has led to a dramatic increase in research aimed at removing BBP from the environment via bioremediation. This review highlights recent progress in the degradation of BBP by pure and mixed bacterial cultures, fungi, and in sludge, sediment, and wastewater. Sonochemical degradation, a unique abiotic remediation technique, and photocatalytic degradation are also discussed. The degradation pathways for BBP are described, and future research directions are considered.

Keywords: Endocrine disrupter; Phthalate; Butyl benzyl phthalate; Biodegradation; Monoesters; Degradation pathway

Synthetic biology approaches in drug discovery and pharmaceutical biotechnology by Heinz Neumann; Petra Neumann-Staubitz (pp. 75-86).

Synthetic biology is the attempt to apply the concepts of engineering to biological systems with the aim to create organisms with new emergent properties. These organisms might have desirable novel biosynthetic capabilities, act as biosensors or help us to understand the intricacies of living systems. This approach has the potential to assist the discovery and production of pharmaceutical compounds at various stages. New sources of bioactive compounds can be created in the form of genetically encoded small molecule libraries. The recombination of individual parts has been employed to design proteins that act as biosensors, which could be used to identify and quantify molecules of interest. New biosynthetic pathways may be designed by stitching together enzymes with desired activities, and genetic code expansion can be used to introduce new functionalities into peptides and proteins to increase their chemical scope and biological stability. This review aims to give an insight into recently developed individual components and modules that might serve as parts in a synthetic biology approach to pharmaceutical biotechnology.

Keywords: Synthetic biology; BioBricks; Natural products; In vivo small molecule libraries; Biosensors; Genetic code expansion

Honey bee pathology: current threats to honey bees and beekeeping by Elke Genersch (pp. 87-97).

Managed honey bees are the most important commercial pollinators of those crops which depend on animal pollination for reproduction and which account for 35% of the global food production. Hence, they are vital for an economic, sustainable agriculture and for food security. In addition, honey bees also pollinate a variety of wild flowers and, therefore, contribute to the biodiversity of many ecosystems. Honey and other hive products are, at least economically and ecologically rather, by-products of beekeeping. Due to this outstanding role of honey bees, severe and inexplicable honey bee colony losses, which have been reported recently to be steadily increasing, have attracted much attention and stimulated many research activities. Although the phenomenon “decline of honey bees” is far from being finally solved, consensus exists that pests and pathogens are the single most important cause of otherwise inexplicable colony losses. This review will focus on selected bee pathogens and parasites which have been demonstrated to be involved in colony losses in different regions of the world and which, therefore, are considered current threats to honey bees and beekeeping.

Keywords: Honey bees; Varroa; Virus; Nosema; European Foulbrood; Colony losses

Current state and perspectives of fungal DNA barcoding and rapid identification procedures by Dominik Begerow; Henrik Nilsson; Martin Unterseher; Wolfgang Maier (pp. 99-108).

Fungal research is experiencing a new wave of methodological improvements that most probably will boost mycology as profoundly as molecular phylogeny has done during the last 15 years. Especially the next generation sequencing technologies can be expected to have a tremendous effect on fungal biodiversity and ecology research. In order to realise the full potential of these exciting techniques by accelerating biodiversity assessments, identification procedures of fungi need to be adapted to the emerging demands of modern large-scale ecological studies. But how should fungal species be identified in the near future? While the answer might seem trivial to most microbiologists, taxonomists working with fungi may have other views. In the present review, we will analyse the state of the art of the so-called barcoding initiatives in the light of fungi, and we will seek to evaluate emerging trends in the field. We will furthermore demonstrate that the usability of DNA barcoding as a major tool for identification of fungi largely depends on the development of high-quality sequence databases that are thoroughly curated by taxonomists and systematists.

Keywords: Fungi; Barcode; ITS rDNA; Taxonomy; Next generation sequencing

Repeated batch fermentation from raw starch using a maltose transporter and amylase expressing diploid yeast strain by Syun-ichi Yamakawa; Ryosuke Yamada; Tsutomu Tanaka; Chiaki Ogino; Akihiko Kondo (pp. 109-115).

We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, α-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that α-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest α-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was α-amylase activity at 6.43 U/ml.

