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Applied Microbiology and Biotechnology (v.86, #6)

Determination of mycotoxins in foods: current state of analytical methods and limitations by Robert Köppen; Matthias Koch; David Siegel; Stefan Merkel; Ronald Maul; Irene Nehls (pp. 1595-1612).

Mycotoxins are natural contaminants produced by a range of fungal species. Their common occurrence in food and feed poses a threat to the health of humans and animals. This threat is caused either by the direct contamination of agricultural commodities or by a “carry-over” of mycotoxins and their metabolites into animal tissues, milk, and eggs after feeding of contaminated hay or corn. As a consequence of their diverse chemical structures and varying physical properties, mycotoxins exhibit a wide range of biological effects. Individual mycotoxins can be genotoxic, mutagenic, carcinogenic, teratogenic, and oestrogenic. To protect consumer health and to reduce economic losses, surveillance and control of mycotoxins in food and feed has become a major objective for producers, regulatory authorities and researchers worldwide. However, the variety of chemical structures makes it impossible to use one single technique for mycotoxin analysis. Hence, a vast number of analytical methods has been developed and validated. The heterogeneity of food matrices combined with the demand for a fast, simultaneous and accurate determination of multiple mycotoxins creates enormous challenges for routine analysis. The most crucial issues will be discussed in this review. These are (1) the collection of representative samples, (2) the performance of classical and emerging analytical methods based on chromatographic or immunochemical techniques, (3) the validation of official methods for enforcement, and (4) the limitations and future prospects of the current methods.

Keywords: Mycotoxins and fungal metabolites; Food; Rapid and validated methods; Regulations; Sampling; Multi-mycotoxin determination

Occurrence, characteristics, and applications of fructosyl amine oxidases (amadoriases) by Zhanglin Lin; Jing Zheng (pp. 1613-1619).

Amadori compounds, formed by the Maillard reaction between reducing sugars (e.g., glucose) and amines (e.g., lysine residues in proteins), are ubiquitous in nature and have been implicated in aging and several chronic diseases. Fructosyl amine oxidases (FAOXs) are a relatively new class of enzymes that cleave amadori compounds and have been found in fungi, yeast, and bacteria. This mini-review summarizes over a dozen of FAOXs with different substrate specificities have been isolated, characterized, and engineered to date. All known FAOX sequences except one have the consensus motif for the ADP-binding βαβ-fold common to all FAD and NAD enzymes, and a recently solved crystal structure provides important clues for this class of enzymes. FAOXs have been explored for applications in diabetes diagnosis, detergents, and food processing. Given that naturally occurring FAOXs can only react directly with small glycated amino acids or short peptides, it is of great interest to engineer and expand the accessibility of the substrate binding sites of these enzymes.

Keywords: Fructosyl amine oxidase; Amadoriase; Occurrence; Characteristics; Applications; Substrate specificity

Biosynthesis and production of polysialic acids in bacteria by Miguel Ángel Ferrero; Leandro Rodríguez Aparicio (pp. 1621-1635).

Polysialic acids (PA) are protective capsular sialohomopolymers present in some bacteria which can invade the mammalian host and cause lethal bacteremia and meningitis. Biosynthesis and translocation of PA to the cell surface are equivalent in different species and bacterial strains which are produced. The diversity in PA structure is derived from the PA linkages and is a consequence of the specific sialyltransferase activities. The monomer acetylation and the polymer length could be important factors in the potential virulence. In vivo PA production is affected by different physical and chemical factors. The temperature of cellular growth strictly regulates PA genesis through a molecular complex and multifactorial mechanism that operate to transcription level.

Keywords: Escherichia coli ; N-acetylneuraminic acid; Neisseria meningitidis ; Polysialic acids

Iron uptake and metabolism in pseudomonads by Pierre Cornelis (pp. 1637-1645).

Pseudomonads are ubiquitous Gram-negative γ proteobacteria known for their extreme versatility and adaptability. Some are plant pathogens (Pseudomonas syringae) which have to survive on the surface of leaves while others can colonize the rhizosphere or survive in soil (Pseudomonas fluorescens, Pseudomonas putida), and one species, Pseudomonas entomophila, is an insect pathogen. The most investigated species, Pseudomonas aeruginosa, is known to be an opportunistic pathogen able to infect plants, nematodes, insects, and mammals, including humans. Like for other bacteria, iron is a key nutrient for pseudomonads. The fluorescent pseudomonads produce siderophores, the best known being the fluorescent high-affinity peptidic pyoverdines. Often diverse secondary siderophores of lower affinity are produced as well (pyochelin, pseudomonin, corrugatins and ornicorrugatins, yersiniabactin, and thioquinolobactin). Reflecting their large capacity of adaptation to changing environment and niche colonization, pseudomonads are able to obtain their iron from heme or from siderophores produced by other microorganisms (xenosiderophores) via the expression of outer membrane TonB-dependent receptors. As expected, iron uptake is exquisitely and hierarchically regulated in these bacteria. In this short review, the diversity of siderophores produced, receptors, and finally the way iron homeostasis is regulated in P. aeruginosa, P. syringae, P. putida, and P. fluorescens, will be presented and, when possible, put in relation with the lifestyle and the ecological niche.

