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Applied Microbiology and Biotechnology (v.79, #3)

Perspectives for biotechnological production of biodiesel and impacts by Wei Du; Wei Li; Ting Sun; Xin Chen; Dehua Liu (pp. 331-337).

In recent years, biological ways for biodiesel production have drawn an increasing attention and compared to chemical approaches, lipase-mediated alcoholysis for biodiesel production has many advantages. Currently, there are extensive reports about enzyme-mediated alcoholysis for biodiesel production, and based on the application forms of biocatalyst, the related research can be classified into immobilized lipase, whole cell catalyst, and liquid lipase-mediated alcoholysis for biodiesel production, respectively. This mini-review is focusing on the study of the aforementioned three forms of biocatalyst for biodiesel production, as well as its impacts and prospects.

Keywords: Biodiesel; Lipase; Whole cell catalyst; Liquid lipase; Immobilized lipase

The yeast Kluyveromyces marxianus and its biotechnological potential by Gustavo Graciano Fonseca; Elmar Heinzle; Christoph Wittmann; Andreas K. Gombert (pp. 339-354).

Strains belonging to the yeast species Kluyveromyces marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. As a consequence, several different biotechnological applications have been investigated with this yeast: production of enzymes (β-galactosidase, β-glucosidase, inulinase, and polygalacturonases, among others), of single-cell protein, of aroma compounds, and of ethanol (including high-temperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production. Compared to its congener and model organism, Kluyveromyces lactis, the accumulated knowledge on K. marxianus is much smaller and spread over a number of different strains. Although there is no publicly available genome sequence for this species, 20% of the CBS 712 strain genome was randomly sequenced (Llorente et al. in FEBS Lett 487:71–75, 2000). In spite of these facts, K. marxianus can envisage a great biotechnological future because of some of its qualities, such as a broad substrate spectrum, thermotolerance, high growth rates, and less tendency to ferment when exposed to sugar excess, when compared to K. lactis. To increase our knowledge on the biology of this species and to enable the potential applications to be converted into industrial practice, a more systematic approach, including the careful choice of (a) reference strain(s) by the scientific community, would certainly be of great value.

Keywords: Kluyveromyces marxianus ; Yeast biotechnology; Yeast physiology; Yeast taxonomy

A chemoenzymatic approach to the synthesis of enantiomerically pure (S)-3-hydroxy-γ-butyrolactone by Sang-Hyun Lee; Oh-Jin Park; Hong-Sun Uh (pp. 355-362).

Optically pure (S)-3-hydroxy-γ-butyrolactone, an important chiral building block in the pharmaceutical industry, was synthesized from l-malic acid by combining a selective hydrogenation and a lipase-catalyzed hydrolysis. Lipase from Candida rugosa was found to be the most efficient enzyme for the hydrolysis of (S)-β-benzoyloxy-γ-butyrolactone. The use of organic solvent-aqueous two-phase system was employed to extract benzoic acid generated from enzymatic hydrolysis of the substrate. Tert-butyl methyl ether as an organic solvent was effective to extract the reaction product, benzoic acid, and stably maintained the enzyme activity of Lipase OF immobilized on polymeric supports Amberlite® XAD-7. The immobilization made the recovery of the product simpler and prevented the formation of the emulsion. The pH adjustment was unnecessary with the immobilized Lipase OF. The scale-up of the enzymatic hydrolysis of S-BBL at 1,850-kg scale was carried out without problems to give 728.5kg of S-HGB at 80% isolated yield. The scale-up results are similar to those of bench scale reactions. Racemic (R,S)-β-benzoyloxy-γ-butyrolactone was prepared from d-, l-malic acid and was found to be hydrolyzed nonselectively by the enzyme.

Keywords: Lipases; Hydrolysis; (S)-3-hydroxy-γ-butyrolactone; Protection and deprotection; Two-phase; Candida rugosa

Construction of two lux-tagged Hg²⁺-specific biosensors and their luminescence performance by Ya-Juan Fu; Wen-Li Chen; Qiao-Yun Huang (pp. 363-370).

