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Applied Microbiology and Biotechnology (v.75, #3)

Fermentation process development for the production of medium-chain-length poly-3-hyroxyalkanoates by Zhiyong Sun; Juliana A. Ramsay; Martin Guay; Bruce A. Ramsay (pp. 475-485).

This paper presents a review of the existing fermentation processes for the production of medium-chain-length poly-3-hydroxyalkanoates (MCL-PHAs). These biodegradable polymers are usually produced most efficiently from structurally related carbon sources such as alkanes and alkanoic acids. Unlike alkanoic acids, alkanes exhibit little toxicity but their low aqueous solubility limits their use in high density culture. Alkanoic acids pose little mass transfer difficulty, but their toxicity requires that their concentration be well controlled. Using presently available technology, large-scale production of MCL-PHA from octane has been reported to cost from US $5 to 10 per kilogram, with expenditures almost evenly divided between carbon source, fermentation process, and the separation process. However, MCL-PHAs, even some with functional groups in their subunits, can also be produced from cheaper unrelated carbon sources, such as glucose. Metabolic engineering and other approaches should also allow increased PHA cellular content to be achieved. These approaches, as well as a better understanding of fermentation kinetics, will likely result in increased productivity and lower production costs.

Histone deacetylases—an important class of cellular regulators with a variety of functions by Christian Hildmann; Daniel Riester; Andreas Schwienhorst (pp. 487-497).

The elucidation of mechanisms of chromatin remodeling, particular transcriptional activation, and repression by histone acetylation and deacetylation has shed light on the role of histone deacetylases (HDAC) as a new kind of therapeutic target for human cancer treatment. HDACs, in general, act as components of large corepressor complexes that prevent the transcription of several tumor suppression genes. In addition, they appear to be also involved in the deacetylation of nonhistone proteins. This paper reviews the most recent insights into the diverse biological roles of HDACs as well as the evolution of this important protein family.

Histone deacetylase inhibitors—turning epigenic mechanisms of gene regulation into tools of therapeutic intervention in malignant and other diseases by Daniel Riester; Christian Hildmann; Andreas Schwienhorst (pp. 499-514).

Histone deacetylase inhibitors reside among the most promising targeted anticancer agents that are potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells. In October 2006, the US Food and Drug Administration approved the first drug of this new class, vorinostat (1, Zolinza, Merck). Several histone deacetylase (HDAC) inhibitors more are in clinical trials. HDAC inhibitors have shown significant activity against a variety of hematological and solid tumors at doses that are well tolerated by patients, both in monotherapy as well as in combination therapy with other drugs. This paper reviews the most recent developments in HDAC inhibitor design, particularly in the context of anticancer therapy, and other possible pharmaceutical applications.

Keywords: Histone deacetylase inhibitors; Epigenetics; Cancer drug design; Hydroxamates; Benzamides; Clinical trials

A bioreactor model system specifically designed for Tetrahymena growth and cholesterol removal from milk by D. G. Noseda; H. G. Gentili; M. L. Nani; A. Nusblat; A. Tiedtke; J. Florin-Christensen; C. B. Nudel (pp. 515-520).

This work describes the configuration and operation of a bioreactor system especially designed for Tetrahymena cultivation and its use for milk improvement, particularly cholesterol elimination by the action of this cell. An advantage of the proposed method is the re-use of the growth medium; thus, the medium is used twice to provide two batches of Tetrahymena biomass without the need of further inoculation. This makes the procedure of producing the cell biomass faster and more economical. Cells are concentrated in the culture vessels by sedimentation at room temperature and then transferred to milk suspensions, where they can further grow for at least one generation with the benefit of reducing steeply cholesterol level. Milk treated according to this process is separated from the biomass by centrifugation. Under these conditions, less than 5% of the cells remain in the milk, and cholesterol elimination amounts to 75 ± 10% of that initially present. No changes in sensorial properties of the milk, such as clotting or butyric odor, were observed as a result of this treatment. In addition, the bioreactor allows the aseptic recovery of the spent growth medium, which contains diverse enzymes of interest, and the cell pellets, to exploit particular lipids like phosphonolipids, abundant poly-unsaturated fatty acids and co-enzyme Q₈.