Keywords: Yeast; Amylase; Raw starch; Ethanol fermentation; Repeated batch fermentation

Evaluation of the biocompatibile ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite pretreatment of corn cob for improved saccharification by Qiang Li; Xinglin Jiang; Yucai He; Liangzhi Li; Mo Xian; Jianming Yang (pp. 117-126).

Ionic liquid (IL) pretreatment of lignocellulose materials is a promising process in biomass conversion to renewable biofuel. More in-depth research involving environment-friendly IL is much needed to explore pretreatment green route. In our case, IL 1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) was chosen as an environment-friendly solvent to pretreat corn cob in view of its biocompatibility with both lignocellulose solubility and cellulase activity. The pretreatment/saccharification process and in situ saccharification process involving [Mmim]DMP were efficiently performed in bioconversion of corn cob to sugars, and more than 70% saccharification rates were obtained. Furthermore, the fermentability of reducing sugars obtained from the hydrolyzates was evaluated using Rhodococcus opacus strain ACCC41043 (R. opacus). High lipid production 41–43% of cell dry matter was obtained after 30 h of cultivation. GC/MS analysis indicated that lipids from R. opacus contained mainly long-chain fatty acids with four major constituent/oleic acid, stearic acid, palmitic acid, palmitoleic acid which are good candidates for biodiesel. These elucidated that corn cob pretreated by IL [Mmim]DMP did not bring negative effects on saccharification, cell growth, and accumulation of lipid of R. opacus. In conclusion, the IL [Mmim]DMP shows promise as green pretreatment solvent for cellulosic materials.

Keywords: Corn cob; Biocompatibility; Ionic liquids pretreatment; 1-Methyl-3-methylimidazolium dimethylphosphite; Enzymatic hydrolysis; Microbial lipid

Fermentation and metabolic characteristics of Gluconacetobacter oboediens for different carbon sources by Dayanidhi Sarkar; Masahiro Yabusaki; Yuta Hasebe; Pei Yee Ho; Shuji Kohmoto; Takayuki Kaga; Kazuyuki Shimizu (pp. 127-136).

The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h⁻¹. The ¹³C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO₂ production rate and the biomass concentration decreased, where the ¹³C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as the feed gluconate concentration was increased.

Keywords: Gluconacetobacter oboediens ; Chemostat culture; Enzyme activity; ¹³C-labeling experiment; Metabolic flux analysis; Acetate fermentation

Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures by Jiang-Lin Zhao; Li-Gang Zhou; Jian-Yong Wu (pp. 137-144).

This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co²⁺, Ag⁺, Cd²⁺), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g^-1 versus 0.2 mg g^-1 in control). The stimulating effect was concentration-dependent, most significant at 25 μM of Ag and Cd and 100 mg l^-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5 g l^-1 versus 8.9 g l^-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.

Keywords: Salvia miltiorrhiza ; Cell culture; Tanshinones; Elicitors; Stress response

Development of a fermentation process based on a defined medium for the production of pregallidermin, a nontoxic precursor of the lantibiotic gallidermin by Giovanni Medaglia; Sven Panke (pp. 145-157).

In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum ΔP, which produces pregallidermin (PGDM), a nontoxic precursor of the lantibiotic gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum ΔP. The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum ΔP was grown up to a biomass concentration of 67 g l⁻¹ and produced 1.95 g l⁻¹ PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based gallidermin production processes (Kempf et al. 2000). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tü 3928 was only 0.08 mM.

Keywords: Defined medium; Staphylococcus; Gallidermin; Lantibiotic; Fermentation; Product inhibition

Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation by Toru Jojima; Miho Fujii; Eiji Mori; Masayuki Inui; Hideaki Yukawa (pp. 159-165).

Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD⁺ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l⁻¹ of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.

Keywords: Corynebacterium glutamicum ; Oxygen deprivation; Amino acid production; Environmentally friendly process

Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems by Markus Michael Müller; Barbara Hörmann; Christoph Syldatk; Rudolf Hausmann (pp. 167-174).