Keywords: Pseudomonas ; Iron; Heme; Siderophores; Pyoverdine; Fur regulation

Properties and applications of microbial β-D-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium by Douglas B. Jordan; Kurt Wagschal (pp. 1647-1658).

Xylan 1,4-β-D-xylosidase catalyzes hydrolysis of non-reducing end xylose residues from xylooligosaccharides. The enzyme is currently used in combination with β-xylanases in several large-scale processes for improving baking properties of bread dough, improving digestibility of animal feed, production of d-xylose for xylitol manufacture, and deinking of recycled paper. On a grander scale, the enzyme could find employment alongside cellulases and other hemicellulases in hydrolyzing lignocellulosic biomass so that reaction product monosaccharides can be fermented to biofuels such as ethanol and butanol. Catalytically efficient enzyme, performing under saccharification reactor conditions, is critical to the feasibility of enzymatic saccharification processes. This is particularly important for β-xylosidase which would catalyze breakage of more glycosidic bonds of hemicellulose than any other hemicellulase. In this paper, we review applications and properties of the enzyme with emphasis on the catalytically efficient β-d-xylosidase from Selenomonas ruminantium and its potential use in saccharification of lignocellulosic biomass for producing biofuels.

Keywords: β-Xylosidase; Protein engineering; Glycoside hydrolase; Bioenergy; GH43; Performance

Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes by Leland S. Pierson III; Elizabeth A. Pierson (pp. 1659-1670).

Phenazines constitute a large group of nitrogen-containing heterocyclic compounds produced by a diverse range of bacteria. Both natural and synthetic phenazine derivatives are studied due their impacts on bacterial interactions and biotechnological processes. Phenazines serve as electron shuttles to alternate terminal acceptors, modify cellular redox states, act as cell signals that regulate patterns of gene expression, contribute to biofilm formation and architecture, and enhance bacterial survival. Phenazines have diverse effects on eukaryotic hosts and host tissues, including the modification of multiple host cellular responses. In plants, phenazines also may influence growth and elicit induced systemic resistance. Here, we discuss emerging evidence that phenazines play multiple roles for the producing organism and contribute to their behavior and ecological fitness.

Keywords: Phenazine; Secondary metabolite; Electron shuttling; Antibiotic; Biofilm

Occurrence, fate, and biodegradation of estrogens in sewage and manure by Sarah Combalbert; Guillermina Hernandez-Raquet (pp. 1671-1692).

The estrogens estrone (E1), 17α-estradiol (E2α), 17β-estradiol (E2β), and estriol (E3) are natural sex hormones produced by humans and animals. In addition, there are some synthetic estrogens, such as 17α-ethinylestradiol (EE2), used for contraception purposes. These compounds are able to produce endocrine disruption in living organisms at nanogram-per-liter levels. In both humans and animals, estrogens are excreted in urine and feces, reaching the natural environment through discharge from sewage treatment plants (STP) and manure disposal units. In STPs, hormone removal depends on the type of treatment process and on different parameters such as the hydraulic and sludge retention times. Thus, hormone elimination rates vary from 0% to 90% in different STPs. Animals are also an important source of estrogens in the environment. Indeed, animals produce high concentrations of hormones which will end up in manure which is typically spread on land. Hence, waste-borne animal hormones may transfer these pollutants to the soil. The purpose of this review is to highlight the significance for both health and the environment of pollution by estrogens and critically review the existing knowledge on their fate and removal in different treatment processes. Relevant information on the microbial degradation of hormones and metabolic pathways is also included.

Keywords: Estrogens; Sewage; Manure; Treatment processes; Biodegradation; Endocrine disruption

Occurrence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons by Efraín Manilla-Pérez; Alvin Brian Lange; Stephan Hetzler; Alexander Steinbüchel (pp. 1693-1706).

Petroleum (or crude oil) is a complex mixture of hydrocarbons. Annually, millions of tons of crude petroleum oil enter the marine environment from either natural or anthropogenic sources. Hydrocarbon-degrading bacteria (HDB) are able to assimilate and metabolize hydrocarbons present in petroleum. Crude oil pollution constitutes a temporary condition of carbon excess coupled to a limited availability of nitrogen that prompts marine oil-degrading bacteria to accumulate storage compounds. Storage lipid compounds such as polyhydroxyalkanoates (PHAs), triacylglycerols (TAGs), or wax esters (WEs) constitute the main accumulated lipophilic substances by bacteria under such unbalanced growth conditions. The importance of these compounds as end-products or precursors to produce interesting biotechnologically relevant chemicals has already been recognized. In this review, we analyze the occurrence and accumulation of lipid storage in marine hydrocarbonoclastic bacteria. We further discuss briefly the production and export of lipophilic compounds by bacteria belonging to the Alcanivorax genus, which became a model strain of an unusual group of obligate hydrocarbonoclastic bacteria (OHCB) and discuss the possibility to produce neutral lipids using A. borkumensis SK2.

Keywords: Hydrocarbonoclastic bacteria; Alcanivorax ; Storage lipids; Oil pollution

A review: recent advances and future prospects of taxol-producing endophytic fungi by Xuanwei Zhou; Huifang Zhu; Lu Liu; Juan Lin; Kexuan Tang (pp. 1707-1717).

