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Applied Microbiology and Biotechnology (v.81, #4)

The dual role of bacteriocins as anti- and probiotics by O. Gillor; A. Etzion; M. A. Riley (pp. 591-606).

Bacteria employed in probiotic applications help to maintain or restore a host’s natural microbial floral. The ability of probiotic bacteria to successfully outcompete undesired species is often due to, or enhanced by, the production of potent antimicrobial toxins. The most commonly encountered of these are bacteriocins, a large and functionally diverse family of antimicrobials found in all major lineages of Bacteria. Recent studies reveal that these proteinaceous toxins play a critical role in mediating competitive dynamics between bacterial strains and closely related species. The potential use of bacteriocin-producing strains as probiotic and bioprotective agents has recently received increased attention. This review will report on recent efforts involving the use of such strains, with a particular focus on emerging probiotic therapies for humans, livestock, and aquaculture.

Keywords: Bacteriocin; Probiotic; Oral cavity; Gastrointestinal tract; Vagina; Livestock

Ionic liquids in biotechnology: applications and perspectives for biotransformations by Christoph Roosen; Pia Müller; Lasse Greiner (pp. 607-614).

Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature.

Keywords: Ionic liquids; Biocatalysis; Biphasic biocatalysis; Non-conventional media; Biotransformation

Strategies for PHA production by mixed cultures and renewable waste materials by Luisa S. Serafim; Paulo C. Lemos; Maria G. E. Albuquerque; Maria A. M. Reis (pp. 615-628).

Production of polyhydroxyalkanoates (PHA) by mixed cultures has been widely studied in the last decade. Storage of PHA by mixed microbial cultures occurs under transient conditions of carbon or oxygen availability, known respectively as aerobic dynamic feeding and anaerobic/aerobic process. In these processes, PHA-accumulating organisms, which are quite diverse in terms of phenotype, are selected by the dynamic operating conditions imposed to the reactor. The stability of these processes during long-time operation and the similarity of the polymer physical/chemical properties to the one produced by pure cultures were demonstrated. This process could be implemented at industrial scale, providing that some technological aspects are solved. This review summarizes the relevant research carried out with mixed cultures for PHA production, with main focus on the use of wastes or industrial surplus as feedstocks. Basic concepts, regarding the metabolism and microbiology, and technological approaches, with emphasis on the kind of feedstock and reactor operating conditions for culture selection and PHA accumulation, are described. Challenges for the process optimization are also discussed.

Keywords: PHA; Mixed cultures; Aerobic dynamic feeding (ADF); Anaerobic/aerobic (AN/AE); Renewable waste resources

Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans by Yanqun Li; Mark Horsman; Bei Wang; Nan Wu; Christopher Q. Lan (pp. 629-636).

Microalgal lipids are the oils of future for sustainable biodiesel production. However, relatively high production costs due to low lipid productivity have been one of the major obstacles impeding their commercial production. We studied the effects of nitrogen sources and their concentrations on cell growth and lipid accumulation of Neochloris oleoabundans, one of the most promising oil-rich microalgal species. While the highest lipid cell content of 0.40 g/g was obtained at the lowest sodium nitrate concentration (3 mM), a remarkable lipid productivity of 0.133 g l⁻¹ day⁻¹ was achieved at 5 mM with a lipid cell content of 0.34 g/g and a biomass productivity of 0.40 g l⁻¹ day⁻¹. The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g/l and a biomass productivity of 0.63 g l⁻¹ day⁻¹. It was observed that cell growth continued after the exhaustion of external nitrogen pool, hypothetically supported by the consumption of intracellular nitrogen pools such as chlorophyll molecules. The relationship among nitrate depletion, cell growth, lipid cell content, and cell chlorophyll content are discussed.

Keywords: Lipid; Microalgal oils; Microalgae; Biofuel; Biodiesel; Nitrogen-starvation

Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production by Shinji Hama; Sriappareddy Tamalampudi; Yuya Suzuki; Ayumi Yoshida; Hideki Fukuda; Akihiko Kondo (pp. 637-645).

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

Keywords: Whole-cell biocatalyst; Biomass support particles; Filamentous fungi; Fusarium heterosporum lipase; Methanolysis

Conversion of puerarin into its 7-O-glycoside derivatives by Microbacterium oxydans (CGMCC 1788) to improve its water solubility and pharmacokinetic properties by Jie-rong Jiang; Sheng Yuan; Juan-fang Ding; Shou-chuang Zhu; Hai-dong Xu; Ting Chen; Xiao-dong Cong; Wen-ping Xu; Hui Ye; Yi-jun Dai (pp. 647-657).