Keywords: Tetrahymena ; Bioreactor system; Milk treatment; Cholesterol removal

Production of different types of mannosylerythritol lipids as biosurfactants by the newly isolated yeast strains belonging to the genus Pseudozyma by Masaaki Konishi; Tomotake Morita; Tokuma Fukuoka; Tomohiro Imura; Koji Kakugawa; Dai Kitamoto (pp. 521-531).

Mannosylerythritol lipids (MEL), which are abundantly secreted by yeasts, are one of the most promising biosurfactants known. To obtain various types of MEL and to attain a broad range of applications for them, screening of novel producers was undertaken. Thirteen strains of yeasts were successfully isolated as potential MEL producers; they showed high production yields of MEL of around 20 g l⁻¹ from 40 g l⁻¹ of soybean oil. Based on the taxonomical study, all the strains were classified to be the genus Pseudozyma. It is interesting to note that they were categorized into three groups according to their production patterns of MEL. The first group, which included 11 strains taxonomically closely related to high-level MEL producers such as Pseudozyma antarctica and Pseudozyma aphidis, mainly produced 4-O-[(4′,6′-di-O-acetyl-2′,3′-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-A) together with 4-O-[(6′-mono-O-acetyl-2′,3′-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-B) and 4-O-[(4′-mono-O-acetyl-2′,3′-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-C) as the minor components. The second group of one strain, which was related to Pseudozyma tsukubaensis, predominantly produced MEL-B. The third group of one strain, which was closely related to Pseudozyma hubeiensis, mainly produced MEL-C; this is the first observation of the efficient production of MEL-C from soybean oil. Moreover, the major fatty acids of the obtained MEL-C were C₆, C₁₂, and C₁₆ acids, and were considerably different from those of the other MEL hitherto reported. The biosynthetic manner for MEL is thus likely to significantly vary among the Pseudozyma strains; the newly isolated strains would enable us to attain a large-scale production of MEL and to obtain various types of MEL with different hydrophobic structures.

Keywords: Mannosylerythritol lipid; Pseudozyma ; Glycolipid; Biosurfactant

Improvement of tolerance to freeze–thaw stress of baker’s yeast by cultivation with soy peptides by Shingo Izawa; Kayo Ikeda; Nobuyuki Takahashi; Yoshiharu Inoue (pp. 533-537).

The tolerance to freeze–thaw stress of yeast cells is critical for frozen-dough technology in the baking industry. In this study, we examined the effects of soy peptides on the freeze–thaw stress tolerance of yeast cells. We found that the cells cultured with soy peptides acquired improved tolerance to freeze–thaw stress and retained high leavening ability in dough after frozen storage for 7 days. The final quality of bread regarding its volume and texture was also improved by using yeast cells cultured with soy peptides. These findings promote the utilization of soy peptides as ingredients of culture media to improve the quality of baker’s yeast.

Keywords: Freeze–thaw stress; Soy peptides; Baker’s yeast; Frozen-dough technology

Screening, purification, and identification of annexin B1 mutants with high phosphatidylserine-binding activity and reduced immunogenicity by Fang Wang; Quan-Yong Luo; Ying He; Shu-Han Sun (pp. 539-548).

Annexin B1 has many potential biomedical applications based on its high affinity for negatively charged phospholipid (phosphatidylserine, PS) in the presence of physiological concentrations of calcium. Low immunogenicity is prerequisite for the in vivo application of a nonhuman protein as a novel-imaging agent. In the present study, three sequence-deleted mutants with different numbers of functional domains were designed and expressed according to the predicted three-dimensional structure of annexin B1. The mutants of annexin B1, as well as the wild-type annexin B1, were expressed as Glutathione-S-transferase (GST)-fusion proteins. Two mutants with their purity above 80% could be obtained after one-step primary purification procedure on basis of the PS-binding activity. The immunogenicity of the two mutants was evaluated in mice by detecting the titers of elicited antigen-specific IgG. A member of three mutants of annexin B1, M12, which involved N-terminal amino-acid sequence and double functional domain I and II of annexin B1, was finally selected to detect apoptosis that is due to its lowest immunogenicity among the candidate mutants. Flourescein isothiocyanate-labeled M12 could bind the outer membranes of apoptotic cells and discriminate apoptotic cells in the early stage from necrotic cells when used with propidium iodide. ^99mTc-labeled M12 could recognize the apoptotic hepatocytes induced by anti-Fas antibody treatment. Our data in vitro and in vivo demonstrated that M12 could be applied as a promising agent for the detection of apoptosis.