Two Hg²⁺-specific biosensors were constructed using bacterial luciferase as reporter gene and plasmid-free Pseudomonas putida X4 and Enterobacter aerogenes NTG-01 as host strains. The performance of X4 biosensor was compared with that of NTG-01 biosensor in the same assay conditions. The maximum bioluminescence for X4 (pmerRluxCDABE-Kan) biosensor was found during the midexponential phase and that for NTG-01 (pmerRluxCDABE-Kan) was at the late exponential phase. The shortest induction time of two biosensors was 30 min. The maximum light signal output for NTG-01 and X4 sensors was observed at the incubation time of 5 and 4 h, respectively. The lowest detectable concentration of mercury by the two biosensors were both of 100 pM at 28°C, pH 7 and an initial cell number of 10⁶ CFU ml⁻¹. Cd²⁺, Zn²⁺, Co²⁺, Cu²⁺, and Pb^{2 +} ions at nanomolar level did not interfere with the measurement by the biosensors. These results show that the sensitivity of the two biosensors is sufficient for the detection of Hg²⁺ under most contaminated environments.

Keywords: LuxCDABE ; Biosensor; Mercury; Luminescence

Effect of mixed organic substrate on α-tocopherol production by Euglena gracilis in photoheterotrophic culture by Tomoya Fujita; Hideki Aoyagi; James C. Ogbonna; Hideo Tanaka (pp. 371-378).

Effects of organic carbon sources on cell growth and α-tocopherol productivity in wild and chloroplast-deficient W₁₄ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, α-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W₁₄ZUL strain. For effective α-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, α-tocopherol productivity per culture broth was 3.89 × 10⁻² mg l⁻¹ h⁻¹, which was higher than the value obtained without addition of organic carbon source (0.92 × 10⁻² mg l⁻¹ h⁻¹). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the α-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 × 10⁻² mg l⁻¹ h⁻¹.

Keywords: Euglena gracilis ; Tocopherol; Photoheterotrophic culture; Microalgae; Chloroplast; Mitochondria

Application of biocathode in microbial fuel cells: cell performance and microbial community by Guo-Wei Chen; Soo-Jung Choi; Tae-Ho Lee; Gil-Young Lee; Jae-Hwan Cha; Chang-Won Kim (pp. 379-388).

Instead of the utilization of artificial redox mediators or other catalysts, a biocathode has been applied in a two-chamber microbial fuel cell in this study, and the cell performance and microbial community were analyzed. After a 2-month startup, the microorganisms of each compartment in microbial fuel cell were well developed, and the output of microbial fuel cell increased and became stable gradually, in terms of electricity generation. At 20 ml/min flow rate of the cathodic influent, the maximum power density reached 19.53 W/m³, while the corresponding current and cell voltage were 15.36 mA and 223 mV at an external resistor of 14.9 Ω, respectively. With the development of microorganisms in both compartments, the internal resistance decreased from initial 40.2 to 14.0 Ω, too. Microbial community analysis demonstrated that five major groups of the clones were categorized among those 26 clone types derived from the cathode microorganisms. Betaproteobacteria was the most abundant division with 50.0% (37 of 74) of the sequenced clones in the cathode compartment, followed by 21.6% (16 of 74) Bacteroidetes, 9.5% (7 of 74) Alphaproteobacteria, 8.1% (6 of 74) Chlorobi, 4.1% (3 of 74) Deltaproteobacteria, 4.1% (3 of 74) Actinobacteria, and 2.6% (2 of 74) Gammaproteobacteria.

Keywords: Microbial fuel cells; Biocathode; Cell performance; Microbial community

High-yield growth and magnetosome formation by Magnetospirillum gryphiswaldense MSR-1 in an oxygen-controlled fermentor supplied solely with air by Jian-Bo Sun; Feng Zhao; Tao Tang; Wei Jiang; Jie-sheng Tian; Ying Li; Ji-Lun Li (pp. 389-397).