Rhamnolipids are biosurfactants with interesting physico-chemical properties. However, the main obstacles towards an economic production are low productivity, high raw-material costs, relatively expensive downstream processing, and a lack of understanding the rhamnolipid production regulation in bioreactor systems. This study shows that the sequenced Pseudomonas aeruginosa strain PAO1 is able to produce high quantities of rhamnolipid during 30 L batch bioreactor cultivations with sunflower oil as sole carbon source and nitrogen limiting conditions. Thus PAO1 could be an appropriate model for rhamnolipid production in pilot plant bioreactor systems. In contrast to well-established production strains, PAO1 allows knowledge-based systems biotechnological process development combined with the frequently used heuristic bioengineering approach. The maximum rhamnolipid concentration obtained was 39 g/L after 90 h of cultivation. The volumetric productivity of 0.43 g/Lh was comparable with previous described production strains. The specific rhamnolipid productivity showed a maximum between 40 and 70 h of process time of 0.088 g_RL/g_BDMh. At the same time interval, a shift of the molar di- to mono-rhamnolipid ratio from 1:1 to about 2:1 was observed. PAO1 not only seems to be an appropriate model, but surprisingly has the potential as a strain of choice for actual biotechnological rhamnolipid production.

Keywords: Rhamnolipids; Pseudomonas aeruginosa PAO1; Glycolipids; Renewable resources; Systems biotechnology; Biosurfactants

Glutathione accumulation in ethanol-stat fed-batch culture of Saccharomyces cerevisiae with a switch to cysteine feeding by Ildar Nisamedtinov; Kaspar Kevvai; Kerti Orumets; Jari J. Rautio; Toomas Paalme (pp. 175-183).

Shot-wise supplementation of cysteine to a yeast culture is a common means of promoting glutathione (GSH) production. In the present work, we study the accumulation kinetics of cysteine, γ-glutamylcysteine, and GSH and the expression of genes involved in GSH and sulfur metabolism in ethanol-stat fed-batch cultures as a result of switching to a medium enriched with cysteine and glycine. Supplementation in this fashion resulted in a rapid but short-term increase in the rate of GSH synthesis, while the expression of GSH1 decreased. Expression of GSH1 and GSH synthesis rate were observed to revert close to the base level after a few hours. These results indicate that, under such conditions, the control of GSH synthesis at higher concentrations occurred at the enzymatic, rather than the transcriptional level. The incorporation of cysteine into GSH was limited to ∼40% of the theoretical yield, due to its requirement as a source of sulfur for protein synthesis under conditions whereby the sulfate assimilation pathway is down-regulated. This was supported by the expression profiles of genes involved in cysteine and homocysteine interconversion.

Keywords: Glutathione; Cysteine; Ethanol-stat; Fed-batch; TRAC; Saccharomyces cerevisiae

Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity by Yun Hee Choi; Hye Jeong Choi; Dooil Kim; Ki-Nam Uhm; Hyung-Kwoun Kim (pp. 185-193).

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.

Keywords: Antidepressant drugs; Chiral intermediate; Docking model; Enantioselectivity; Reductase

Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran by Kofi Abokitse; Meiqun Wu; Hélène Bergeron; Stephan Grosse; Peter C. K. Lau (pp. 195-203).

A putative α/β hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75°C, the enzyme retained at least 95% of its original activity for over 80 min; at 80°C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.

Keywords: Biorefinery; Platform chemicals; Antioxidant; Phenolic acid; ORAC; Genome mining

Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis by Hee-Jung Moon; Manish Tiwari; Marimuthu Jeya; Jung-Kul Lee (pp. 205-214).

Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to d-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide of 266 amino acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated to be ∼28 kDa by sodium dodecyl sulfate-polyacrylamide gel and ∼58 KDa with gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 9.5 and 65°C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a k _cat of 4.83 s⁻¹ for NADH (k _cat/K _m = 27.3 s⁻¹ mM⁻¹) and k _cat of 2.79 s⁻¹ for NADPH (k _cat/K _m = 10.8 s⁻¹ mM⁻¹). Homology modeling and docking studies of NAD⁺ and NADP⁺ into the active site of ZmRDH shed light on the dual coenzyme specificity of ZmRDH.

Keywords: Characterization; Coenzyme specificity; Zymomonas mobilis ; Ribitol dehydrogenase; Ribulose

Characterization of a Δ12-fatty acid desaturase gene from Ceriporiopsis subvermispora, a selective lignin-degrading fungus by Takahito Watanabe; Saeko Tsuda; Hiroshi Nishimura; Yoichi Honda; Takashi Watanabe (pp. 215-224).