Microbacterium oxydans strain NJ 6 isolated from soil samples converted puerarin into two novel compounds, puerarin-7-O-glucoside and puerarin-7-O-isomaltoside, via an unreported O-glycosylation of the phenolic hydroxyl group at the 7-position of puerarin. Sucrose, maltotriose, and maltose could be used as glucosyl donors for glycosylation of puerarin, but uridine-diphosphate glucose, glucose, fructose, lactose, cyclodextrin, and starch could not. Regardless of the position of B-ring in the (iso)flavonoids core structure, the glycosylation of the phenolic hydroxyl group at the 7-position of (iso)flavonoids was governed by the presence or absence of a glucosyl residue at 8-C. The apparent solubility of puerarin-7-O-glucoside and puerarin-7-O-isomaltoside was approximately 18 and 100 times that of natural puerarin, respectively. Like parent puerarin, puerarin-7-O-glucoside maintained its physiological ability to relax the contractions of isolated rat thoracic aortic rings in vitro induced by phenylephrine. However, puerarin-7-O-glucoside was able to maintain higher plasma concentrations and have a longer mean residence time in the blood than the parent puerarin.

Keywords: Puerarin; Glycosylation; Microbacterium oxydans ; Water solubility; Vasorelaxing effect

Renewable sustainable biocatalyzed electricity production in a photosynthetic algal microbial fuel cell (PAMFC) by David P. B. T. B. Strik; Hilde Terlouw; Hubertus V. M. Hamelers; Cees J. N. Buisman (pp. 659-668).

Electricity production via solar energy capturing by living higher plants and microalgae in combination with microbial fuel cells are attractive because these systems promise to generate useful energy in a renewable, sustainable, and efficient manner. This study describes the proof of principle of a photosynthetic algal microbial fuel cell (PAMFC) based on naturally selected algae and electrochemically active microorganisms in an open system and without addition of instable or toxic mediators. The developed solar-powered PAMFC produced continuously over 100 days renewable biocatalyzed electricity. The sustainable performance of the PAMFC resulted in a maximum current density of 539 mA/m² projected anode surface area and a maximum power production of 110 mW/m² surface area photobioreactor. The energy recovery of the PAMFC can be increased by optimization of the photobioreactor, by reducing the competition from non-electrochemically active microorganisms, by increasing the electrode surface and establishment of a further-enriched biofilm. Since the objective is to produce net renewable energy with algae, future research should also focus on the development of low energy input PAMFCs. This is because current algae production systems have energy inputs similar to the energy present in the outcoming valuable products.

Keywords: Alga; Bioenergy; Electricity; Microbial fuel cell; Photobioreator; Renewable

Marmorin, a new ribosome inactivating protein with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from the mushroom Hypsizigus marmoreus by Jack H. Wong; H. X. Wang; T. B. Ng (pp. 669-674).

Ribosome inactivating proteins (RIPs) are enzymes that inactivate ribosomes by eliminating one or more adenosine residues from rRNA, a 9,567-Da RIP with a novel N-terminal sequence was isolated from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The protein was unadsorbed on DEAE–cellulose, adsorbed on Affi-gel blue gel, and appeared as a single peak upon gel filtration on Superdex 75. The protein, designated as marmorin, inhibited proliferation of hepatoma Hep G2 cells and breast cancer MCF-7 cells, HIV-1 reverse transcriptase activity, and translation in the rabbit reticulocyte lysate system with an IC₅₀ of 0.15 μM, 5 μM, 30 μM, and 0.7 nM, respectively. Compared to RIPs from hairy gourd, bitter gourd, ridge gourd, garden pea, and the mushroom Flammulina velutipes, marmorin was more potent in its antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF-7) cells, similar in inhibitory potency toward HIV-1 reverse transcriptase (with the exception that it was more potent than ridge gourd RIP and bitter gourd RIP), and less potent in translation-inhibitory potency. Marmorin was devoid of antifungal, protease, RNase, mitogenic, anti-mitogenic, nitric oxide-inducing, hemagglutinating, and trypsin inhibitory activities.

Keywords: Ribosome inactivating; Mushroom; Hypsizigus marmoreus ; Antiproliferative

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation by M. Téllez-Téllez; F. J. Fernández; A. M. Montiel-González; C. Sánchez; G. Díaz-Godínez (pp. 675-679).

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.

Keywords: Enzymes; Fermentation; Laccases; Pleurotus

Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli by Hikaru Nakazawa; Katsunori Okada; Ryota Kobayashi; Tetsuya Kubota; Tomoko Onodera; Nobuhiro Ochiai; Naoki Omata; Wataru Ogasawara; Hirofumi Okada; Yasushi Morikawa (pp. 681-689).

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.

Keywords: Endoglucanase; Trichoderma reesei ; Heterologous expression; Low temperature; Rosetta-gami B (DE3) pLacI

Simultaneous utilization of d-cellobiose, d-glucose, and d-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions by Miho Sasaki; Toru Jojima; Masayuki Inui; Hideaki Yukawa (pp. 691-699).