Magnetotactic bacteria are difficult to grow under defined conditions in culture, which has presented a major obstacle to commercial application of magnetosomes. We studied the relationships among the cell growth, magnetosome formation, dissolved oxygen concentration (DO), and the ability to supply oxygen to the cells. Mass culture of Magnetospirillum gryphiswaldense MSR-1 for the production of magnetosomes was established in a 42-L fermentor under the following conditions: (1) sterile air was the sole gas supplied in the fermentor, and DO could be regulated at any level below 10% saturation by cascading the stir rate to DO, (2) to resolve the paradoxical situation that the cell growth requires higher DO whereas magnetosome formation requires low DO below the detectable range of regular oxygen electrode, DO was controlled to optimal level using the change of cell growth rate, rather than reading from the highly sensitive oxygen electrode, as the signal for determining appropriate DO, and (3) timing and rate of supplying the substrates were determined by measuring cell density and Na-lactate concentration. Under these conditions, cell density (OD₅₆₅) of strain MSR-1 reached 7.24 after 60-h culture in a 42-L fermentor, and cell yield (dry weight) was 2.17 g/L, the highest yield so far being reported. The yield of magnetosomes (dry weight) was 41.7 mg/L and 16.7 mg/L/day, which were 2.8 and 2.7 times higher than the previously reported yields.

Keywords: Magnetospirillum gryphiswaldense ; Magnetosome; Oxygen-controlled; Fermentor; Submerged culture

Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37 by Ritu Sareen; Prashant Mishra (pp. 399-405).

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with α-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50°C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-l-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.

Keywords: Purification; Organic solvent tolerance; Transesterification; Protease; Bacillus licheniformis

Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum by Didem Sutay Kocabas; Ufuk Bakir; Simon E. V. Phillips; Michael J. McPherson; Zumrut B. Ogel (pp. 407-415).

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 ± 0.2 and 10.1 ± 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and l-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.

Keywords: Scytalidium thermophilum ; Humicola insolens ; Catalase; Phenol oxidase; Catechol oxidase; Bifunctional enzyme

Identification of functionally important amino acids in a novel indigo-producing oxygenase from Rhodococcus sp. strain T104 by Na Ra Kwon; Jong-Chan Chae; Ki Young Choi; Miyoun Yoo; Gerben J. Zylstra; Young Min Kim; Beom Sik Kang; Eungbin Kim (pp. 417-422).

A novel indigo-producing oxygenase gene, designated ipoA (1,197 bp) was characterized from Rhodococcus sp. strain T104. Three indigo-negative mutations (A58V, P59L, and G251D) were obtained through random mutagenesis using an E. coli mutator strain. Subsequent saturation mutagenesis resulted in the identification of nine and three amino acid substitutions that restore activity in the A58V and P59L mutants, respectively. Activity was not restored in the G251D mutation by any other amino acids. Interestingly, activity in the A58V mutant, where a methyl group is only replaced by an isopropyl side chain, is restored by a variety of amino acids, including polar ones. A molecular modeling study suggests that the residues at positions 58, 59, and 251 of the T104 IpoA enzyme are far from the active site, indicating that the mutations must alter the overall structure of the enzyme.

Keywords: Rhodococcus; Oxygenase; Indole; Indigo

Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody by Meng Cao; Peng Cao; Huaijiang Yan; Fang Ren; Wuguang Lu; Yunlong Hu; Shuangquan Zhang (pp. 423-431).

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.

Keywords: Anti-BAFF antibody; Enhanced green fluorescent protein (EGFP); Bacterial expression; Autoimmune diseases

Enzymatic properties of cellobiose 2-epimerase from Ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme by Shigeaki Ito; Hidenori Taguchi; Shigeki Hamada; Shinpei Kawauchi; Hiroyuki Ito; Takeshi Senoura; Jun Watanabe; Megumi Nishimukai; Susumu Ito; Hirokazu Matsui (pp. 433-441).

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30°C and pH 7.5, and the enzyme activity was inhibited by Al³⁺, Fe³⁺, Co²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Ag⁺, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-β-d-galactopyranosyl-d-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The K _m and k _cat/K _m values toward lactose were 33 mM and 1.6 s⁻¹ mM⁻¹, and those toward cellobiose were 13.8 mM and 4.6 s⁻¹ mM⁻¹, respectively. N-Acetyl-d-glucosamine, uridine 5′-diphosphate-glucose, d-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.