Ceriporiopsis subvermispora, a white-rot fungus, is characterized as one of the best biopulping fungi because it can degrade lignin selectively without serious damage to cellulose. We previously demonstrated that during the early stage of wood decay, this fungus produces large amounts of linoleic acid (18:2n-6) and degrades lignin by manganese peroxidase-catalyzed lipid peroxidation. In this study, we cloned a Δ12-fatty acid desaturase gene absolutely essential for the biosynthesis of linoleic acid as the main substrate for lipid peroxidation. This gene designated Cs-fad2 encodes a protein with three histidine-rich domains and four membrane-spanning domains characteristic of other Δ12-fatty acid desaturases. Moreover, we heterologously expressed Cs-fad2 in Saccharomyces cerevisiae lacking Δ12-fatty acid desaturase, and detected the de novo biosynthesis of linoleic acid by gas chromatography–mass spectrometry analysis. We also investigated transcription of Cs-fad2 under various conditions. The transcription was activated and repressed in the presence of a lignin fragment and exogenous fatty acids, respectively. These results may shed light on the molecular relationship between fatty acid metabolism and selective lignin degradation in C. subvermispora.

Keywords: Fatty acid desaturase; Lignin degradation; White-rot fungus; Lipid peroxidation

Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4 by Yingying Zheng; Yanfen Xue; Yueling Zhang; Cheng Zhou; Ulrich Schwaneberg; Yanhe Ma (pp. 225-233).

A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 °C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 °C for 6 h. TtcGA can hydrolyze both α-1, 4- and α-1, 6-glycosidic linkages in various α-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca²⁺, Mn²⁺, Co²⁺, Mg²⁺, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn²⁺, Pb²⁺, Cu²⁺, and EDTA.

Keywords: Glucoamylase; Thermoanaerobacter tengcongensis ; Recombinant expression; Thermostable

Enzymatic fragmentation of the antimicrobial peptides casocidin and isracidin by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus by George A. Somkuti; Moushumi Paul (pp. 235-242).

The cumulative effect of peptidase and protease activities associated with cells of Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB) was evaluated on the milk protein-based antimicrobial peptides casocidin and isracidin. Reaction mixtures of casocidin or isracidin and nonproliferating mid-log cells of these essential yogurt starter cultures were individually incubated for up to 4 h at pH 4.5 and 7.0, and samples removed at various time points were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF/TOF-MS. Both casocidin and isracidin remained largely unchanged following exposure to cell suspensions of ST or LB strains at pH 4.5. Casocidin was extensively degraded by both ST and LB strains at pH 7.0, whereas isracidin remained largely intact after incubation for 4 h with ST strains but was degraded by exposure to LB strains. The results showed the feasibility of using the bovine casein-based peptides casocidin and isracidin as food grade antimicrobial supplements to impart fermented dairy foods additional protection against bacterial contamination. The structural integrity and efficacy of these biodefensive peptides may be preserved by timing their addition near the end of the fermentation of yogurt-like dairy foods (at or below pH 4.5), when conditions for bacterial proteolytic activity become unfavorable.

Keywords: Streptococcus thermophilus ; Lactobacillus delbrueckii ssp. bulgaricus ; RP-HPLC; Casocidin; Isracidin

Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects by Jingjing Liu; Guixin Yan; Changlong Shu; Can Zhao; Chunqin Liu; Fuping Song; Lin Zhou; Junlan Ma; Jie Zhang; Dafang Huang (pp. 243-249).

A newly engineered strain, denominated BIOT185, was constructed through integrating the cry8Ca2 gene into the endogenous plasmid of BT185 (contains cry8Ea1) by homologous recombination. The thermosensitive plasmid vector was eliminated by the rising temperature of recombinant cultures. No antibiotic gene or other unnecessary genes were introduced to the new strain. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry8Ca2 gene was expressed normally and produced a 130-kDa protein in the BIOT185 strain. Bioassay results showed that the new strain had high toxicity to the pests Anomala corpulenta and Holotrichia parallela, which often damage the same fields.

