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Applied Microbiology and Biotechnology (v.83, #2)

Rapid sampling devices for metabolic engineering applications by Friederike Schädel; Ezequiel Franco-Lara (pp. 199-208).

A number of rapid sampling devices for metabolic engineering applications have been developed over the last years with the purpose of the estimation of in vivo metabolic concentrations and dynamics. This review outlines the designs and characteristics as well as the developments and changes in diverse approaches over the years. Primary performance parameters for these constructions are sampling time and rate and, for an accurate representation of the in vivo condition in cells, the reproducibility of results and easy handling throughout the sampling operation.

Keywords: Metabolic profiling; Quenching; Rapid sampling; Sequential sampling; Stimulus response experiments

Towards tailor-made oligosaccharides—chemo-enzymatic approaches by enzyme and substrate engineering by Arne Homann; Jürgen Seibel (pp. 209-216).

Carbohydrate structures have been identified in eukaryotic and prokaryotic cells as glycoconjugates with communication skills. Their recently discussed role in various diseases has attracted high attention in the development of simple and convenient methods for oligosaccharide synthesis. In this review, recent approaches combining nature’s power for the design of tailor made biocatalysts by enzyme engineering and substrate engineering will be presented. These strategies lead to highly efficient and selective glycosylation reactions. The introduced concept shall be a first step in the direction to a glycosylation toolbox which paves the way for the tailor-made synthesis of designed carbohydrate structures.

Keywords: Tailor-made oligosaccharides; Enzyme engineering; Substrate engineering; Glucosyltransferase; Fructosyltransferase

A flow injection analysis system with encapsulated high-density Saccharomyces cerevisiae cells for rapid determination of biochemical oxygen demand by Kyo Seong Seo; Kwang Ho Choo; Ho Nam Chang; Joong Kon Park (pp. 217-223).

The biochemical oxygen demand (BOD) determination was studied using a novel flow injection analysis (FIA) system with encapsulated Saccharomyces cerevisiae cells and an oxygen electrode and was compared with conventional 5-day BOD tests. S. cerevisiae cells were packed in a calcium alginate capsule at a dry cell weight of 250 g/l of capsule core. The level of dissolved oxygen (DO) was reduced due to the enhanced respiratory activity of the microbial cells when the injected nutrient passed through the bioreactor. The decrease in DO (ΔDO) was intensified with the amount of microbial cells packed in the bioreactor. However, the specific ΔDO decreased as the amount of cells loaded in the bioreactor increased. The ΔDO value was dependent on the pH and temperature of the mobile phase and reached its maximum value at 35°C and pH 7–8. Also, ΔDO became larger at longer response times as the flow rate of the mobile phase decreased. The measurement of ΔDO was repeated more than six times consecutively using a 20-ppm standard glucose and glutamic acid solution, which confirmed the reproducibility with a standard deviation of 0.95%. A strong linear correlation between ΔDO and BOD was also observed. The 5-day BOD values of actual water and wastewater samples were in accordance with the BOD values obtained by this FIA method using encapsulated S. cerevisiae cells. Unlike the cell-immobilized bead system, there was no contamination of the bioreactor resulting from any leak of yeast cells from the sensor capsules during BOD measurements.

Keywords: BOD sensor; FIA; Biochemical oxygen demand; Encapsulation; High cell density; Saccharomyces cerevisiae

Isolation of a novel high erythritol-producing Pseudozyma tsukubaensis and scale-up of erythritol fermentation to industrial level by Marimuthu Jeya; Kyoung-Mi Lee; Manish Kumar Tiwari; Jung-Soo Kim; Paramasamy Gunasekaran; Sang-Yong Kim; In-Won Kim; Jung-Kul Lee (pp. 225-231).

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.

Keywords: Dissolved oxygen; Erythritol; Fed-batch culture; Pseudozyma tsukubaensis ; Scale-up production

Paclitaxel production using co-culture of Taxus suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor by Yong-Cheng Li; Wen-Yi Tao; Long Cheng (pp. 233-239).