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF 317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF 317A–bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.

Keywords: Corynebacterium glutamicum ; Genomic integration; d-Xylose; d-Cellobiose; Simultaneous utilization

The absence of effect of gene copy number and mRNA level on the amount of mAb secretion from mammalian cells by Hannes Reisinger; Willibald Steinfellner; Beate Stern; Hermann Katinger; Renate Kunert (pp. 701-710).

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

Keywords: CHO cells; GCN; Monoclonal antibody; qPCR; Protein-free transfection; Signal peptide

Construction of an Aspergillus oryzae cell-surface display system using a putative GPI-anchored protein by Takashi Adachi; Junji Ito; Kouji Kawata; Masahiro Kaya; Hiroki Ishida; Hiroshi Sahara; Yoji Hata; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 711-719).

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.

Keywords: Aspergillus oryzae ; Cell-surface display; GPI-anchored protein

Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones by Rainer Wardenga; Frank Hollmann; Oliver Thum; Uwe Bornscheuer (pp. 721-729).

Efficient recombinant expression of N-acyl-l-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL–GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.

Keywords: N-acyl-l-aminoacylase; Synthetic gene; Codon usage; Soluble expression; Inclusion body; Chaperones; StrepTag

Identifying Escherichia coli genes involved in intrinsic multidrug resistance by Miao Duo; Shuyu Hou; Dacheng Ren (pp. 731-741).

Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.

Keywords: Escherichia coli ; Multidrug resistance; Transposon mutagenesis

Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae by Z. Lewis Liu; Jaewoong Moon; Brad J. Andersh; Patricia J. Slininger; Scott Weber (pp. 743-753).

Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors generated from biomass pretreatment using dilute acid hydrolysis that interfere with yeast growth and subsequent fermentation. Few yeast strains tolerant to inhibitors are available. In this study, we report a tolerant strain, Saccharomyces cerevisiae NRRL Y-50049, which has enhanced biotransformation ability to convert furfural to furan methanol (FM), HMF to furan di-methanol (FDM), and produce a normal yield of ethanol. Our recent identification of HMF and development of protocol to synthesize the HMF metabolic conversion product FDM allowed studies on fermentation metabolic kinetics in the presence of HMF and furfural. Individual gene-encoding enzymes possessing aldehyde reduction activities demonstrated cofactor preference for NADH or NADPH. However, protein extract from whole yeast cells showed equally strong aldehyde reduction activities coupled with either cofactor. Deletion of a single candidate gene did not affect yeast growth in the presence of the inhibitors. Our results suggest that detoxification of furfural and HMF by the ethanologenic yeast S. cerevisiae strain Y-50049 likely involves multiple gene mediated NAD(P)H-dependent aldehyde reduction. Conversion pathways of furfural and HMF relevant to glycolysis and ethanol production were refined based on our findings in this study.

Keywords: Biomass conversion inhibitor; Biotransformation; Metabolic pathways; Stress tolerance; Yeast

Isolation and structural characterisation of two antibacterial free fatty acids from the marine diatom, Phaeodactylum tricornutum by Andrew P. Desbois; Tomas Lebl; Liming Yan; Valerie J. Smith (pp. 755-764).

One solution to the global crisis of antibiotic resistance is the discovery of novel antimicrobial compounds for clinical application. Marine organisms are an attractive and, as yet, relatively untapped resource of new natural products. Cell extracts from the marine diatom, Phaeodactylum tricornutum, have antibacterial activity and the fatty acid, eicosapentaenoic acid (EPA), has been identified as one compound responsible for this activity. During the isolation of EPA, it became apparent that the extracts contained further antibacterial compounds. The present study was undertaken to isolate these additional antibacterial factors using silica column chromatography and reverse-phase high-performance liquid chromatography. Two antibacterial fractions, each containing a pure compound, were isolated and their chemical structures were investigated by mass spectrometry and nuclear magnetic resonance spectroscopy. The antibacterial compounds were identified as the monounsaturated fatty acid (9Z)-hexadecenoic acid (palmitoleic acid; C16:1 n-7) and the relatively unusual polyunsaturated fatty acid (6Z, 9Z, 12Z)-hexadecatrienoic acid (HTA; C16:3 n-4). Both are active against Gram-positive bacteria with HTA further inhibitory to the growth of the Gram-negative marine pathogen, Listonella anguillarum. Palmitoleic acid is active at micro-molar concentrations, kills bacteria rapidly, and is highly active against multidrug-resistant Staphylococcus aureus. These free fatty acids warrant further investigation as a new potential therapy for drug-resistant infections.

Keywords: Antimicrobial; (6Z ; 9Z ; 12Z)-hexadecatrienoic acid; Palmitoleic acid; Micro-alga; Phaeodactylum tricornutum ; MRSA.