Keywords: Bacillus thuringiensis ; Engineered strain; Homologous recombination; Anomala corpulenta ; Holotrichia parallela

A novel family 9 β-1,3(4)-glucanase from thermoacidophilic Alicyclobacillus sp. A4 with potential applications in the brewing industry by Yingguo Bai; Jianshe Wang; Zhifang Zhang; Pengjun Shi; Huiying Luo; Huoqing Huang; Chunliang Luo; Bin Yao (pp. 251-259).

An endo-β-1,3(4)-glucanase gene, Agl9A, was cloned from Alicyclobacillus sp. A4 and expressed in Pichia pastoris. Its deduced amino acid sequence shared the highest identity (48%) with an endo-β-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A exhibited relatively wide substrate specificity, including lichenan (109%), barley β-glucan (100%), CMC-Na (15.02%), and laminarin (6.19%). The optimal conditions for Agl9A activity were pH 5.8 and 55°C. The enzyme was stable over a broad pH range (>60% activity retained after 1-h incubation at pH 3.8–11.2) and at 60°C (>70% activity retained after 1-h incubation). Agl9A was highly resistant to various neutral proteases (e.g., trypsin, α-chymotrypsin, and collagenase) and Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A (20 U/ml) or a commercial xylanase (200 U/ml) reduced the filtration rate (26.71% and 20.21%, respectively) and viscosity (6.12% and 4.78%, respectively); furthermore, combined use of Agl9A (10 U/ml) and the xylanase (100 U/ml) even more effectively reduced the filtration rate (31.73%) and viscosity (8.79%). These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.

Keywords: β-1,3(4)-glucanase; Alicyclobacillus sp.; Malting and brewing; Glycoside hydrolase family 9

Use of an inducible promoter for antibiotic production in a heterologous host by Volker Dangel; Lucia Westrich; Margaret C. M. Smith; Lutz Heide; Bertolt Gust (pp. 261-269).

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L⁻¹ of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25–2.0 mg L⁻¹). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.

Keywords: Novobiocin; Inducible promoter; Genetic engineering; Streptomyces

Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes by Yujin Cao; Jianming Yang; Mo Xian; Xin Xu; Wei Liu (pp. 271-280).

Biodiesel is an interesting alternative energy source and is used as substitute for petroleum-based diesel. Microorganisms have been used for biodiesel production due to their significant environmental and economic benefits. However, few researches have investigated the regulation of fatty acid composition of these microbial diesels. Fatty acid biosynthesis in Escherichia coli has provided a paradigm for other bacteria and plants. By overexpressing two genes (fabA and fabB) associated with unsaturated fatty acid (UFA) synthesis in E. coli, we have engineered an efficient producer of UFAs. Saturated fatty acid (SFA) contents decreased from 50.2% (the control strain) to 34.6% (the recombinant strain overexpressing fabA and fabB simultaneously) and the ratio of cis-vaccenate (18:1Δ11), a major UFA in E. coli, reached 51.1% in this recombinant strain. When an Arabidopsis thaliana thioesterase (AtFatA) was coexpressed with these two genes, 0.19 mmol l⁻¹ fatty acids was produced by this E. coli strain after 18-h culture under shake-flask conditions. Free fatty acids made up about 37.5% of total fatty acid concentration in this final engineered strain carrying fabA, fabB, and AtFatA, and the ratio of UFA/SFA reached 2.3:1. This approach offers a means to improve the fatty acid composition of microdiesel and might pave the way for production of biodiesel equivalents using engineered microorganisms in the near future.

Keywords: AtFatA ; Biodiesel; fabA ; fabB ; Unsaturated fatty acid

Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol by Yan Liu; Qing Wei; Shu-Jun Wang; Hong Liu; Ning-Yi Zhou (pp. 281-287).

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.

Keywords: Non-antibiotic resistance gene marker; Methyl parathion; ortho-Nitrophenol; para-Nitrophenol; Simultaneous degradation

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori by Xingwei Xiang; Rui Yang; Shaofang Yu; Cuiping Cao; Aiqin Guo; Lin Chen; Xiaofeng Wu; Weizheng Cui; J. L. Cenis (pp. 289-295).

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.