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.

Keywords: Biochemical engineering; Co-bioreactor; Co-culture; Endophytic fungi; Paclitaxel; Plant cell suspension culture

Litre-scale microbial fuel cells operated in a complete loop by Peter Clauwaert; Schalla Mulenga; Peter Aelterman; Willy Verstraete (pp. 241-247).

Using the anode effluent to compensate the alkalinization in a bio-cathode has recently been proposed as a way to operate a microbial fuel cell (MFC) in a continuous and pH neutral way. In this research, we successfully demonstrated that the operation of a MFC without any pH adjustments is possible by completing the liquid loop over cathode and anode. During the complete loop operation, a stable current production of 23.2 ± 2.5 A m⁻³ MFC was obtained, even in the presence of 3.2–5.2 mg O₂ L⁻¹ in the anode. The use of current collectors and subdivided electrical circuitries for relative large 2.5-L-scale MFCs resulted in ohmic cell resistances in the order of 1.4–1.7 mΩ m³ MFC, which were comparable to values of ten times smaller MFCs. Nevertheless, the bio-cathode activity still needs to be improved significantly with a factor 10–50 in order achieve desirable current densities of 1,000 A m⁻³ MFC.

Keywords: Energy recovery; Bio-fuel cell; Bio-catalysed cathode; Bio-electrochemical system; Wastewater

A novel beta-propeller phytase from Pedobacter nyackensis MJ11 CGMCC 2503 with potential as an aquatic feed additive by Huoqing Huang; Na Shao; Yaru Wang; Huiying Luo; Peilong Yang; Zhigang Zhou; Zhichun Zhan; Bin Yao (pp. 249-259).

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.

Keywords: Aquaculture; Phytate; Beta-propeller phytase; Pedobacter nyackensis ; Bacillus phytases

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B by Sanni P. Voutilainen; Harry Boer; Marika Alapuranen; Janne Jänis; Jari Vehmaanperä; Anu Koivula (pp. 261-272).

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT _m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT _m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT _m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.

Keywords: Site-directed mutagenesis; Cellulase; Saccharomyces cerevisiae expression; Protein engineering; Cellulose

Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5 by Jung-Soo Kim; Manish Kumar Tiwari; Hee-Jung Moon; Marimuthu Jeya; Thangadurai Ramu; Deok-Kun Oh; In-Won Kim; Jung-Kul Lee (pp. 273-283).

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.

Keywords: Cloning; Dinitrile; Aliphatic nitrilase; Pseudomonas fluorescens ; Substrate specificity

Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola by Ah-Reum Joo; Marimuthu Jeya; Kyoung-Mi Lee; Won-Il Sim; Jung-Soo Kim; In-Won Kim; Yeong-Suk Kim; Deok-Kun Oh; Paramasamy Gunasekaran; Jung-Kul Lee (pp. 285-294).

An efficient ß-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50°C. The enzyme showed high substrate specificity and high catalytic efficiency (k _cat = 2,990 s⁻¹, K _m = 1.76 mM, k _cat/K _m = 1,700 mM⁻¹ s⁻¹) for p-nitrophenyl-β-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.

Keywords: Catalytic efficiency; Enzyme purification; Fomitopsis pinicola ; β-1,4-glucosidase; Isolation; Glycoside hydrolase family 3

Substrate specificity of a glucose-6-phosphate isomerase from Pyrococcus furiosus for monosaccharides by Ran-Young Yoon; Soo-Jin Yeom; Chang-Su Park; Deok-Kun Oh (pp. 295-303).

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.

Keywords: Glucose-6-phosphate isomerase; Isomerization; Pyrococcus furiosus ; Rare sugars; Substrate specificity; l-Talose

fabC of Streptomyces lydicus involvement in the biosynthesis of streptolydigin by Guang-Rong Zhao; Ting Luo; Yong-Jin Zhou; Xin Jiang; Bin Qiao; Feng-Ming Yu; Ying-Jin Yuan (pp. 305-313).