Keywords: Annexin B1; Immunogenicity; Apoptosis imaging; ^99mTc

Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties by Michiko Kato; Jun Fuchimoto; Takanori Tanino; Akihiko Kondo; Hideki Fukuda; Mitsuyoshi Ueda (pp. 549-555).

To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C. antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by α-agglutinin as the anchor protein for easy handling and stability of the enzyme. When mCALB was displayed on the yeast cell surface, it showed a preference for short chain fatty acids, an advantage for producing flavors; although when Rhizopus oryzae lipase (ROL) was displayed, the substrate specificity was for middle chain lengths. When the thermal stability of mCALB on the cell surface was compared with that of ROL on a cell surface, T _1/2, the temperature required to give a residual activity of 50% for heat treatment of 30 min, was 60°C for mCALB and 44°C for ROL indicating that mCALB displayed on cell surface has a higher thermal stability. Furthermore, the activity of the displayed mCALB against p-nitrophenyl butyrate was 25-fold higher than that of soluble CALB, as reported previously. These findings suggest that mCALB-displaying yeast is more practical for industrial use as the whole-cell biocatalyst.

Keywords: Candida antarctica lipase B; Mutation; Yeast cell surface engineering; Substrate specificity

Purification and cloning of a novel serine protease from the nematode-trapping fungus Dactylellina varietas and its potential roles in infection against nematodes by Jinkui Yang; Lianming Liang; Ying Zhang; Juan Li; Lin Zhang; Fengping Ye; Zhongwei Gan; Ke-Qin Zhang (pp. 557-565).

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5°C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method.

Keywords: Dactylellina varietas ; Protease; Purification; Gene cloning; Nematicidal activity

Cloning of srfA operon from Bacillus subtilis C9 and its expression in E. coli by Young-Ki Lee; Byung-Dae Yoon; Jung-Hoon Yoon; Seung-Goo Lee; Jae Jun Song; Jong-Guk Kim; Hee-Mock Oh; Hee-Sik Kim (pp. 567-572).

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R _f value of 0.52 from B. subtilis C9 or authentic surfactin (R _f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.

Keywords: Bacillus subtilis ; LA-PCR; srfA operon; Surfactin

Field study results on the probability and risk of a horizontal gene transfer from transgenic herbicide-resistant oilseed rape pollen to gut bacteria of bees by Kathrin I. Mohr; Christoph C. Tebbe (pp. 573-582).

Bees are specifically subjected to intimate contacts with transgenic plants due to their feeding activities on pollen. In this study, the probability and ecological risk of a gene transfer from pollen to gut bacteria of bees was investigated with larvae of Apis mellifera (honeybee), Bombus terrestris (bumblebee), and Osmia bicornis (red mason bee), all collected at a flowering transgenic oilseed rape field. The plants were genetically engineered with the pat-gene, conferring resistance against glufosinate (syn. phosphinothricin), a glutamine-synthetase inhibitor in plants and microorganisms. Ninety-six bacterial strains were isolated and characterized by 16S rRNA gene sequencing, revealing that Firmicutes represented 58% of the isolates, Actinobacteria 31%, and Proteobacteria 11%, respectively. Of all isolates, 40% were resistant to 1 mM glufosinate, and 11% even to 10 mM. Resistant phenotypes were found in all phylogenetic groups. None of the resistant phenotypes carried the recombinant pat-gene in its genome. The threshold of detecting gene transfer in this field study was relatively insensitive due to the high background of natural glufosinate resistance. However, the broad occurrence of glufosinate-resistant bacteria from different phylogenetic groups suggests that rare events of horizontal gene transfer will not add significantly to natural bacterial glufosinate resistance.

