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Applied Microbiology and Biotechnology (v.77, #1)

Recent advances using green and red fluorescent protein variants by Annette Müller-Taubenberger; Kurt I. Anderson (pp. 1-12).

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent “two-color” advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.

Keywords: BiFC; Fluorescent protein; FRET; GFP; Microscopy; RFP; Photoactivation; Two-photon

Properties, production, and applications of camelid single-domain antibody fragments by M. M. Harmsen; H. J. De Haard (pp. 13-22).

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

Keywords: Single-domain; Microbial production; Yeast; Glycosylation

Biofuels from microbes by Dominik Antoni; Vladimir V. Zverlov; Wolfgang H. Schwarz (pp. 23-35).

Today, biomass covers about 10% of the world’s primary energy demand. Against a backdrop of rising crude oil prices, depletion of resources, political instability in producing countries and environmental challenges, besides efficiency and intelligent use, only biomass has the potential to replace the supply of an energy hungry civilisation. Plant biomass is an abundant and renewable source of energy-rich carbohydrates which can be efficiently converted by microbes into biofuels, of which, only bioethanol is produced on an industrial scale today. Biomethane is produced on a large scale, but is not yet utilised for transportation. Biobutanol is on the agenda of several companies and may be used in the near future as a supplement for gasoline, diesel and kerosene, as well as contributing to the partially biological production of butyl-t-butylether, BTBE as does bioethanol today with ETBE. Biohydrogen, biomethanol and microbially made biodiesel still require further development. This paper reviews microbially made biofuels which have potential to replace our present day fuels, either alone, by blending, or by chemical conversion. It also summarises the history of biofuels and provides insight into the actual production in various countries, reviewing their policies and adaptivity to the energy challenges of foreseeable future.

Keywords: Biofuel; Sustainability; Fermentation; Biomass; Substrate; Energy

Effect on epidermal cell of soybean protein-degraded products and structural determination of the root hair promoting peptide by Yoshiki Matsumiya; Sayoko Sumiyoshi; Takuma Matsukura; Motoki Kubo (pp. 37-43).

Peptide(s) produced from degraded soybean protein by an alkaline protease from Bacillus circulans HA12 (degraded soybean-meal products; DSP) increased the number of both the root hair cells (trichoblasts) and hairless cells (atrichoblasts) of Brassica rapa by about 4.4 times and 1.9 times, respectively. To identify the root hair-promoting peptide(s) in DSP, the origin protein of the root hair-promoting peptide(s) was identified as Kunitz trypsin inhibitor (KTI). The root hair-promoting peptide in the degraded products of KTI was purified and produced a signal of 1,198.2 Da with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) analysis. A search of the amino acid sequence of KTI located the peptide GGIRAAPTGNER, which had a molecular weight identical to 1,198.2 Da. The peptide GGIRAAPTGNER was chemically synthesized, and the synthetic peptide possessed root hair-promoting activity. Thus, it is concluded that this peptide in DSP is the foreign bioactive peptide promoting the differentiation of root hairs.

Keywords: Bacillus; Degraded soybean-meal products; Root hair; Bioactive peptide; Plant growth

Control of chemoselectivity of microbial reaction with resin adsorbent: enhancement of Baeyer–Villiger oxidation over reduction by Mikio Fujii; Hiroyuki Akita; Yoshiteru Ida; Toshiya Nakagawa; Kaoru Nakamura (pp. 45-51).

Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer–Villiger (BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded, and BV oxidation was scarcely found in the absence of the polymer.

Keywords: Baeyer–Villiger; Microbial; Reduction; Oxidation; XAD; Resin

Production of ascorbic acid glucoside by alginate-entrapped mycelia of Aspergillus niger by Hsin-Ju Hsieh; Kai-Yu Tung; Giridhar R. Nair; I-Ming Chu; Wen-Teng Wu (pp. 53-60).

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.

Keywords: Glucosylation; Ascorbic acid glucoside; Aspergillus niger ; Fixed bed reactor; Calcium alginate; Entrapped mycelia

Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 by Tania I. Georgieva; Birgitte K. Ahring (pp. 61-68).