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.

Keywords: Streptolydigin; Fatty acid biosynthetic gene cluster; Metabolism; Acyl carrier protein; Streptomyces lydicus ; Polyketide

Molecular mechanism of SugR-mediated sugar-dependent expression of the ldhA gene encoding l-lactate dehydrogenase in Corynebacterium glutamicum by Koichi Toyoda; Haruhiko Teramoto; Masayuki Inui; Hideaki Yukawa (pp. 315-327).

This paper reports on the transcriptional regulation mechanism of the Corynebacterium glutamicum ldhA gene encoding l-lactate dehydrogenase responsible for production of l-lactate. DNA affinity purification allowed us to identify SugR, a global repressor of genes involved in sugar uptake and glycolysis, as a protein binding to the ldhA promoter region. Whereas ldhA gene expression and ldhA promoter activity were completely repressed during growth of wild-type cells in the absence of sugar, no such repression was observed in sugR mutant cells, indicating that SugR represses ldhA transcription. Electrophoretic mobility shift assays and DNase I footprint analyses revealed that two direct repeats, centered at position-17 with respect to the transcriptional start point, are required for SugR binding to the ldhA promoter and that fructose-1-phosphate (F-1-P) is the strongest negative effector of repressor activity of SugR. Furthermore, the ldhA promoter activity during growth on either fructose or sucrose, under which F-1-P is generated, is higher than on glucose, supporting the results of the in vitro binding assays. Thus, C. glutamicum ldhA is repressed by SugR in the absence of sugar and the expression level is dependent on the extent of derepression, which varies in response to sugars provided.

Keywords: Corynebacterium glutamicum ; Lactate dehydrogenase; Transcriptional regulation

Chaperone-aided expression of LipA and LplA followed by the increase in α-lipoic acid production by Hee-Jung Moon; Marimuthu Jeya; In-Sik Yu; Jung-Hwan Ji; Deok-Kun Oh; Jung-Kul Lee (pp. 329-337).

α-Lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32–111%, respectively.

Keywords: Chaperone; Expression; Lipoate synthase; Lipoic acid; Pseudomonas fluorescens

Assessment of the core cryparin promoter from Cryphonectria parasitica for heterologous expression in filamentous fungi by Bo-Ra Kwon; Myoung-Ju Kim; Jin-A Park; Hea-Jong Chung; Jung-Mi Kim; Seung-Moon Park; Sung-Hwan Yun; Moon-Sik Yang; Dae-Hyuk Kim (pp. 339-348).

Cryparin is an abundant cell-wall-associated hydrophobin of Cryphonectria parasitica. Although cryparin is encoded as a single copy gene, it is the most abundant protein produced by this fungus when grown in liquid culture. Studies to characterize the transcriptional regulatory element(s) found that the fragment between nt −188 and the start codon was the minimal but sufficient promoter element for expression of the cryparin gene. To explore the possibility of using this small fragment as a minimal core promoter, three different chimeric reporter genes were constructed using a bacterial hygromycin B resistance gene (hph), an inducible laccase of C. parasitica, and glucose oxidase of Aspergillus niger to examine the promoter properties of the fragment. When using C. parasitica as an expression host, the 188-bp fragment functioned as a promoter for the expression of all three reporter genes. Moreover, a high level of expression was further confirmed by measuring the relative amount of transcripts of hph and an internal control gene, glyceraldehyde-3-phosphate dehydrogenase, using quantitative real-time polymerase chain reaction. The 188-bp fragment also showed promoter activity in other fungi, Gibberella zeae, A. niger, and Aspergillus nidulans, which suggests that this fragment can be applied as a strong core promoter for heterologous gene expression in various fungi.