Keywords: Gut bacteria; Bees; Glufosinate; Horizontal gene transfer; Field study; pat-gene

The pydA–pydB fusion gene produces an active dioxygenase–hydrolase that degrades 3-hydroxy-4-pyridone, an intermediate of mimosine metabolism by Jonathan D. Awaya; Chad Walton; Dulal Borthakur (pp. 583-588).

The objective of this research was to construct a pydA–pydB hybrid gene that encodes a functional dioxygenase–hydrolase (PydA–PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA–PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pydB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA–PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E. coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.

Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism by Kiriko Murakami; Eriko Tao; Yuki Ito; Minetaka Sugiyama; Yoshinobu Kaneko; Satoshi Harashima; Takahiro Sumiya; Atsushi Nakamura; Masafumi Nishizawa (pp. 589-597).

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain. Further systematic deletion could lead to the formation of a eukaryotic cell with a minimum set of genes exhibiting appropriately altered regulation for enhanced metabolite production.

The development of a flagellin surface display expression system in a moderate thermophile, Bacillus halodurans Alk36 by Michael Crampton; Eldie Berger; Sharon Reid; Maureen Louw (pp. 599-607).

This study relates to the development of an alkaliphilic, thermotolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium continuously over-produces flagellin. To harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene, which codes for flagellin, was inactivated on the chromosome giving rise to the B. halodurans BhFC01 mutant. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-1 subtype C gp120 epitope were, respectively, incorporated into this site as in-frame fusions. The peptides were found to be successfully displayed on the cell surface and functional through metal binding and immunological studies, respectively.

Keywords: Flagellin; Cell surface display; Bacillus halodurans ; Fusion protein; Integration

Long serial analysis of gene expression for transcriptome profiling during the initiation of ligninolytic enzymes production in Phanerochaete chrysosporium by Masahiko Minami; Orie Kureha; Mari Mori; Hisatoshi Kamitsuji; Kazumi Suzuki; Toshikazu Irie (pp. 609-618).

To analyze the transcriptome profile during the initiation of manganese peroxidase (MnP) and lignin peroxidase (LiP) production in Phanerochaete chrysosporium, we constructed long serial analysis of gene expression (LongSAGE) libraries. A total of 13,666 tags (the number of cumulative counted tags) that included 6,945 unique tags (the number of distinct tags) were isolated from the day-3 culture, which just started the enzymes production and was 24 h after veratryl alcohol addition and oxygen-purge into the culture (day-2 culture). A total of 12,402 tags that included 6,396 unique tags were isolated from the day-2 culture, in which the activity of enzymes is not detected. The comparison of the two libraries suggested that 38 genes showed significant (p ≤ 0.01) fourfold or greater upregulation; this included the MnP gene (mnp2, mnp3) and LiP H8 gene. On the other hand, 43 genes showed significant (p ≤ 0.01) fourfold or greater downregulation. This LongSAGE analysis found many new candidate genes regulating the enzymes production.

Keywords: Basidiomycetes; Ligninolytic; SAGE; Transcriptome; Biodegradation

Synergistic antidigestion effect of Lactobacillus rhamnosus and bovine colostrums in simulated gastrointestinal tract (in vitro) by Wei Hua; Xu Yang; Xiong Yonghua; Xu Feng; Liu Gengpin (pp. 619-626).