Keywords: Cellobiose 2-epimerase; Ruminococcus albus ; Epilactose; Prebiotics; Bifidobacteria; Tight junction

Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance by Mina Okochi; Kei Kanie; Masaki Kurimoto; Masafumi Yohda; Hiroyuki Honda (pp. 443-449).

Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this work, we characterized the organic solvent tolerance of Escherichia coli cells that overexpress prefoldin and group II chaperonin from a hyperthermophilic archeaum, Pyrococcus horikoshii OT3. The colony-forming efficiency of E. coli cells overexpressing prefoldin increased by 1,000-fold and decreased the accumulation of intracellular organic solvent. The effect was impaired by deletions of the region responsible for the chaperone function of prefoldin. Therefore, we concluded that prefoldin endows E. coli cells by preventing accumulation of intracellular organic solvent through its molecular chaperone activity.

Keywords: Organic solvent tolerance; Prefoldin; Chaperone activity

The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production by C. Ireland; N. Peekhaus; P. Lu; R. Sangari; A. Zhang; P. Masurekar; Z. An (pp. 451-459).

Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp⁻/hy^r) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.

Keywords: Trytophan synthetase; Nodulisporium sp; Nodulisporic acid A

Analysis of expressed sequence tags (ESTs) from Lentinula edodes by Takumi Suizu; Guo-Lei Zhou; Yasuo Oowatari; Makoto Kawamukai (pp. 461-470).

The 1,031 expressed sequence tags (ESTs) from the basidiomycete Lentinula edodes were generated as a pilot experiment to see distribution of genes expressed in L. edodes. Among them, genes for hydrophobin, which are specifically found in filamentous fungi, were the most frequently obtained ESTs (33 times), suggesting that they are highly expressed in L. edodes. In addition to known hydrophobin 1 and 2 types, our analysis revealed the existence of novel types of hydrophobin, which we named hydrophobin 3, 4, and 5. The second and the third most highly obtained ESTs were phosphatidylserine decarboxylase and formate dehydrogenase, which were obtained eight and seven times, respectively. It should be noted that two important genes (argonaute and RNA-dependent RNA polymerase) involved in the RNAi pathway were found, suggesting a future application for gene knock-down by RNA interference. The 53 ESTs were identical with the sequences already reported in L. edodes. The 433 ESTs were found to show significant sequence similarity (E value <1 × 10⁻⁵) with the proteins reported (or predicted) in other species. In total, 387,952bp were sequenced and registered in DDBJ/GenBank (accession number BJ998097–BJ999127).

Keywords: Lentinula edodes ; cDNA; EST

Corynebacterium glutamicum tailored for high-yield L-valine production by Bastian Blombach; Mark E. Schreiner; Tobias Bartek; Marco Oldiges; Bernhard J. Eikmanns (pp. 471-479).

We recently engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of L-valine from glucose by inactivation of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. Based on the first generation of pyruvate-dehydrogenase-complex-deficient C. glutamicum strains, a second generation of high-yield L-valine producers was constructed by successive deletion of the genes encoding pyruvate:quinone oxidoreductase, phosphoglucose isomerase, and pyruvate carboxylase and overexpression of ilvBNCE. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 410 mM (48 g/l) L-valine, showed a maximum yield of 0.75 to 0.86 mol/mol (0.49 to 0.56 g/g) of glucose in the production phase and, in contrast to the first generation strains, excreted neither pyruvate nor any other by-product tested.

Keywords: Corynebacterium glutamicum ; L-valine production; Pyruvate dehydrogenase complex; Pyruvate:quinone oxidoreductase; Phosphoglucose isomerase; Pyruvate carboxylase

Lactic fermentation of cellobiose by a yeast strain displaying β-glucosidase on the cell surface by Kenro Tokuhiro; Nobuhiro Ishida; Akihiko Kondo; Haruo Takahashi (pp. 481-488).