Dilute sulfuric acid pretreated corn stover is potential feedstock of industrial interest for second generation fuel ethanol production. However, the toxicity of corn stover hydrolysate (PCS) has been a challenge for fermentation by recombinant xylose fermenting organisms. In this work, the thermophilic anaerobic bacterial strain Thermoanaerobacter BG1L1 was assessed for its ability to ferment undetoxified PCS hydrolysate in a continuous immobilized reactor system at 70°C. The tested strain showed significant resistance to PCS, and substrate concentrations up to 15% total solids (TS) were fermented yielding ethanol of 0.39–0.42 g/g-sugars consumed. Xylose was nearly completely utilized (89–98%) for PCS up to 10% TS, whereas at 15% TS, xylose conversion was lowered to 67%. The reactor was operated continuously for 135 days, and no contamination was seen without the use of any agent for preventing bacterial infections. This study demonstrated that the use of immobilized thermophilic anaerobic bacteria for continuous ethanol fermentation could be promising in a commercial ethanol process in terms of system stability to process hardiness and reactor contamination. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol.

Keywords: Continuous fermentation; Corn stover; Fluidized-bed reactor; Hemicellulose; Thermophilic anaerobic bacteria; Xylose

Heterotrophic high-cell-density fed-batch and continuous-flow cultures of Galdieria sulphuraria and production of phycocyanin by Olav Sune Graverholt; Niels Thomas Eriksen (pp. 69-75).

Production of biomass and phycocyanin (PC) were investigated in highly pigmented variants of the unicellular rhodophyte Galdieria sulphuraria, which maintained high specific pigment concentrations when grown heterotrophically in darkness. The parental culture, G. sulphuraria 074G was grown on solidified growth media, and intensely coloured colonies were isolated and grown in high-cell-density fed-batch and continuous-flow cultures. These cultures contained 80–110 g L⁻¹ biomass and 1.4–2.9 g L⁻¹ PC. The volumetric PC production rates were 0.5–0.9 g L⁻¹ day⁻¹. The PC production rates were 11–21 times higher than previously reported for heterotrophic G. sulphuraria 074G grown on glucose and 20–287 times higher than found in phototrophic cultures of Spirulina platensis, the organism presently used for commercial production of PC.

Keywords: Galdieria sulphuraria ; Heterotrophic; High-cell-density culture; Fed-batch culture; Continuous-flow culture; Phycocyanin

Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain Verticillium sp. P9 by Monika Kasieczka-Burnecka; Karina Kuc; Halina Kalinowska; Monika Knap; Marianna Turkiewicz (pp. 77-89).

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg²⁺and Br⁻ ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn²⁺, Cu²⁺, K⁺, Cd²⁺, Ag⁺, Fe³⁺, Mn²⁺, Co²⁺, Hg²⁺, Pb²⁺ and Sn²⁺), $${ ext{CO}}_{ ext{3}}^{{ ext{2}} - }$$ anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E _a of these reactions and temperature dependence (at 0–30°C) of k _cat, k _cat/K _m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k _cat and k _cat/K _m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.

Keywords: Antarctic cold-adapted extracellular tannases; Verticillium sp.

Membrane-bound l- and d-lactate dehydrogenase activities of a newly isolated Pseudomonas stutzeri strain by Cuiqing Ma; Chao Gao; Jianhua Qiu; Jianrong Hao; Wenwen Liu; Ailong Wang; Yinan Zhang; Min Wang; Ping Xu (pp. 91-98).

Pseudomonas stutzeri SDM was newly isolated from soil, and two stereospecific NAD-independent lactate dehydrogenase (iLDH) activities were detected in membrane of the cells cultured in a medium containing dl-lactate as the sole carbon source. Neither enzyme activities was constitutive, but both of them might be induced by either enantiomer of lactate. P. stutzeri SDM preferred to utilize lactate to growth, when both l-lactate and glucose were available, and the consumption of glucose was observed only after lactate had been exhausted. The Michaelis–Menten constant for l-lactate was higher than that for d-lactate. The l-iLDH activity was more stable at 55°C, while the d-iLDH activity was lost. Both enzymes exhibited different solubilization with different detergents and different oxidation rates with different electron acceptors. Combining activity staining and previous proteomic analysis, the results suggest that there are two separate enzymes in P. stutzeri SDM, which play an important role in converting lactate to pyruvate.