In the urgent search for more effective ways to treat cancer, new extraction methods of taxol from endophytic fungus have demonstrated high potential in increasing the efficiency of taxol extraction for more efficient and sustainable production of taxol and cancer treatment products. This paper summarizes recent advances in taxol-producing endophytic fungi, both in China and abroad, in the following areas: isolation and identification of endophytic fungi types, extraction and detection methods of endophytic taxol in plants, and improved efficiency of the extraction process. With the advancement of science and technology, new techniques in biotechnology, such as fungal strain improvement and recombining technique and microbial fermentation engineering, have increased the extraction yield from taxol-producing fungi, thereby improved the overall efficiency of taxol production.

Keywords: Taxol; Endophytic fungi; Isolation; Identification; Meliorate

Unraveling polyketide synthesis in members of the genus Aspergillus by Yi-Ming Chiang; Berl R. Oakley; Nancy P. Keller; Clay C. C. Wang (pp. 1719-1736).

Aspergillus species have the ability to produce a wide range of secondary metabolites including polyketides that are generated by multi-domain polyketide synthases (PKSs). Recent biochemical studies using dissected single or multiple domains from PKSs have provided deep insight into how these PKSs control the structural outcome. Moreover, the recent genome sequencing of several species has greatly facilitated the understanding of the biosynthetic pathways for these secondary metabolites. In this review, we will highlight the current knowledge regarding polyketide biosynthesis in Aspergillus based on the domain architecture of non-reducing, highly reducing, and partially reducing PKSs, and PKS-non-ribosomal peptide synthetases.

Keywords: Secondary metabolites; Fungi; Polyketide; Nonribosomal peptides

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain by I. Geetha; A. M. Manonmani; K. P. Paily (pp. 1737-1744).

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC₅₀ dose of 1 µg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13–C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.

Keywords: Bacillus subtilis ; Surfactin; Mosquitoes; Pupicidal metabolite; Biocontrol agent

Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations by Peter Ödman; Claus Lindvald Johansen; Lisbeth Olsson; Krist V. Gernaey; Anna Eliasson Lantz (pp. 1745-1759).

Fed-batch cultivations of Streptomyces coelicolor, producing the antibiotic actinorhodin, were monitored online by multiwavelength fluorescence spectroscopy and off-gas analysis. Partial least squares (PLS), locally weighted regression, and multilinear PLS (N-PLS) models were built for prediction of biomass and substrate (casamino acids) concentrations, respectively. The effect of combination of fluorescence and gas analyzer data as well as of different variable selection methods was investigated. Improved prediction models were obtained by combination of data from the two sensors and by variable selection using a genetic algorithm, interval PLS, and the principal variables method, respectively. A stepwise variable elimination method was applied to the three-way fluorescence data, resulting in simpler and more accurate N-PLS models. The prediction models were validated using leave-one-batch-out cross-validation, and the best models had root mean square error of cross-validation values of 1.02 g l⁻¹ biomass and 0.8 g l⁻¹ total amino acids, respectively. The fluorescence data were also explored by parallel factor analysis. The analysis revealed four spectral profiles present in the fluorescence data, three of which were identified as pyridoxine, NAD(P)H, and flavin nucleotides, respectively.

Keywords: Bioprocess monitoring; Genetic algorithm; iPLS; LWR; Multiwavelength fluorescence; PLS; Principal variables

Optimization of phenazine-1-carboxylic acid production by a gacA/qscR-inactivated Pseudomonas sp. M18GQ harboring pME6032Phz using response surface methodology by Quan Zhou; Jinjiang Su; Haixiang Jiang; Xianqing Huang; Yuquan Xu (pp. 1761-1773).

Phenazine-1-carboxylic acid (PCA) production was enhanced in Pseudomonas sp. M18 wild strain and its mutants carrying recombinant pME6032Phz for phz gene cluster overexpression, among which Pseudomonas sp. strain M18GQ/pME6032Phz, a gacA and qscR double gene chromosomally inactivated mutant harboring pME6032Phz, showed the highest PCA yield. The conditions for fermentation and isopropyl-β-d-1-thiogalactopyranoside (IPTG) induction were optimized for strain M18GQ/pME6032Phz in shake flask experiments. A one-factor-at-a-time approach, followed by a fractional factorial design identified soybean meal, corn steep liquor, and ethanol as statistically significant factors. Optimal concentrations and mutual interactions of the factors were then determined by the method of steepest ascent and by response surface methodology based on the center composite design. The predicted PCA production was 6,335.2 mg/l after 60 h fermentation in the optimal medium of 65.02 g soybean meal, 15.36 g corn steep liquor, 12 g glucose, 21.70 ml ethanol, and 1 g MgSO₄ per liter in the flask fermentations, with induction of 1.0 mmol/l IPTG 24 h after inoculation. In an experimental validation under these conditions, the maximum PCA production was 6,365.0 mg/l. This represents a ∼60% increase over production by strain M18GQ in optimal conditions. The negative effect of plasmid pME6032 on the expression of chromosomally located phz gene cluster was found in Pseudomonas sp. M18GQ, and the possible reason was discussed in the text.