Probiotics and bovine colostrums had been proven to be beneficial for human health. Lactobacillus rhamnosus ZDY114 and anti-Helicobacter pylori bovine colostrums were used for the preparation of microecological additives, and their synergistic antidigestion effect in the simulated gastrointestinal tract (in vitro) was investigated. Either L. rhamnosus or purified IgG from immune colostrums was very sensitive in simulated gastric environment and slightly sensitive in simulated intestinal tract. No viable counts were recovered from the solution of dissolved freeze-dried powder (7.14 log₁₀ CFU/ml) of L. rhamnosus when digested at pH 3.0 with pepsin for 30 min. Activity of purified IgGs from immune colostrums could not be detected when digested at pH 3.0 with pepsin for 30 min; 29% titer could be detected when digested at pH 8.0 with trypsin for 5 h. The IgGs in nonpurified immune colostrums presented stronger resistance against gastrointestinal digestion than purified IgGs. Moreover, the combination of L. rhamnosus ZDY114 and immune colostrums strengthened their antidigestion ability. Even under pH 3.0, 4.0 with pepsin, the titer of anti-HP IgG maintained above 123 and 83.3%, respectively. Similarly, that titer was above 93.3% when digested at pH 8.0 with trypsin for 4.5 h. In conclusion, L. rhamnosus and anti-Helicobacter pylori bovine colostrums had synergistic antidigestion effect in simulated gastrointestinal tract (in vitro).

Keywords: Lactobacillus rhamnosus ; Helicobacter pylori ; Bovine colostrums; Simulated gastrointestinal tract; Antidigestion

Chromate reduction by Burkholderia cepacia MCMB-821,isolated from the pristine habitat of alkaline crater lake by Revati Wani; K. M. Kodam; K. R. Gawai; P. K. Dhakephalkar (pp. 627-632).

The Cr(VI)-reducing bacterial strain MCMB-821 was isolated from the alkaline crater lake of Lonar and was identified as Burkholderia cepacia. MCMB-821 was resistant to 1,000-ppm Cr(VI) and reduced 98% of the 75 ppm Cr(VI) within 36 h at pH 9.0 in the presence of 2% salt and lactose as the electron donor. The chromate-reducing efficiency of MCMB-821 was comparable under both aerobic as well as anaerobic conditions. Electron paramagnetic resonance spectroscopy data suggested that MCMB-821 reduced Cr(VI) to Cr(III) via the formation of transient Cr(V) intermediate. The chromate-reducing ability of MCMB-821 was suppressed in the presence of membrane inhibitors and enhanced in the presence of 2,4-dinitrophenol, suggesting the involvement of electron transport chain in the Cr(VI) bioreduction.

Keywords: Burkholderia ; Chromate reduction; Resistance and optimization of bioreduction

Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation by Giovanni Vigliotta; Michele Di Giacomo; Elisabetta Carata; Domenica R. Massardo; Salvatore M. Tredici; Daniele Silvestro; Marianna Paolino; Paola Pontieri; Luigi Del Giudice; Dino Parente; Pietro Alifano (pp. 633-645).

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.

Keywords: Debaryomyces hansenii ; Tobacco fermentation; Nitrogen metabolism; Assimilatory NAD(P)H:nitrite reductase

Reduction and partial degradation mechanisms of naphthylaminesulfonic azo dye amaranth by Shewanella decolorationis S12 by Yiguo Hong; Jun Guo; Zhicheng Xu; Cuiyun Mo; Meiying Xu; Guoping Sun (pp. 647-654).

Reduction and biodegradation mechanisms of naphthylaminesulfonic azo dye amaranth using a newly isolated Shewanella decolorationis strain S12 were investigated. Under anaerobic conditions, amaranth was reduced by strain S12, and a stoichiometric amount of two reduction products RP-1 and RP-2 were generated. UV/visible spectrophotometric and high performance liquid chromatography (HPLC) analysis indicated that RP-1 and RP-2 were 1-aminenaphthylene -4-sulfonic acid and 1-aminenaphthylene-2-hydroxy-3, 6-disulfonic acid. The result strongly supports a mechanism of azo dye reduction by the process via the reductive cleavage of the azo bond to form corresponding aromatic amines. The result of HPLC analyses revealed that these aromatic amines were not able to be mineralized by strain S12 under anaerobic conditions. But after re-aeration of the decolorized culture, RP-2 was mineralized completely by this microorganism, but the consumption of RP-1 was not observed. Ames test showed that amaranth had mutagenic but no cytotoxic potential. The mutagenic potential was relieved after the anaerobic treatment with strain S12 as the mutagenic effect of the two reduction products from amaranth was not detected by Ames test. Thus, the ability of strain S12 to reduce and partially mineralize the naphthylaminesulfonic azo dye efficiently was demonstrated, which can potentially be used to biodegrade and detoxify wastewater containing azo dyes using an alternating anaerobic/aerobic treatment procedure.