Keywords: Pseudomonas stutzeri ; Lactate dehydrogenase; Expression; Location; Biochemical properties

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in Escherichia coli by Sehoon Kim; Lawrence H. Cheung; Weihe Zhang; Michael G. Rosenblum (pp. 99-106).

The immunocytokine scFvMEL/TNF is a fusion protein composed of tumor necrosis factor (TNF-α) and a single-chain Fv antibody scFvMEL targeting the melanoma-associated gp240 antigen. The fusion protein containing thioredoxin and a hexa-histidine tag was expressed in two Escherichia coli host cells, AD494 (DE3) pLysS and T7 Express I ^q. The cell growth and expression level of target protein, His-tagged scFvMEL/TNF, were highly dependent on the induction temperature, inducer types and host strains. The ratio of insoluble to soluble target proteins was found to be controllable and could be minimized using cold shock conditions at less than 18°C. The total productivity of soluble target protein was further improved by high cell density cultivation using a DO-STAT feeding strategy. The scFvMEL/TNF purified under their conditions was specifically cytotoxic to gp240-antigen positive melanoma A375-M cells as previously described.

Keywords: scFvMEL/TNF; Single-chain antibody; Melanoma; Lactose induction; Escherichia coli ; Fed-batch culture

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, Vibrio sp. GMD509 by Sang-Yi Park; Jun-Tae Kim; Sung Gyun Kang; Jung-Hee Woo; Jung-Hyun Lee; Hyoung-Tae Choi; Sang-Jin Kim (pp. 107-115).

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.

Keywords: Screening; Lipase/esterase; Dienelactone hydrolase; Vibrio ; Marine microorganism

Coexpression of rumen microbial β-glucanase and xylanase genes in Lactobacillus reuteri by Je-Ruei Liu; Bi Yu; Xin Zhao; Kuo-Joan Cheng (pp. 117-124).

The aim of this study was to clone and coexpress two rumen fibrolytic enzyme genes in Lactobacillus reuteri. The ability of the genetically modified strain to degrade β-glucan and xylan was evaluated. The Fibrobacter succinogenes β-glucanase (1,3–1,4-β-d-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene and the Neocallimastix patriciarum xylanase gene, xynCDBFV, were constructed to coexpress and secrete under control of the Lactococcus lactis lacA promoter and its secretion signal and then transformed into L. reuteri Pg4, a strain isolated from the gastrointestinal tract of broiler chickens. The transformed L. reuteri strain acquired the capacity to break down soluble β-glucan and xylan. The introduction of the recombinant plasmids and production of β-glucanase and xylanase did not affect cell growth. To the best of our knowledge, this is the first report of coexpression of rumen microbial fibrolytic enzyme genes in L. reuteri.

Keywords: Rumen microbe; β-Glucanase; Xylanase; Lactobacillus reuteri

The ABC transporter Tba of Amycolatopsis balhimycina is required for efficient export of the glycopeptide antibiotic balhimycin by R. Menges; G. Muth; W. Wohlleben; E. Stegmann (pp. 125-134).

All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His₆-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.

Keywords: Vancomycin; Antibiotic resistance; Combinatorial biosynthesis; Actinomycetales

CYP201A2, a cytochrome P450 from Rhodopseudomonas palustris, plays a key role in the biodegradation of tributyl phosphate by Cécile Berne; David Pignol; Jérôme Lavergne; Daniel Garcia (pp. 135-144).

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in nuclear fuel processing and chemical industries. Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosynthetic conditions. This study shows that this O₂- and NADPH/FMNH₂-dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain. The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype. The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (K _d ≈ 65 μM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP.

Keywords: Bioremediation; Cytochrome P450; Organophosphorus

Assimilable nitrogen utilisation and production of volatile and non-volatile compounds in chemically defined medium by Saccharomyces cerevisiae wine yeasts by M. Vilanova; M. Ugliano; C. Varela; T. Siebert; I. S. Pretorius; P. A. Henschke (pp. 145-157).