Keywords: Phenazine-1-carboxylic acid; Overexpression; Pseudomonas sp. M18GQ; Response surface methodology; Optimization

Recombinant expression of BTA hydrolase in Streptomyces rimosus and catalytic analysis on polyesters by surface plasmon resonance by Nitat Sinsereekul; Thidarat Wangkam; Arinthip Thamchaipenet; Toemsak Srikhirin; Lily Eurwilaichitr; Verawat Champreda (pp. 1775-1784).

A recombinant polyester-degrading hydrolase from Thermobifida sp. BCC23166 targeting on aliphatic-aromatic copolyester (rTfH) was produced in Streptomyces rimosus R7. rTfH was expressed by induction with thiostrepton as a C-terminal His₆ fusion from the native gene sequence under the control of tipA promoter and purified from the culture supernatant to high homogeneity by a single step affinity purification on Ni-Sepharose matrix. The enzyme worked optimally at 50–55°C and showed esterase activity on C3-C16 p-nitrophenyl alkanoates with a specific activity of 76.5 U/mg on p-nitrophenyl palmitate. Study of rTfH catalysis on surface degradation of polyester films using surface plasmon resonance analysis revealed that the degradation rates were in the order of poly-ε-caprolactone > Ecoflex^® > polyhydroxybutyrate. Efficient hydrolysis of Ecoflex^® by rTfH was observed in mild alkaline conditions, with the highest activity at pH 8.0 and ionic strength at 250 mM sodium chloride, with the maximal specific activity of 0.79 mg⁻¹min⁻¹mg⁻¹ protein. Under the optimal conditions, rTfH showed a remarkable 110-time higher specific activity on Ecoflex^® in comparison to a lipase from Thermomyces lanuginosus, while less difference in degradation efficiency of the two enzymes was observed on the aliphatic polyesters, suggesting greater specificities of rTfH to the aliphatic-aromatic copolyester. This study demonstrated the use of streptomycetes as an alternative expression system for production of the multi-polyester-degrading enzyme of actinomycete origin and provided insights on its catalytic properties on surface degradation contributing to further biotechnological application of this enzyme.

Keywords: Aliphatic-aromatic copolyester; Ecoflex^® ; Hydrolase; Streptomyces rimosus ; Surface plasmon resonance; Thermobifida sp.

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum by Jake Tewalt; Jonathan Schilling (pp. 1785-1793).

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using β-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.

Keywords: Ethanol; Cellulase; Bioprocessing; Gloeophyllum trabeum

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein by Nagesh K. Tripathi; Jyoti Shukla; Karttik C. Biswal; P. V. Lakshmana Rao (pp. 1795-1803).

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.

Keywords: Japanese encephalitis; Escherichia coli ; Media optimization; Cultivation; ELISA

Inhibition of oxidative stress-induced amyloid β formation in NT2 neurons by culture filtrate of a strain of Streptomyces antibioticus by Bahareh Eftekharzadeh; Javad Hamedi; Fatemeh Mohammadipanah; Fariba Khodagholi; Nader Maghsoudi; Hans Peter Klenk (pp. 1805-1811).

Actinomycetes isolated from Iran soil habitats were tested for the capacity to produce compounds which can protect neurons from cell death generated by oxidative stress in NT2 neurons. Confirmation of our initial hit was accomplished via the determination of amyloid β level using the enzyme-linked immunosorbent assay test. The most interesting amyloid β formation inhibitor discovered in our study was a secondary metabolite which was produced by strain HM45. This bioactive strain was identified as a strain of Streptomyces antibioticus DSM 40234 using polyphasic approach. The strain HM45 was deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen as S. antibioticus DSM 41955 and University of Tehran Microorganisms Sollection as S. antibioticus UTMC 00105. This work is the first report on efficiency of an actinomycete metabolite in prohibition of neurons death caused by amyloid β formation.

Keywords: Streptomyces ; Secondary metabolites; Alzheimer's disease; Amyloid beta; NT2 neurons

Purification, cloning, and overexpression of an alcohol dehydrogenase from Nocardia globerula reducing aliphatic ketones and bulky ketoesters by Julia Parkot; Harald Gröger; Werner Hummel (pp. 1813-1820).

For the huge amount of chiral chemicals and precursors that can potentially be produced by biocatalysis, there is a tremendous need of enzymes with new substrate spectra, higher enantioselectivity, and increased activity. In this paper, a highly active alcohol dehydrogenase is presented isolated from Nocardia globerula that shows a unique substrate spectrum toward different prochiral aliphatic ketones and bulky ketoesters as well as thioesters. For example, the enzyme reduced ethyl 4-chloro-3-oxo butanoate with an ee >99% to (S)-4-chloro-3-hydroxy butanoate. Very interesting is also the fact that 3-oxobutanoic acid tert-butylthioester is reduced with 49.4% of the maximal activity while the corresponding tert-butyloxyester is not reduced at all. Furthermore, it has to be mentioned that acetophenone, a standard substrate for many known alcohol dehydrogenases, is not reduced by this enzyme. The enzyme was purified from wild-type N. globerula cells, and the corresponding 915-bp-long gene was determined, cloned, expressed in Escherichia coli, and applied in biotransformations. The N. globerula alcohol dehydrogenase is a tetramer of about 135 kDa in size as determined from gel filtration. Its sequence is related to several hypothetical 3-hydroxyacyl-CoA dehydrogenases whose sequences were derived by whole-genome sequencing from bacterial sources as well as known mammalian 3-hydroxyacyl-CoA dehydrogenases and ß-hydroxyacyl-CoA dehydrogenases from different clostridiae.