Keywords: Cryphonectria parasitica ; Hypovirulence; Promoter analysis; Cryparin

In vitro model to study the modulation of the mucin-adhered bacterial community by Pieter Van den Abbeele; Charlotte Grootaert; Sam Possemiers; Willy Verstraete; Kim Verbeken; Tom Van de Wiele (pp. 349-359).

There is an upsurge of interest in gastro-intestinal microbiology to improve the balance between positive and negative commensals. Mucosal bacteria make closer contact with the host than luminal ones and can therefore have a stronger health impact. An in vitro adhesion assay was developed to study the mucin colonization of bacteria from the mixed microbial communities of the Simulator of the Human Intestinal Microbial Ecosystem. Adhesion capacity differed substantially between bacteria and decreased from lactobacilli over fecal coliforms, bifidobacteria, and clostridia to total anaerobes. Lactobacillus rhamnosus GG adhered most selectively. Further, intestinal water lowered adhesion compared to phosphate-buffered saline. By processing the data to an Adhesion-Related Prebiotic Index, it was found that intestinal water stimulated adherence of positive commensals. Arabinoxylans decreased the adhesion capacity matrix independently, whereas inulin had less or no influence. Measurements of bacterial surface tension, surface hydrophobicity, liquid surface tension, and viscosity showed that bacterial adhesion to mucin agar is a matter of both non-specific and specific interactions. The developed methodology can be useful for the characterization of the relevant but barely investigated mucin-associated bacterial community in health and disease (e.g., IBD) as well as for its modulation with functional foods like prebiotics.

Keywords: SHIME; Mucus; Microflora; Gut; Colon

Identification of α-d-glucose-1-phosphate cytidylyltransferase involved in Ebosin biosynthesis of Streptomyces sp. 139 by Xiao-Qiang Qi; Qing-Li Sun; Li-Ping Bai; Jun-Jie Shan; Yang Zhang; Ren Zhang; Yuan Li (pp. 361-368).

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139 has antagonist activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. Its biosynthesis gene cluster (ste) has been identified. In this study, gene ste17 was expressed in Escherichia coli BL21 and the recombinant protein was purified. With CTP and α-d-glucose-1-phosphate as substrates, the recombinant Ste17 protein was found capable of catalyzing the production of CDP-d-glucose and pyrophosphate, demonstrating its identity as an α-d-glucose-1-phosphate–cytidylyltransferase (CDP-d-glucose synthase). To investigate the function of ste17 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition of exopolysaccharide (EPS) produced by the mutant Streptomyces sp. 139 (ste17 ⁻) was found significantly altered from that of Ebosin, with glucose becoming undetectable. This gene knockout also negatively affected the antagonist activity for IL-1R of EPS. These results indicate that the CDP-d-glucose synthase encoded by ste17 gene is involved in the formation of nucleotide sugar (CDP-d-glucose) as glucose precursor in Ebosin biosynthesis.

Keywords: ste17 gene; CDP-d-glucose synthase; Gene disruption; Biosynthesis of exopolysaccharide; Streptomyces

Transformation of iron sulfide to greigite by nitrite produced by oil field bacteria by Shiping Lin; Federico Krause; Gerrit Voordouw (pp. 369-376).