Keywords: Bombyx mori ; Baculovirus; Polyhedra; Oral infection

Identification and characterization of genes involved in naphthalene degradation in Rhodococcus opacus R7 by Patrizia Di Gennaro; Paola Terreni; Gianmarco Masi; Silvia Botti; Francesca De Ferra; Giuseppina Bestetti (pp. 297-308).

Rhodococcus opacus R7 is a naphthalene-degrading microorganism which is also able to grow on o-xylene. This work describes the isolation and analysis of two new genomic regions in which genes involved in naphthalene (nar gene cluster) and salicylate (gen gene cluster) degradation are located. In the nar gene cluster we found: two genes encoding the large (narAa) and the small (narAb) components of the naphthalene dioxygenase, three genes (rub1, rub2, rub1bis) encoding three rubredoxins, an orf (orf7) associated to the complex encoding a protein of unknown function, two regulatory genes (narR1, narR2), a gene (narB) encoding the naphthalene dihydrodiol dehydrogenase and six orfs (orf1, orf2, orf3, orf4, orf5, orf6) encoding proteins of unknown function. In the gen gene cluster, we found the following genes: two genes encoding the salicylate CoA ligase and the salicylate CoA synthetase (genA and genB), respectively, a gene (genC) encoding a salicylate hydroxylase, a gene (genH) encoding a gentisate 1,2-dioxygenase, a gene (genI) encoding a 3-maleylpyruvate isomerase, and a gene (genL) encoding a protein of unknown function. The transcription of some genes of R. opacus R7 strain grown on different substrates was also investigated to evaluate the expression of the two gene clusters after cDNA preparations.

Keywords: Rhodococcus ; Naphthalene degradation; Catabolic genes; RT-PCR

Chemoinformatics-assisted development of new anti-biofilm compounds by Anna Dürig; Irene Kouskoumvekaki; Rebecca M. Vejborg; Per Klemm (pp. 309-317).

Bacterial biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Here, we use a novel cross-disciplinary approach combining microbiology and chemoinformatics to identify new and efficient anti-biofilm drugs. We found that ellagic acid (present in green tea) significantly inhibited biofilm formation of Streptococcus dysgalactiae. Based on ellagic acid, we performed in silico screening of the Chinese Natural Product Database to predict a 2nd-generation list of compounds with similar characteristics. One of these, esculetin, proved to be more efficient in preventing biofilm formation by Staphylococcus aureus. From esculetin a 3rd-generation list of compounds was predicted. One of them, fisetin, was even better to abolish biofilm formation than the two parent compounds. Fisetin dramatically inhibited biofilm formation of both S. aureus and S. dysgalactiae. The compounds did not affect planktonic growth in concentrations where they affected biofilm formation and appeared to be specific antagonists of biofilms. Arguably, since all three compounds are natural ingredients of dietary plants, they should be well-tolerated by humans. Our results indicate that such small plant components, with bacterial lifestyle altering properties are promising candidates for novel generations of antimicrobial drugs. The study underlines the potential in combining chemoinformatics and biofilm research.

Keywords: Anti-biofilm drugs; Bacterial biofilms; Chemoinformatics; Dietary plant components; Staphylococcus aureus ; Streptococcus dysgalactiae

Searching for monooxygenases and hydrolases in bacteria from an extreme environment by Georgiana F. da Cruz; Célio F. F. Angolini; Luciana G. de Oliveira; Patrícia F. Lopes; Suzan P. de Vasconcellos; Elaine Crespim; Valéria M. de Oliveira; Eugênio V. dos Santos Neto; Anita J. Marsaioli (pp. 319-329).

Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities.

Keywords: Oil reservoirs; Alcohol dehydrogenases; Monooxygenases; Hydrolases; High-throughput screening; Bacterial 16S rRNA genes; Petroleum microorganisms

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry by Yingying Wang; Lieve Claeys; David van der Ha; Willy Verstraete; Nico Boon (pp. 331-341).