Keywords: Alcohol dehydrogenase; Biocatalysis; 3-Hydroxyacyl-CoA dehydrogenase; Nocardia globerula

Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas by Sarika Sharma; Farrah Gul Khan; Ghulam Nabi Qazi (pp. 1821-1828).

The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 °C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.

Keywords: Metagenome; Amylase; Northwestern Himalayas

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases by Huiying Luo; Jun Yang; Jiang Li; Pengjun Shi; Huoqing Huang; Yingguo Bai; Yunliu Fan; Bin Yao (pp. 1829-1839).

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-β-d-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 °C, maintained more than 60% of maximal activity when assayed at pH 1.5–4.0, and had good thermal stability at 60 °C and remained stable at pH 1.0–6.0. The enzyme activity was enhanced in the presence of Ni²⁺ and β-mercaptoethanol and inhibited by some metal irons (Hg²⁺, Cu²⁺, Pb²⁺, Mn²⁺, Li⁺, and Fe³⁺) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-d-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg⁻¹, respectively. The apparent K _m and V _max values for beechwood xylan were 5.6 mg ml⁻¹ and 3,622 μmol min⁻¹ mg⁻¹, respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.

Keywords: Acidic xylanase; Glycosyl hydrolase family 30; Bispora sp. MEY-1; Pichia pastoris

Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17 by Dockyu Kim; Ki Young Choi; Miyoun Yoo; Jung Nam Choi; Choong Hwan Lee; Gerben J. Zylstra; Beom Sik Kang; Eungbin Kim (pp. 1841-1847).

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography–mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.

Keywords: Rhodococcus ; o-Xylene dioxygenase; Benzylic hydroxylation; Meta effect

Study of thermostable chitinases from Oerskovia xanthineolytica NCIM 2839 by Shailesh R. Waghmare; Jai S. Ghosh (pp. 1849-1856).

The mesophilic organism, Oerskovia xanthineolytica NCIM 2839, was adapted to grow at moderate thermophilic temperatures. At these elevated temperatures, it was found to produce two thermostable chitinases—C1 and C2. These were purified by ion exchange chromatography using DEAE cellulose. The chitinases C1 and C2 were found to be stable in a pH range from 3.0 to 9.0 with 7.5 and 8.0 being the optimum pH, respectively. The optimum temperatures of the activities of C1 and C2 were 50 and 55°C, respectively. These were activated by Mn⁺⁺ and Cu⁺⁺and inactivated by Hg⁺⁺. This is first report of an extracellular thermostable chitinase being produced by O. xanthineolytica NCIM 2839.

Keywords: Chitinase; Oerskovia ; Thermostability; N-Acetylglucosamine; Chitin

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN by Pratibha Dheeran; Sachin Kumar; Yogesh K. Jaiswal; Dilip K. Adhikari (pp. 1857-1866).

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K m and V ma_x were 36 mg ml⁻¹ and 222 µmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.

Keywords: Amylase; Geobacillus sp.; Hyperthermostable; Characterization; Metalloenzyme; Cassava

Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce by Yoichi Toyokawa; Hiroaki Takahara; Alissara Reungsang; Masakazu Fukuta; Yuki Hachimine; Shinjiro Tachibana; Masaaki Yasuda (pp. 1867-1875).

A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50°C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.

Keywords: Bacillus licheniformis ; Fish sauce; Halotolerant; Serine proteinase; Thermotolerant

Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide by Michael Caspers; Ulf Brockmeier; Christian Degering; Thorsten Eggert; Roland Freudl (pp. 1877-1885).

Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous proteins. However, the yields of the desired target proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2–7) of the signal peptide of the Bacillus subtilis α-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous proteins.

Keywords: Heterologous protein secretion; Signal peptide; Saturation mutagenesis; Bacillus subtilis

Methylglyoxal activates Gcn2 to phosphorylate eIF2α independently of the TOR pathway in Saccharomyces cerevisiae by Wataru Nomura; Kazuhiro Maeta; Keiko Kita; Shingo Izawa; Yoshiharu Inoue (pp. 1887-1894).

Methylglyoxal is a ubiquitous 2-oxoaldehyde derived from glycolysis. Previously, we have reported that methylglyoxal attenuates the rate of overall protein synthesis in Saccharomyces cerevisiae through phosphorylation of the α subunit of translation initiation factor 2 (eIF2α) in a Gcn2-dependent manner. Phosphorylation of eIF2α impedes the formation of a translation initiation complex, and subsequently, overall protein synthesis is reduced. Uncharged tRNA plays an important role in the activation of Gcn2, although we found that MG treatment did not elevate the levels of uncharged tRNA. Rapamycin, a potent inhibitor of TOR kinase, is known to induce phosphorylation of eIF2α without affecting the levels of uncharged tRNA. We determined the correlation between methylglyoxal and TOR kinase activity and found that phosphorylation of eIF2α by methylglyoxal occurred independently of the target of rapamycin (TOR) pathway.