Biological breakdown of denitrifying sulfide removal process in high-rate expanded granular bed reactor by Chuan Chen; Aijie Wang; Nanqi Ren; Hongjing Kan; Duu-Jong Lee (pp. 765-770).

This work conducted a denitrifying sulfide removal (DSR) test in an expanded granular sludge bed (EGSB) reactor at sustainable loadings of 6.09 kg m⁻³ day⁻¹ for sulfide, 3.11 kg m⁻³ day⁻¹ for nitrate–nitrogen, and 3.27 kg m⁻¹ day⁻¹ for acetate–carbon with >93% efficiency, which is significantly higher than those reported in literature. Strains Pseudomonas sp., Nitrincola sp., and Azoarcus sp. very likely yield heterotrophs. Strains Thermothrix sp. and Sulfurovum sp. are the autotrophs required for the proposed high-rate EGSB-DSR system. The EGSB-DSR reactor experienced two biological breakdowns, one at loadings of 4.87, 2.13, and 1.82 kg m⁻³ day⁻¹; reactor function was restored by increasing nitrate and acetate loadings. Another breakdown occurred at loadings of up to 8.00, 4.08, and 4.50 kg m⁻¹ day⁻¹; the heterotrophic denitrification pathway declined faster than the autotrophic pathway. The mechanism of DSR breakdown is as follows. High sulfide concentration inhibits heterotrophic denitrifiers, and the system therefore accumulates nitrite. Autotrophic denitrifiers are then inhibited by the accumulated nitrite, thereby leading to breakdown of the DSR process.

Keywords: Denitrifying sulfide removal; EGSB; Microbial community; Breakdown

Successions of bacterial community in composting cow dung wastes with or without hyperthermophilic pre-treatment by Takeshi Yamada; Atsushi Suzuki; Hideyo Ueda; Yasuichi Ueda; Keisuke Miyauchi; Ginro Endo (pp. 771-781).

Comparative analyses of bacterial community successions in the composting materials were done for a conventional windrow post-treatment (WPOT) process with the hyperthermophilic pre-treatment (HTPRT) and simple windrow composting (SWC; without the HTPRT). Multidimensional scaling profiles based on data of terminal restriction fragment length polymorphisms of the bacterial population in the samples of every 7 days composting material and analyses of the 16S rRNA gene-based clone library of the 7 and 21 days composting materials suggested that bacterial communities of the composting materials differed much between these two processes until the 35 days of composting, whereas that they were closely related to each other at the final composting stage (42 days of composting). Detailed phylogenetic analysis clarified that all WPOT clone libraries contained many clones of the lineages of aerobic bacteria (for example, bacilli). However, the most abundant clones retrieved from all SWC materials were affiliated with a clone cluster closely related to identified and classified members of the phylum Firmicutes that have strictly anaerobic metabolism pathways. From these results, we conclude that the HTPRT process contributed to easily establish an aerobic ecosystem from the early stage to the final stage of WPOT composting with plowing the materials only once a week.

Keywords: Composting; Cow dung waste; Hyperthermophilic pre-treatment; Terminal restriction fragment length polymorphisms; Multidimensional scaling analysis; 16S rRNA gene-based clone library

Synergistic effect of laccase mediators on pentachlorophenol removal by Ganoderma lucidum laccase by Jong-Rok Jeon; Kumarasamy Murugesan; Young-Mo Kim; Eun-Ju Kim; Yoon-Seok Chang (pp. 783-790).

Laccases have low redox potentials limiting their environmental and industrial applications. The use of laccase mediators has proven to be an effective approach for overcoming the low redox potentials. However, knowledge about the role played by the mediator cocktails in such a laccase-mediator system (LMS) is scarce. Here, we assembled different dual-agent mediator cocktails containing 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), vanillin, and/or acetovanillone, and compared their mediating capabilities with those of each individual mediator alone in oxidation of pentachlorophenol (PCP) by Ganoderma lucidum laccase. Cocktails containing ABTS and either vanillin or acetovanillone strongly promoted PCP removal compared to the use of each mediator alone. The removal enhancement was correlated with mediator molar ratios of the cocktails and incubation times. Analysis of the kinetic constants for each mediator compound showed that G. lucidum laccase was very prone to react with ABTS rather than vanillin and acetovanillone in the cocktails. Moreover, the presence of the ABTS radical (ABTS^+•) and vanillin or acetovanillone significantly enhanced PCP removal concomitant with electron transfer from vanillin or acetovanillone to ABTS^+•. These results strongly suggest that vanillin and acetovanillone mediate the reaction between ABTS and PCP via multiple sequential electron transfers among laccase and its mediators.

Keywords: Laccase-mediator system; Ganoderma lucidum laccase; Pentachlorophenol

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Applied Microbiology and Biotechnology (v.81, #4)

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Applied Microbiology and Biotechnology (v.81, #4)