The present study reports the disinfection effects of chemically and electrochemically dosed chlorine on two models for typical water-borne bacteria (Escherichia coli and Legionella beliardensis) by plating and flow cytometry (FCM) in combination with different fluorescence dyes. The residual effect on various cell functions, including cultivability, esterase activity, membrane polarization, and integrity, was tested at different free chlorine concentrations. In comparison, chemical disinfection yielded on average 60% more E. coli cells entering the viable but nonculturable (VBNC) state than electrochemical disinfection. Here, VBNC is defined as those cells with intact cell membrane but which cannot be cultured on solid nutrient agar plates. L. beliardensis was about five times more resistant to chlorine disinfection than E. coli. The results also suggested the two methods result in different disinfection mechanisms on L. beliardensis, i.e., chemically dosed chlorine targeted cell membrane integrity before enzyme activity, while electrochemically dosed chlorine acted the other way round. In addition, both bacteria lost the integrity of their cell membranes at three times lower chlorine concentration over a longer contact time (i.e., 40 vs. 10 min) by the chemical method. Our results showed that FCM is an appropriate tool to evaluate the effects of water disinfection and the percentage of cells in VBNC in a matter of hours. Electrochemical disinfection is suggested to be a favorable alternative for chemical disinfection.

Keywords: Disinfection; E. coli ; Legionella ; Chlorination; Flow cytometry; VBNC

An optimized RNA amplification method for prokaryotic expression profiling analysis by Feng-Lin Cao; Han-Hua Liu; Ya-Hui Wang; Yu Liu; Xiao-Yu Zhang; Jian-Qing Zhao; Yi-Min Sun; Jin Zhou; Liang Zhang (pp. 343-352).

DNA microarray technology has been extensively used for gene expression analysis of both eukaryotic and prokaryotic organisms. For eukaryotic gene expression profiling, the poly(A)-based reverse transcription of messenger RNA (mRNA) followed by T7 RNA polymerase-based in vitro transcription is generally required to produce enough RNA targets for hybridization with the microarray chips. However, the same method cannot be directly applied to prokaryotic mRNAs due to the lack of poly(A) sequences at the 3′ ends. Conventional methods usually require large amounts of starting RNAs and lead to high background noise. Recently developed amplification methods enable smaller amounts of prokaryotic RNA to be used from samples with species-specific primers, oligo(dT) primers, or random primers. In this study, three target preparation methods, including the direct labeling, polyadenylation-involved oligo-dT priming, and random priming amplification (respectively referred to as DL, PAOD, and RPA hereafter) were evaluated through expression profiling of a heat shock model of Escherichia coli. The PAOD method was found to be more sensitive and more specific in differential gene expression measurements than either DL and RPA, even when the E. coli RNA was only a small proportion of the simulated eukaryotic host RNA. The results suggest that PAOD is the preferred target preparation method for prokaryotic transcriptome.

Keywords: Microarray analysis; Prokaryotic expression; RNA amplification; Target preparation method

Microbial community dynamics in anaerobic bioreactors and algal tanks treating piggery wastewater by Sayali S. Patil; Martin S. Kumar; Andrew S. Ball (pp. 353-363).

Integrated biosystem is becoming a major aspect of wastewater management practice. Microbial communities in piggery wastewater sampled from anaerobic (thermophilic and mesophilic) and aerobic digesters (algal tanks) during waste remediation were analyzed by culture-independent techniques based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The use of Muyzer's 314F-GC, 518R bacterial primers, and archaeal A934F, 1309R primers followed by partial 16s rDNA sequence analysis of the main bands from DGGE revealed the presence of unknown and as yet uncultured microorganisms but also showed functional and ecologically significant denitrifying, acetogenic bacteria along with autotrophic, hydrogenotrophic, and acetoclastic methanogen archaea. Thermophilic digesters were dominated by γ-Proteobacteria, Methanothermobacter sp., while mesophilic digesters showed dominance by Firmicutes, uncultured bacteria, Methanosarcina, and Methanoculleus genera. Under aerobic conditions within algal tanks, pH rose from 7.17 to 9.32, with a significant decrease in total ammonia nitrogen, chemical oxygen demand, and soluble phosphorus levels. PCR-DGGE proved a useful tool for investigating the dynamics of microbial community in the bio-processing of piggery wastewater. Knowledge of the microbial communities involved in digestion of piggery wastewater will allow optimization of integrated biosystem by removing the main pollutants like inorganic ammonium-nitrogen, phosphorus, and pathogens from intensive farming system.