Polycyclic aromatic hydrocarbon-degrading Mycobacterium isolates: their association with plant roots by R. Child; C. D. Miller; Y. Liang; G. Narasimham; J. Chatterton; P. Harrison; R. C. Sims; D. Britt; A. J. Anderson (pp. 655-663).

Five environmental mycobacterium isolates that degrade polycyclic aromatic hydrocarbons (PAHs) were associated with barley root surfaces after growth of the seedlings from inoculated seed. Mycobacterium cells were detected along the total root length for four of these isolates. These PAH-degrading mycobacterium strains had hydrophilic cell surfaces, whereas one strain, MCS, that was hydrophobic had reduced association along the root length with no cells being detected from the root tips. The root-tip-competent strain, KMS, was competitive for its root association in the presence of the root-colonizing pseudomonad, Pseudomonas putida KT2440. All mycobacterium strains utilized simple sugars (fructose, glucose) and the trisaccharide 6-kestose, present in barley root washes, for planktonic growth, but they differed in their potential for biofilm formation under in vitro conditions. Mineralization of pyrene by the KMS strain occurred when the components in the barley root wash were amended with labeled pyrene suggesting to us that mineralization could occur in plant rhizospheres containing such mycobacterium strains.

Keywords: Polycyclic aromatic hydrocarbons; Root colonization; Mycobacterium; Remediation

Anaerobic biodegradation of pentachlorophenol in a fixed-film reactor inoculated with polluted sediment from Santos–São Vicente Estuary, Brazil by F. T. Saia; M. H. R. Z. Damianovic; E. B. M. Cattony; G. Brucha; E. Foresti; R. F. Vazoller (pp. 665-672).

This paper discusses the results of pentachlorophenol (PCP) anaerobic biodegradation in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor operated under methanogenic and halophylic conditions. The system was inoculated with autochthonous microorganisms taken from a site in the Santos-São Vicente Estuary (state of São Paulo, Brazil) severely contaminated with PCP, phenolic compounds, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and heavy metals. The inoculum was previously enriched for methanogenesis activity by changing glucose concentrations and under halophylic condition. PCP was added to the HAIB reactor as sodium salt (NaPCP) at an initial concentration of 5 mg l⁻¹ and increased to 13, 15, and 21 mg l⁻¹. Organic matter removal efficiency ranged from 77 to 100%. PCP removal efficiency was 100%. Denaturing gradient gel electrophoresis profile showed changes in the structure of Bacteria domain, which was associated with NaPCP and glucose amendments. The diversity of Archaea remained unaltered during the different phases. Scanning electron microscope examinations showed that cells morphologically resembling Methanosarcina and Methanosaeta predominated in the biofilm. These cells were detected by fluorescence in situ hybridization with the Methanosarcinales (MSMX860) specific probe. The results are of great importance in planning the estuary’s restoration by using anaerobic technology and autochthonous microorganisms for bioremediation.

Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge by Herto Dwi Ariesyady; Tsukasa Ito; Kazumi Yoshiguchi; Satoshi Okabe (pp. 673-683).

The phylogenetic and functional diversity of syntrophic propionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR–FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52–62% of the total active POB under all the propionate concentrations tested (0.5–15 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobacter accounted for 16–31% of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration.

Keywords: Anaerobic digestion; Propionate-oxidizing bacteria; MAR–FISH; Functional diversity

Oxygen diffusion and consumption in active aerobic granules of heterogeneous structure by Z. C. Chiu; M. Y. Chen; D. J. Lee; C. H. Wang; J. Y. Lai (pp. 685-691).