Keywords: Methylglyoxal; TORC1; Gcn2; eIF2α; S. cerevisiae ; Rapamycin

Synthesis of functional dipeptide carnosine from nonprotected amino acids using carnosinase-displaying yeast cells by Chiaki Inaba; Shinsuke Higuchi; Hironobu Morisaka; Kouichi Kuroda; Mitsuyoshi Ueda (pp. 1895-1902).

Carnosine (β-alanyl-l-histidine) is one of the bioactive dipeptides and has antioxidant, antiglycation, and cytoplasmic buffering properties. In this study, to synthesize carnosine from nonprotected amino acids as substrates, we cloned the carnosinase (CN1) gene and constructed a whole-cell biocatalyst displaying CN1 on the yeast cell surface with α-agglutinin as the anchor protein. The display of CN1 was confirmed by immunofluorescent labeling, and CN1-displaying yeast cells showed hydrolytic activity for carnosine. When carnosine was synthesized by the reverse reaction of CN1, organic solvents were added to the reaction mixture to reduce the water content. The CN1-displaying yeast cells were lyophilized and examined for organic solvent tolerance. Results showed that the CN1-displaying yeast cells retained their original hydrolytic activity in hydrophobic organic solvents. In the hydrophobic organic solvents and hydrophobic ionic liquids, the CN1-displaying yeast cells catalyzed carnosine synthesis, and carnosine was synthesized from nonprotected amino acids in only one step. The results of this research suggest that the whole-cell biocatalyst displaying CN1 on the yeast cell surface can be used to synthesize carnosine with ease and convenience.

Keywords: Carnosine; Whole-cell biocatalyst; Carnosinase (CN1); Functional dipeptide; Cell surface engineering; Cell surface display

Proteomic characterization of lignocellulose-degrading enzymes secreted by Phanerochaete carnosa grown on spruce and microcrystalline cellulose by Sonam Mahajan; Emma R. Master (pp. 1903-1914).

Proteins secreted by the white-rot, softwood-degrading fungus Phanerochaete carnosa during growth on cellulose and spruce were analyzed using tandem mass spectrometry and de novo sequencing. Homology-driven proteomics was applied to compare P. carnosa peptide sequences to proteins in Phanerochaete chrysosporium using MS BLAST and non-gapped alignment. In this way, 665 and 365 peptides from cellulose and spruce cultivations, respectively, were annotated. Predicted activities included endoglucanases from glycoside hydrolase (GH) families 5, 16, and 61, cellobiohydrolases from GH6 and GH7, GH3 β-glucosidases, xylanases from GH10 and GH11, GH2 β-mannosidases, and debranching hemicellulases from GH43 and CE15. Peptides corresponding to glyoxal oxidases, peroxidases, and glycopeptides that could participate in lignin degradation were also detected. Overall, predicted activities detected in extracellular filtrates of cellulose and spruce cultures were similar, suggesting that the adaptation of P. carnosa to growth on lignocellulose might result from fine tuning the expression of similar enzyme families.

Keywords: Phanerochaete carnosa ; Homology-driven proteomics; Carbohydrate-active enzymes; Lignin peroxidases

Transcriptome shifts in response to furfural and acetic acid in Saccharomyces cerevisiae by Bing-Zhi Li; Ying-Jin Yuan (pp. 1915-1924).

Furfural and acetic acid are two prevalent inhibitors to microorganisms during cellulosic ethanol production, but molecular mechanisms of tolerance to these inhibitors are still unclear. In this study, genome-wide transcriptional responses to furfural and acetic acid were investigated in Saccharomyces cerevisiae using microarray analysis. We found that 103 and 227 genes were differentially expressed in the response to furfural and acetic acid, respectively. Furfural downregulated genes related to transcriptional control and translational control, while it upregulated stress-responsive genes. Furthermore, furfural also interrupted the transcription of genes involved in metabolism of essential chemicals, such as etrahydrofolate, spermidine, spermine, and riboflavin monophosphate. Acetic acid downregulated genes encoding mitochondrial ribosomal proteins and genes involved in carbohydrate metabolism and regulation and upregulated genes related to amino acid metabolism. The results revealed that furfural and acetic acid had effects on multiple aspects of cellular metabolism on the transcriptional level and that mitochondria might play important roles in response to both furfural and acetic acid. This research has provided insights into molecular response to furfural and acetic acid in S. cerevisiae, and it will be helpful to construct more resistant strains for cellulosic ethanol production.

Keywords: Furfural; Acetic acid; Lignocellulose; Bioethanol; Microarray

Effect of stress pretreatment on survival of probiotic bacteria in gastrointestinal tract simulator by Ingrid Sumeri; Liisa Arike; Jelena Stekolštšikova; Riin Uusna; Signe Adamberg; Kaarel Adamberg; Toomas Paalme (pp. 1925-1931).