Surveys conducted worldwide have shown that a significant proportion of grape musts are suboptimal for yeast nutrients, especially assimilable nitrogen. Nitrogen deficiencies are linked to slow and stuck fermentations and sulphidic off-flavour formation. Nitrogen supplementation of grape musts has become common practice; however, almost no information is available on the effects of nitrogen supplementation on wine flavour. In this study, the effect of ammonium supplementation of a synthetic medium over a wide range of nitrogen values on the production of volatile and non-volatile compounds by two high-nitrogen-demand wine fermentation strains of Saccharomyces cerevisiae was determined. To facilitate this investigation, a simplified chemically defined medium that resembles the nutrient composition of grape juice was used. Analysis of variance revealed that ammonium supplementation had significant effects on the concentration of residual sugar, L-malic acid, acetic acid and glycerol but not the ethanol concentration. While choice of yeast strain significantly affected half of the aroma compounds measured, nitrogen concentrations affected 23 compounds, including medium-chain alcohols and fatty acids and their esters. Principal component analysis showed that branched-chain fatty acids and their esters were associated with low nitrogen concentrations, whereas medium-chain fatty esters and acetic acid were associated with high nitrogen concentrations.

Keywords: Wine yeast; Fermentation; Nitrogen; Saccharomyces cerevisiae; Aroma compounds; Flavour compounds

Distribution of free and esterified ergosterols in the medicinal fungus Ganoderma lucidum by Jian-Ping Yuan; Jiang-Hai Wang; Xin Liu (pp. 159-165).

The fruiting bodies, spores, and lipid from the spores of Ganoderma lucidum have been widely used for medicinal purpose in China. Ergosterol content may be a suitable marker for evaluating the quality of ganoderma spore and ganoderma spore lipid (GSL) products. A gradient reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of free and esterified ergosterols in G. lucidum. The contents of free and esterified ergosterols in the different parts (the stipe, pileus, tubes, and spores) of G. lucidum and GSL were determined. The results showed that total ergosterol levels in the stipe, pileus, tubes, and spores of G. lucidum were between 0.8 and 1.6 mg/g. The relative abundances of free to esterified ergosterol were different in the different parts of G. lucidum. The spores and the tubes, the hymenophore tissue that contains the spore-producing cells, have a considerably higher percentage of ergosteryl esters (41.9 and 39.7% of total ergosterol) in comparison with the pileus and stipe tissues (3.6 and 6.2%).

Keywords: Ganoderma lucidum ; Free and esterified ergosterols; Spores; Hymenophore; Stipe; Pileus

Polyphosphate kinase genes from full-scale activated sludge plants by Katherine D. McMahon; Suzan Yilmaz; Shaomei He; Daniel L. Gall; David Jenkins; Jay D. Keasling (pp. 167-173).

The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the polyphosphate kinase gene (ppk1) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1, to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses.

Keywords: Enhanced biological phosphorus removal; Activated sludge; Rhodocyclus ; Accumulibacter phosphatis; Polyphosphate kinase

Effects of aeration intensity on formation of phenol-fed aerobic granules and extracellular polymeric substances by Sunil S. Adav; Duu-Jong Lee; J. Y. Lai (pp. 175-182).

Effect of air aeration intensities on granule formation and extracellular polymeric substances content in three identical sequential batch reactors were investigated. The excitation–emission–matrix spectra and multiple staining and confocal laser scanning microscope revealed proteins, polysaccharides, lipids, and humic substances in the sludge and granule samples. Seed sludge flocs were compacted at low aeration rate, with produced extracellular polymeric substances of 50.2–76.7 mg g⁻¹ of proteins, 50.2–77.3 mg g⁻¹ carbohydrates and 74 mg g⁻¹ humic substances. High aeration rate accelerated formation of 1.0–1.5 mm granules with smooth outer surface. The corresponding quantities of extracellular polymeric substances were 309–537 mg g⁻¹ of proteins, 61–109 mg g⁻¹ carbohydrates, 49–92 mg g⁻¹ humic substances, and 49–68 mg g⁻¹ lipids. Intermediate aeration rate produced 3.0–3.5 mm granules with surface filaments. Reactor failure occurred with overgrowth of filaments, probably owing to the deficiency of nutrient in liquid phase. No correlation was noted between extracellular polymeric substances composition and the proliferation of filamentous microorganisms on granule surface.