Keywords: Integrated biosystem; 16S rDNA polymerase chain reaction (PCR); Denaturing gradient gel electrophoresis analysis (DGGE); Microbial community

The trans/cis ratio of unsaturated fatty acids is not applicable as biomarker for environmental stress in case of long-term contaminated habitats by Janett Fischer; Frieder Schauer; Hermann J. Heipieper (pp. 365-371).

Cis-trans isomerization of unsaturated fatty acids is a crucial adaptive reaction of Pseudomonas and Vibrio species to toxic organic compounds or other environmental stress factors. In order to test the long-term performance of this adaptive mechanism as well as to assess its application as biomarker for environmental contamination studies were performed in batch cultures and in continuously running sand columns, simulating long-term contamination with bisphenol A (BPA). In short-term grown batch cultures a high correlation between trans/cis ratio and added BPA concentration and toxicity was observed. In contrary, this did not occur in the case of long-term sand columns. An increase in trans/cis ratio of unsaturated fatty acids only appeared in a limited period of time. Afterwards the trans/cis ratio reached the values measured for non-stressed cultures. Cis-trans isomerization is only an urgent response mechanism that is later substituted by other adaptive mechanisms. Therefore, it can be concluded that the trans/cis ratio of unsaturated fatty acids was shown not to be an appropriate biomarker for durable stress in the environment.

Keywords: trans/cis ratio of unsaturated fatty acids; Pseudomonas putida P8; Biomarker; Adaptation; Bisphenol A; Sand columns

Formation of soluble microbial products by activated sludge under anoxic conditions by Wen-Ming Xie; Bing-Jie Ni; Raymond J. Zeng; Guo-Ping Sheng; Han-Qing Yu; Jing Song; De-Zhi Le; Xue-Jun Bi; Chang-Qing Liu; Min Yang (pp. 373-382).

In this work, both experimental and modeling approaches are used to explore the formation of soluble microbial products (SMP) by activated sludge under anoxic conditions. With substrate consumption, the SMP concentration increases gradually. Utilization associated products (UAP) are the main fraction of SMP when substrate is present; whereas biomass associated products (BAP) are the major content of SMP as substrate is completely consumed. The fraction of the accumulated SMP accounts for 3-4% of initial organic substrate. Three dimensional excitation emission matrix analysis results indicate that the SMP concentration increases in the denitrification process. The accumulation of nitrite up to 22.6 mg/l under anoxic conditions has no significant effect on the SMP formation. With a consideration of SMP formation under anoxic conditions, an ASM3-based denitrification model is developed. The results show that the developed model is able to capture the relationship between the SMP formation and the substrate consumption by activated sludge in the denitrification process.

Keywords: Activated sludge; C/N; Denitrification; Modeling; Soluble microbial products (SMP)

Electricity generation from glucose by a Klebsiella sp. in microbial fuel cells by Xue Xia; Xiao-xin Cao; Peng Liang; Xia Huang; Su-ping Yang; Gen-gui Zhao (pp. 383-390).

As electrochemically active bacteria play an important role in microbial fuel cells (MFCs), it is necessary to get a comprehensive understanding of their electrogenesis mechanisms. In this study, a new electrochemically active bacterium, Klebsiella sp. ME17, was employed into an “H” typed MFC for electrogenesis, with glucose as the electron donor. The maximum power density was 1,209 mW/m² at a resistance of 340 Ω and the maximum current was 1.47 mA. Given the original anode medium, fresh medium, and the supernatant of the anode medium in the same MFC, respectively, the polarization curves illustrated that the strain produced mediators to promote extracellular electron transfer. The anode medium supernatant was electrochemically active, based on cyclic voltammogram, and the supernatant was very likely to contain quinone-like substances, as indicated by spectrophotometric and excitation–emission matrix fluorescence spectroscopy analysis. Further investigation on the color and ultraviolet absorbance at 254 nm of the filtered anode medium showed that the redox states of mediators strongly associated with the electricity generation states in MFCs.

Keywords: Microbial fuel cell; Glucose; Klebsiella sp. ME17; Mediator; Quinone-like substance

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Applied Microbiology and Biotechnology (v.87, #1)

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Applied Microbiology and Biotechnology (v.87, #1)