Nitrate, injected into oil fields, can oxidize sulfide formed by sulfate-reducing bacteria (SRB) through the action of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB). When reservoir rock contains siderite (FeCO₃), the sulfide formed is immobilized as iron sulfide minerals, e.g. mackinawite (FeS). The aim of our study was to determine the extent to which oil field NR-SOB can oxidize or transform FeS. Because no NR-SOB capable of growth with FeS were isolated, the well-characterized oil field isolate Sulfurimonas sp. strain CVO was used. When strain CVO was presented with a mixture of chemically formed FeS and dissolved sulfide (HS⁻), it only oxidized the HS⁻. The FeS remained acid soluble and non-magnetic indicating that it was not transformed. In contrast, when the FeS was formed by adding FeCl₂ to a culture of SRB which gradually produced sulfide, precipitating FeS, and to which strain CVO and nitrate were subsequently added, transformation of the FeS to a magnetic, less acid-soluble form was observed. X-ray diffraction and energy-dispersive spectrometry indicated the transformed mineral to be greigite (Fe₃S₄). Addition of nitrite to cultures of SRB, containing microbially formed FeS, was similarly effective. Nitrite reacts chemically with HS⁻ to form polysulfide and sulfur (S⁰), which then transforms SRB-formed FeS to greigite, possibly via a sulfur addition pathway (3FeS + S⁰ → Fe₃S₄). Further chemical transformation to pyrite (FeS₂) is expected at higher temperatures (>60°C). Hence, nitrate injection into oil fields may lead to NR-SOB-mediated and chemical mineral transformations, increasing the sulfide-binding capacity of reservoir rock. Because of mineral volume decreases, these transformations may also increase reservoir injectivity.

Keywords: Sulfate-reducing bacteria; Nitrate-reducing sulfide-oxidizing bacteria; Nitrite; Sulfur; Iron sulfide; Greigite; Oil field; Souring

Bioaugmentation of UASB reactors with immobilized Sulfurospirillum barnesii for simultaneous selenate and nitrate removal by Markus Lenz; Anne Marie Enright; Vincent O’Flaherty; Adriaan C. van Aelst; Piet N. L. Lens (pp. 377-388).

Whole-cell immobilization of selenate-respiring Sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 µg Se × L⁻¹) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). Gel-immobilized S. barnesii cells were used to inoculate a mesophilic (30°C) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (COD) × L⁻¹ day⁻¹. Selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 µg Se × L⁻¹ was obtained. Scanning electron microscopy with energy dispersive X-ray (SEM–EDX) analysis revealed spherical bioprecipitates of ≤2 µm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. To validate the bioaugmentation success under microbial competition, gel cubes with immobilized S. barnesii cells were added to an Upflow Anaerobic Sludge Bed (UASB) reactor, resulting in earlier selenate (24 hydraulic retention times (HRTs)) and sulfate (44 HRTs) removal and higher nitrate/nitrite removal efficiencies compared to a non-bioaugmented control reactor. S. barnesii was efficiently immobilized inside the UASB bioreactors as the selenate-reducing activity was maintained during long-term operation (58 days), and molecular analysis showed that S. barnesii was present in both the sludge bed and the effluent. This demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. Eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation.

Keywords: Denitrification; Whole-cell immobilization; Dissimilatory selenium reduction; Bioprecipitation; Drainage water treatment

A new approach to retrieve full lengths of functional genes from soil by PCR-DGGE and metagenome walking by Sho Morimoto; Takeshi Fujii (pp. 389-396).

Metagenomes are a vast genetic resource, and various approaches have been developed to explore them. Here, we present a new approach to retrieve full lengths of functional genes from soil DNA using PCR-denaturing gradient gel electrophoresis (DGGE) followed by metagenome walking. Partial fragments of benzoate 1,2-dioxygenase alpha subunit gene (benA) were detected from a 3-chlorobenzoate (3CB)-dosed soil by PCR-DGGE, and one DGGE band induced by 3CB was used as a target fragment for metagenome walking. The walking retrieved the flanking regions of the target fragment from the soil DNA, resulting in recovery of the full length of benA and also downstream gene (benB). The same strategy retrieved another gene, tfdC, and a complete tfdC and two downstream genes were obtained from the same soil. PCR-DGGE allows screening for target genes based on their potential for degrading contaminants in the environment. This feature provides an advantage over other existing metagenomic approaches.

Keywords: PCR-DGGE; Soil; Metagenome walking; benA ; tfdC

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Applied Microbiology and Biotechnology (v.83, #2)