The effect of stress pretreatment on survival of probiotic Lactobacillus acidophilus La-5, Lactobacillus rhamnosus GG, and Lactobacillus fermentum ME-3 cultures was investigated in the single bioreactor gastrointestinal tract simulator (GITS). The cultures were pregrown in pH-auxostat, subjected to temperature, acid, or bile stress treatment, fast frozen in liquid nitrogen (LN₂), and tested for survival in GITS. After LN₂ freezing the colony forming ability of L. rhamnosus GG and L. fermentum ME-3 nonstressed and stressed cells was well retained (average survival of 75.4 ± 18.3% and 88.0 ± 7.2%, respectively). L. acidophilus La-5 strain showed good survival of auxostat nonstressed cells after fast freezing (94.2 ± 15.0), however the survival of stress pretreated cells was considerably lower (30.8 ± 8.5%). All LN₂ frozen auxostat cultures survived well in the acid phase of the GIT simulation (survival 81 ± 21%); however, after the bile phase, the colony formation ability of L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 decreased by approximately 1.4 ± 0.2, 3.8 ± 0.3, and 3.5 ± 1.2 logarithmic units, respectively. No statistically relevant positive effect of stress pretreatments on survival of LN₂ frozen L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 in GITS was observed.

Keywords: Probiotics; pH-auxostat; GIT simulator; Stress response; Survival

Cell surface properties of five polycyclic aromatic compound-degrading yeast strains by Yanqin Deng; Yu Zhang; Abd El-Latif Hesham; Ruyin Liu; Min Yang (pp. 1933-1939).

To investigate the effects of physiological properties on polycyclic aromatic compound (PAH) degradation, the surface tension and emulsification activities, and cell surface hydrophobicity of five PAH-degrading yeast isolates were compared to Saccharomyces cerevisiae from cultures grown with glucose, hexadecane, or naphthalene as carbon sources. The cell surface hydrophobicity values for the five yeast strains were significantly higher than for S. cerevisiae for all culture conditions, although these were highest with hexadecane and naphthalene. Strains with higher hydrophobicity showed higher rates of naphthalene and phenanthrene degradation, indicating that increased cell hydrophobicity might be an important strategy in PAH degradation for the five strains. Emulsification activities increased for all five yeast strains with naphthalene culturing, although no relationship existed between emulsification activity and PAH degradation rate. Surface tensions were not markedly reduced with naphthalene culturing.

Keywords: PAHs; Yeast; Cell surface hydrophobicity; Emulsification activity; Surface tension

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria by Bryan A. Stubblefield; Kristen E. Howery; Bianca N. Islam; Ariel J. Santiago; Wendy E. Cardenas; Eric S. Gilbert (pp. 1941-1946).

Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.

Keywords: Biofilm; Multispecies biofilm; Biocatalysis; Environmental microbiology; Microbial ecology

Characterization and validation of a chemiluminescent assay based on 1,2-dioxetanes for rapid detection of viable Escherichia coli by Annette S. Bukh; Peter Roslev (pp. 1947-1957).

[(4-methoxy-4(3-β-d-galactose-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0^2,7]tridec-2,7-ene] (“sβ-Gal 102”) and sodium [4-methoxy-4(3-β-d-glucuronic acid-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0^2,7]tridec-2,7-ene] (“sβ-Glucor 102”) are carbohydrate-containing 1,2-dioxetane compounds that produce chemiluminescence upon enzymatic hydrolysis by β-d-galactosidase, and β-d-glucuronidase, respectively. In this study, we have characterized and validated a sensitive detection principle for viable Escherichia coli based on enzymatic cleavage of sβ-Gal 102 and sβ-Glucor 102 (“ColiLight II”). The proposed chemiluminescent assay was optimized with respect to analytical requirements including incubation time, temperature, pH, enzyme induction, and cell permeabilization. The sensitivity and specificity rates of the assay were tested on ten different bacterial genera. The assay was found to be representative based on low coefficients of variations for both accuracy and precision. The analysis time was less than 1 h and the analytical detection limit was 10² to 10³ E. coli cells. In combination with membrane filtration and a brief resuscitation step of 4 h, the proposed assay was capable of detecting low concentrations of stressed E. coli in potable water (<30 CFU 100 ml⁻¹). The proposed chemiluminescent enzyme assay may be used for assessing the metabolic activity of E. coli in oligotrophic environments and for early warning detection of low concentrations of E. coli in water for human consumption.

Keywords: 1,2-dioxetanes; Drinking water; Early warning; β-d-galactosidase; β-d-glucuronidase; Indicator organisms

An improved transconjugation protocol for Bacillus megaterium facilitating a direct genetic knockout by Janine Richhardt; Michael Larsen; Friedhelm Meinhardt (pp. 1959-1965).