The interior structure of aerobic granules is highly heterogeneous, hence, affecting the transport and reaction processes in the granules. The granule structure and the dissolved oxygen profiles were probed at the same granule in the current work for possible estimation of transport and kinetic parameters in the granule. With the tested granules fed by phenol or acetate as carbon source, most inflow oxygen was consumed by an active layer thickness of less than 125 μm on the granule surface. The confocal laser scanning microscopy scans also revealed a surface layer thickness of approximately 100 μm consisting of cells. The diffusivities of oxygen transport and the kinetic constant of oxygen consumption in the active layers only were evaluated. The theoretical models adopted in literature that ignored the contributions of the layered structure of aerobic granule could have overlooked the possible limitations on oxygen transport.

Keywords: Aerobic granule; Oxygen; Diffusivity; Structure; Microelectrode; CLSM

Quantitative analysis of a high-rate hydrogen-producing microbial community in anaerobic agitated granular sludge bed bioreactors using glucose as substrate by Chun-Hsiung Hung; Kuo-Shing Lee; Lu-Hsiu Cheng; Yu-Hsin Huang; Ping-Jei Lin; Jo-Shu Chang (pp. 693-701).

Fermentative H₂ production microbial structure in an agitated granular sludge bed bioreactor was analyzed using fluorescence in situ hybridization (FISH) and polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). This hydrogen-producing system was operated at four different hydraulic retention times (HRTs) of 4, 2, 1, and 0.5 h and with an influent glucose concentration of 20 g chemical oxygen demand/l. According to the PCR-DGGE analysis, bacterial community structures were mainly composed of Clostridium sp. (possibly Clostridium pasteurianum), Klebsiella oxytoca, and Streptococcus sp. Significant increase of Clostridium/total cell ratio (68%) was observed when the reactor was operated under higher influent flow rate. The existence of Streptococcus sp. in the reactor became more important when operated under a short HRT as indicated by the ratio of Streptococcus probe-positive cells to Clostridium probe-positive cells changing from 21% (HRT 4 h) to 38% (HRT 0.5 h). FISH images suggested that Streptococcus cells probably acted as seeds for self-flocculated granule formation. Furthermore, combining the inspections with hydrogen production under different HRTs and their corresponding FISH analysis indicated that K. oxytoca did not directly contribute to H₂ production but possibly played a role in consuming O₂ to create an anaerobic environment for the hydrogen-producing Clostridium.

Keywords: Fermentative hydrogen production; Granular sludge; Bioreactor design; Bacterial community structure; Fluorescence in situ hybridization

Creation of DNA overhangs by using modified DNA overhang cloning method by Wei Li; Feng Gao (pp. 703-709).

DNA overhang cloning (DOC) method was invented by Kevin A. Jarrell and colleagues to create DNA overhangs at the ends of polymerase chain reaction (PCR) fragments. To increase the ligation products of DOC and make it possible to join the ligation products together to yield a larger DNA fragment without amplification by using the secondary PCR, we modified the original method by lengthening RNA–DNA hybrids to 8 bp, using RNase H to digest RNA chains, and so on. We found that the ligation of PCR fragments with DNA overhangs could be improved significantly by using the modified DOC. The ligation products of the modified DOC were rich enough to be separated by using agarose gel and joined together to yield a larger DNA fragment without amplification by using the secondary PCR. The modified DOC is simpler and much more effective than the original one. The creation of DNA overhangs by using complementary DNA oligonucleotides, T4 ligase, and RNase H is simpler and cheaper than that by using reverse transcriptase and RNase H.

Keywords: DNA overhang; Seamless gene engineering; Gene synthesis; RNA–DNA primer; PCR; DNA ligation

Synergistic antidigestion effect of Lactobacillus rhamnosus and bovine colostrums in simulated gastrointestinal tract (in vitro) by Hua Wei; Yang Xu; Yonghua Xiong; Feng Xu; Gengpin Liu (pp. 711-711).

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Applied Microbiology and Biotechnology (v.75, #3)

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Applied Microbiology and Biotechnology (v.75, #3)