The Aspergillus aculeatus β-glucosidase 1 (bgl1) gene was expressed in a lactic-acid-producing Saccharomyces cerevisiae strain to enable lactic fermentation with cellobiose. The recombinant β-glucosidase enzyme was expressed on the yeast cell surface by fusing the mature protein to the C-terminal half region of the α-agglutinin. The β-glucosidase expression plasmids were integrated into the genome. Three strong promoters of S. cerevisiae, the TDH3, PGK1, and PDC1 promoters, were used for β-glucosidase expression. The specific β-glucosidase activity varied with the promoter used and the copy number of the bgl1 gene. The highest activity was obtained with strain PB2 that possessed two copies of the bgl1 gene driven by the PDC1 promoter. PB2 could grow on cellobiose and glucose minimal medium at the same rate. Fermentation experiments were conducted in non-selective-rich media containing 95 g l⁻¹ cellobiose or 100 g l⁻¹ glucose as a carbon source under microaerobic conditions. The maximum rate of l-lactate production by PB2 on cellobiose (2.8 g l⁻¹ h⁻¹) was similar to that on glucose (3.0 g l⁻¹ h⁻¹). This indicates that efficient fermentation of cellobiose to l-lactate can be accomplished using a yeast strain expressing β-glucosidase from a mitotically stable genomic integration plasmid.

Keywords: Yeast; Beta-glucosidase; l-Lactate; Cellobiose; Cellulase

Denitration of 2,4,6-trinitrotoluene by Pseudomonas aeruginosa ESA-5 in the presence of ferrihydrite by Laurent Eyers; Ben Stenuit; Spiros N. Agathos (pp. 489-497).

Denitration of 2,4,6-trinitrotoluene (TNT) was evaluated in oxygen-depleted enrichment cultures. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum and contained TNT as sole nitrogen source. Incubations were carried out in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite, and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. In addition to reduced derivatives of TNT, several unidentified metabolites were detected. Concomitant to a decrease of TNT concentration, a release of nitrite was observed. The concentration of nitrite peaked and then it slowly decreased. In the absence of TNT, the drop in the concentration of nitrite in oxygen-depleted cultures was lower when ferrihydrite was provided, suggesting that ferrihydrite inhibited the utilization of nitrite by P. aeruginosa ESA-5.

Keywords: Biodegradation; Denitration; Trinitrotoluene; Pseudomonas; Ferrihydrite; Nitrite

In situ bacterial colonization of compacted bentonite under deep geological high-level radioactive waste repository conditions by E. Chi Fru; R. Athar (pp. 499-510).

Subsurface microorganisms are expected to invade, colonize, and influence the safety performance of deep geological spent nuclear fuel (SNF) repositories. An understanding of the interactions of subsurface dwelling microbial communities with the storage is thus essential. For this to be achieved, experiments must be conducted under in situ conditions. We investigated the presence of groundwater microorganisms in repository bentonite saturated with groundwater recovered from tests conducted at the Äspö underground Hard Rock Laboratory in Sweden. A 16S ribosomal RNA and dissimilatory bisulfite reductase gene distribution between the bentonite and groundwater samples suggested that the sulfate-reducing bacteria widespread in the aquifers were not common in the clay. Aerophilic bacteria could be cultured from samples run at ≤55°C but not at ≥67°C. Generally, the largely gram-negative groundwater microorganisms were poorly represented in the bentonite while the gram-positive bacteria commonly found in the clay predominated. Thus, bentonite compacted to a density of approximately 2 g cm⁻³ together with elevated temperatures might discourage the mass introduction of the predominantly mesophilic granitic aquifer bacteria into future SNF repositories in the long run.

Keywords: Clay buffer; Spent nuclear fuel; Subsurface; Äspö; Granitic aquifers; Groundwater

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA by Cindy H. Wu; Yu-Chen Hwang; Wonkyu Lee; Ashok Mulchandani; Thomas K. Wood; Marylynn V. Yates; Wilfred Chen (pp. 511-518).

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.

Keywords: FISH; mRNA; Rhizosphere bacteria; In situ gene expression

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Applied Microbiology and Biotechnology (v.79, #3)

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Applied Microbiology and Biotechnology (v.79, #3)