We provide a simple but very efficient transconjugation protocol for Bacillus megaterium. By combining utile attributes of known transconjugation methods (small size of the transferred DNA, close physical contact between donor and recipient cells, and heat treatment of the latter) and by determining the appropriate donor/recipient ratio, mating approaches yielded 5 × 10⁻⁵ transconjugants/recipient. Counter-selection for eliminating Escherichia coli donor cells from the mating mixture was possible by pasteurization in case a wild type sporulation proficient B. megaterium served as the mating partner. For nonsporulating mutants, the sacB gene from Bacillus subtilis coding for levansucrase was successfully employed to select against the E. coli donor. The transfer efficiency, up to 15,000 transconjugants acquirable in a single experiment, sufficed—for the first time in this species—to directly select a gene (uvrA) knockout in a one-step procedure. By making use of a mobilizable B. megaterium suicide vector, ten out of the 40 sampled putative transconjugants displayed the expected UV sensitivity and were found to harbor the suicide vector at the anticipated position. Along with the soon available information arising from current B. megaterium sequencing projects, the possibility to quickly inactivate genetic loci will considerably speed up genetic work with this biotechnologically relevant species.

Keywords: Bacillus megaterium ; Transconjugation; sacB counter-selection; Suicide vector; Direct gene knockout

Effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors by Xin-Hua Wang; Li-Hong Gai; Xue-Fei Sun; Hui-Jun Xie; Ming-Ming Gao; Shu-Guang Wang (pp. 1967-1975).

Copper (Cu(II)) and nickel (Ni(II)) are often encountered in wastewaters. This study investigated the individual toxic effects of long-term addition of Cu(II) and Ni(II) on the biochemical properties of aerobic granules in sequencing batch reactors (SBRs). The biochemical properties of aerobic granules were characterized by extracellular polymeric substances (EPS) content, dehydrogenase activity, microbial community biodiversity, and SBR performance. One SBR was used as a control system, while another two received respective concentration of Cu(II) and Ni(II) equal to 5 mg/L initially and increased to 15 mg/L on day 27. Results showed that the addition of Cu(II) drastically reduced the biomass concentration, bioactivity, and biodiversity of aerobic granules, and certainly deteriorated the treatment performance. The toxic effect of Ni(II) on the biodiversity of aerobic granules was milder and the aerobic granular system elevated the level of Ni(II) toxicity tolerance. Even at a concentration of 15 mg/L, Ni(II) still stimulated the biomass yield and bioactivity of aerobic granules to some extent. The elevated tolerance seemed to be owed to the concentration gradient developed within granules, increased biomass concentration, and promoted EPS production in aerobic granular systems.

Keywords: Aerobic granules; Cu(II); Ni(II); Toxic effects; Biochemical properties

Integrated photocatalytic-biological reactor for accelerated phenol mineralization by Yongming Zhang; Lei Wang; Bruce E. Rittmann (pp. 1977-1985).

An integrated photocatalytic-biological reactor (IPBR) was developed for accelerated phenol degradation and mineralization. In the IPBR, photodegradation and biodegradation occurred simultaneously, but in two separated zones: a piece of mat-glass plate coated with TiO₂ film and illuminated by UV light was connected by internal circulation to a honeycomb ceramic that was the biofilm carrier for biodegradation. This arrangement was designed to give intimate coupling of photocatalysis and biodegradation. Phenol degradation was investigated by following three protocols: photocatlysis with TiO₂ film under ultraviolet light, but no biofilm (photodegradation); biofilm biodegradation with no UV light (biodegradation); and simultaneous photodegradation and biodegradation (intimately coupled photobiodegradation). Photodegradation alone could partly degrade phenol, but was not able to achieve significant mineralization, even with an HRT of 10 h. Biodegradation alone could completely degrade phenol, but it did not mineralize the COD by more than 74%. Photobiodegradation allowed continuous rapid degradation of phenol, but it also led to more complete mineralization of phenol (up to 92%) than the other protocols. The results demonstrate that intimate coupling was achieved by protecting the biofilm from UV and free-radical inhibition. With phenol as the target compound, the main advantage of intimate coupling in the IPBR was increased mineralization, presumably because photocatalysis made soluble microbial products more rapidly biodegradable.

Keywords: Biofilm; Photocatalysis; Bioreactor; Wastewater treatment; Phenol

Strains of internal biofilm in aerobic granular membrane bioreactors by Yu-Chuan Juang; Sunil S. Adav; Duu-Jong Lee (pp. 1987-1993).

This study isolated strains in suspended liquor, the surface fouling layer, and biofilm inside hollow-fiber membranes of a membrane bioreactor (MBR); analyzed their distributions, sizes, surface charges, and growth behaviors; and determined the quantities of extracellular polymeric substances (EPS) secreted by these strains under different organic loadings. Three strains, which may penetrate the microfiltration membranes, were close relatives of the Ralstonia mannitolilytica strain SDV (GenBank Accession No. GU451066), Arthrobacter sp. BJQ-2 (GenBank Accession No. GU451067), and Actinobacterium DS3 (GenBank Accession No. GU451068). Among these three strains, only Arthrobacter sp. developed an internal biofilm. The relatively short length of Arthrobacter sp. minimizes resistance to cells moving through the membrane matrix, thereby enhancing its ability to build a biofilm in the interior surface of membranes.

Keywords: Membrane bioreactor; Internal biofilm; Strains; Arthrobacter

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Applied Microbiology and Biotechnology (v.86, #6)

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Applied Microbiology and Biotechnology (v.86, #6)