Keywords: Aeration; EPS; Granule formation; Filaments

Alkaliphilic and halophilic hydrocarbon-utilizing bacteria from Kuwaiti coasts of the Arabian Gulf by H. Al-Awadhi; Rasha H. D. Sulaiman; Huda M. Mahmoud; S. S. Radwan (pp. 183-186).

Green animate materials from the intertidal zone of the Arabian Gulf coast accommodated more alkaliphilic and halophilic bacteria than inanimate materials. The alkaliphilic oil-utilizing bacteria, as identified by their 16S ribonucleic acid sequences, belonged to the following genera arranged in decreasing frequences: Marinobacter, Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus. The halophilic oil-utilizing bacteria belonged to the genera: Marinobacter, Georgenia, Microbacterium, Stappia, Bacillus, Isoptericola, and Cellulomonas. Most isolates could grow on a wide range of pure n-alkanes and aromatic compounds, as sole sources of carbon and energy. Quantitative gas liquid chromatographic analysis showed that individual isolates attenuated crude oil and representative pure hydrocarbons in culture. The optimum pH for most of the alkaliphilic genera was pH 10, and the optimum salinity for the halophiles ranged between 2.5 and 5% NaCl (w/v). It was concluded that as far as their microbial makeup is concerned, oily alkaline and saline intertidal areas of the Kuwaiti coasts have a self-cleaning potential.

Biodegradation of diphenyl ether and transformation of selected brominated congeners by Sphingomonas sp. PH-07 by Young-Mo Kim; In-Hyun Nam; Kumarasamy Murugesan; Stefan Schmidt; David E. Crowley; Yoon-Seok Chang (pp. 187-194).

Polybrominated diphenyl ethers (PBDEs) are common flame-retardant chemicals that are used in diverse commercial products such as textiles, circuit boards, and plastics. Because of the widespread production and improper disposal of materials that contain PBDEs, there has been an increasing accumulation of these compounds in the environment. The toxicity and bioavailability of PBDEs are variable for different congeners, with some congeners showing dioxin-like activities and estrogenicity. The diphenyl ether-utilizing bacterium Sphingomonas sp. PH-07 was enriched from activated sludge of a wastewater treatment plant. In liquid cultures, this strain mineralized 1 g of diphenyl ether per liter completely within 6 days. The metabolites detected and identified by gas chromatography/mass spectrometry (MS) and electrospray ionization/MS analysis corresponded with a feasible degradative pathway. However, the strain PH-07 even catabolized several brominated congeners such as mono-, di-, and tribrominated diphenyl ethers thereby producing the corresponding metabolites.

Keywords: Biodegradation; Biotransformation; Diphenyl ether; PBDEs (polybrominated diphenyl ethers); Sphingomonas sp.

The effect of calcium ion on the biodegradation of octylphenol polyethoxylates, and the antiandrogenic activity of their biodegradates by Atsushi Shibata; Yoichi Ishimoto; Yosuke Nishizaki; Akifumi Hosoda; Hiromichi Yoshikawa; Hiroto Tamura (pp. 195-201).

Because limes have been used as important fertilizers to neutralize acidified farmland in Japan, our interest in this study was focused on the effect of calcium ion on the biodegradation of octylphenol polyethoxylates (OPEOn) by a pure culture of Pseudomonas putida S5 isolated from a rice paddy field in Japan. In the presence of calcium ion, P. putida S5 accelerated the formation of octylphenol oligoethoxy carboxylates (OPECn) rather than that of octylphenol oligoethoxylates under an aerobic condition, indicating that more soluble biodegradates with terminal carboxyl group may liquate out easily to surface and ground water rather than more hydrophobic biodegradates with shorter ethylene oxide residues. Therefore, the androgen receptor (AR) activity of their degradation products was characterized using an in vitro reporter gene assay. As ethylene oxide chain length decreased, the biodegradates, OPEOn (n < 3), increased their AR antagonist activity. However, OPECn (n < 3) were unable to determine their AR activity because of their cytotoxicity in our reporter gene assay system.

Keywords: Alkylphenol; Androgen receptor; Endocrine disruptor; MDA-kb2; Pseudomonas putida ; Reporter gene assay

Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity by Ermanno Federici; Vanessa Leonardi; Maria A. Giubilei; Daniele Quaratino; Roberta Spaccapelo; Alessandro D’Annibale; Maurizio Petruccioli (pp. 203-211).

Botryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 ± 0.3 × 10⁸ and 8.35 ± 0.6 × 10⁸, respectively, vs 9.2 ± 0.3 × 10⁷). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil.

Keywords: Mycoremediation; Bioaugmentation; Historically contaminated soil; DGGE; Microbial interactions; Aromatic hydrocarbons

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates by Hiroshi Yokoyama; Naoko Moriya; Hideyuki Ohmori; Miyoko Waki; Akifumi Ogino; Yasuo Tanaka (pp. 213-222).

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75°C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

Keywords: Community structure; Hydrogen production; Extreme thermophile; Caldoanaerobacter subterraneus ; Caloramator fervidus

Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT) by Hyesuk Kong; Dmitriy V. Volokhov; Joseph George; Pranvera Ikonomi; Donna Chandler; Christine Anderson; Vladimir Chizhikov (pp. 223-232).

Herein, we present data demonstrating that the application of initial cell culture enrichment could significantly improve mycoplasma testing methods based on the nucleic acid amplification technology (NAT) including a polymerase chain reaction (PCR)/microarray method. The results of the study using Vero cells demonstrated that this cell culture is able (1) to support efficient growth of mycoplasmas of primary interest, i.e., species found to be cell line contaminants, (2) to increase the sensitivity of NAT assay to the detection limits of the conventional broth/agar culture methods, and (3) to reduce the time required for mycoplasma testing fourfold in comparison with the conventional methods. Detection and identification of mycoplasmal agents were conducted using a modified PCR/microarray assay based on genetic differences among Mollicutes in the 16S-23S rRNA intergenic transcribed spacer (ITS). The application of nano-gold/silver enhancement technology instead of previously used fluorescent dyes significantly simplified the readout of microarray results and allowed us to avoid using expensive scanning equipment. This modification has the potential to expand the implementation of microarray techniques into laboratories involved in diagnostic testing of mycoplasma contamination in cell substrates and potentially in other biological and pharmaceutical products.

A fluorescent method for assessing the antimicrobial efficacy of disinfectant against Escherichia coli ATCC 35218 biofilm by Alberto Mariscal; Manuel Carnero-Varo; Mario Gutierrez-Bedmar; Antonio Garcia-Rodriguez; Joaquin Fernandez-Crehuet (pp. 233-240).

In this study, a versatile method was developed to assess biocide efficacy against Escherichia coli biofilm growth on carriers made of five different materials. The glucuronidase activity of live E. coli on a fluorogenic substrate (4-methylumbellyferyl-β-d-glucuronide, MUG) was used as a viability test. Fluorescence emissions from cellular suspensions of E. coli in the test range displayed a linear response with a MUG concentration of 10 μg ml⁻¹. A glucuronidase activity curve with cellular suspensions of E. coli calculated as colony-forming units per milliliter showed a good correlation (0.9487 and 0.917 for 1 and 18 h of incubation, respectively), with counts obtained from biofilm containing this organism; E. coli cultures in suspension were used as standard. Three agents commonly used as disinfectants, sodium hypochlorite, hydrogen peroxide, and ethanol, were tested at use concentrations and at one-half and decimal dilutions. At decimal dilutions, ethanol at 70% proved to be the least active disinfectant on E. coli biofilm. Unlike other methods, our method permits the testing of disinfectant efficacy against biofilm growth on different materials. In preliminary assays, glass, polyvinyl chloride, polypropylene, polycarbonate, and silicon were tested. Because they gave the lowest E. coli counts after 24 and 48 h, glass and polypropylene were the two materials to which biofilm adhered least strongly.

Keywords: Biofilm; Disinfection; Escherichia coli ; Fluorometric method; Glucuronidase

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli by Christoph Reisinger; Alexander Kern; Kateryna Fesko; Helmut Schwab (pp. 241-244).

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the “T-vector” can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3′-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5′-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.

Keywords: PCR-cloning; TA-cloning; Expression plasmid; Recombinant protein expression; Parallel cloning

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Applied Microbiology and Biotechnology (v.77, #1)

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Applied Microbiology and Biotechnology (v.77, #1)