press edited by a. méndez-vilas walker · no part of this book may be reproduced or transmitted in...

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Microbes in the Spotlight:

Recent Progress in the Understanding of Beneficial and Harmful Microorganisms

Edited by A. Méndez-Vilas

BrownWalker Press

Boca Raton

Brown

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Microbes in the Spotlight: Recent Progress in the Understanding of Beneficial and Harmful Microorganisms

Copyright © 2016 Formatex Research Center All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval

system, without written permission from the publisher.

BrownWalker Press Boca Raton, Florida

USA • 2016

ISBN-10: 1-62734-612-0 ISBN-13: 978-1-62734-612-2

www.brownwalker.com

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*The publisher hereby retracts this chapter due to the fact that it was published without the consent of all the authors I

TABLE OF CONTENTS

Introduction IX

Agricultural and environmental microbiology. Biodeterioration, biodegratation, bioremediation

A comparative microbiological assessment of the Isipingo River and Palmiet River in Kwa-Zulu Natal province to elucidate health risks A. Sithebe, G. Singh, I. D. Amoah and T. A. Stenstrom

2

Aerobic degradation of lindane in the presence of bacteria and zero valent iron nanoparticles A.V Menéndez, L.R Osuna, V. Mesa, J.L.R Gallego, H. Sastre and A.I Peláez 7

Altruistic model of N2-fixing microbe-plant symbiosis: evolutionary and agronomic aspects N. Provorov, O. Onishchuk, S. Yurgel, O. Kurchak, E. Chizhevskaya and N. Vorobyov 13

Analysis by PCR / DGGE of bacterial diversity of Oryza sativa, cultivated in southern Brazil and their interaction with pesticides C. Reali and L.M. Fiuza

18

Assessment of diesel and biodiesel multiple degrading potential of fungal isolates from Caatinga soil and mangrove sediments (PE, Brazil) A.B. Souza-Junior, P.N. Santos, A.C.R. Carvalho, H.F. Nobrega, A.A. Antunes, P.R.B. Filizola, M.A. Pele, P.C.V.S. Maia, M.A.C. Luna, C.D.C. Albuquerque and G.M. Campos-Takaki

23

Bacterial Biosensors for Diagnostic Determination of Hydrocarbon in Refined Oil Product’s Contaminated Water Samples Ibrahim Isiaka Hussein, Abdulrasheed Mansur, Moshood Alhaji Yusuf

27

Bacterial diversity and metal reducing bacteria in Australian thermal environments A. C. Greene, M. H. Wright and H. A. Aldosary 32

Bacteriological and Physico-chemical qualities of borehole water in Imo State, Nigeria J.N. Dike-Ndudim, D.O. Dike, I. Ukogo, R.C. Egbuobi, H.C. Nwaigwe and M.O.E. Iwuala 37

Biodegradation of biodiesel and diesel blends J. M. Cruz, R. N. Montagnolli, G. M. Quitério, E. M. T. Claro and E. D. Bidoia 42

*Biodegradation of Bisphenol A by native and modified by linear-dendritic copolymers Laccase from Trametes versicolor M. Brazkova, M. Gerginova, I. Gitsov and A. Krastanov

45

Biodeterioration of wooden churches from Romania. Case studies: The church from Amărăşti, Vâlcea County I. Gomoiu, M.I. Enache, R. Cojoc, I. Mohanu and D. Mohanu

51

Biological activity of essential oils in the mycelial growth of the entompathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Isaria sp. M. Mireles-Martínez, J.E. Navarro-Sáenz, J.M. Villegas-Mendoza, A.D. Paz-González, G. Rivera and N.M. Rosas-García

56

Changes of bacterial community in soil contaminated with lubricant oil after bioremediation by PCR-DGGE P.R.M. Lopes, R.N. Montagnolli, J.M. Cruz, T. Gumiere, A. Durrer, P.A.M. Andrade, F.D. Andreote and E. D. Bidoia

60

Degradation of carbon steel AISI 1020 coupons immersed in blends containing 90% Diesel B6 and 10% Seawater, and 90% Diesel B30 and 10% Seawater T. C. da S. Cruz, E. de J. Argolo, S. H. de Oliveira, R. G. C. da Silva, V. B. de Queiroz, M. A. G. A. Lima, S. L. Urtiga Filho, F. P. de França

65

Diesel degradation by Aspergillus sp. D.D. Lira, P.A. Silva, L.N.A. Neto, M.A. Barbosa, E.B.S. Nogueira, M.J.S. Fernandes, C.M.S. Motta and N.B. Gusmão

70

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Effect of salinity on diesel biodegradation by Candida lipolytica UCP 0988 H.F. Nobrega, F. Seger, A.M.A.B. Correia, M.L.O.M.F. Henriques, G.M. Campos-Takaki and C.D.C. Albuquerque

75

Endophytic bacteria isolated from two varieties of Oryza sativa crops in southern Brazil Lidia Mariana Fiuza, Taís Vargas Garcia, Diouneia Lisiane Berlitz and Neiva Knaak

80

Hydrocarbon biodegradation in coastal environments: Bacterial characterization and biostimulation treatments L.R. Osuna, C. Méndez-García, V. Mesa, A.V. Menéndez, A.I. Peláez and J.R. Gallego

86

Identification of multi-drug resistant bacteria isolated from industrial sources J. Campos Guillén, I. Arvizu Hernández, L. Ávalos Esparza, G.H. Jones and S. Romero Gómez

91

Kinetic and thermodynamic models applied to the removal of Acid Blue 161 dye by Saccharomyces cerevisiae from effluents G. Dilarri, H. B. Pecora, E. J. R. Almeida, G. C. Santos, A. A. Menegario and C. R. Corso

96

Laccase activity in black chanterelle mushroom and its importance in forestry Ezzatollah Keyhani

101

Optimization of Extraction of Difenoconazole from Soil for UPLC-MS Analysis M.N. Filimon, V. Ostafe and A. Isvoran

106

Physiological response of bacteria consortium to the presence of intact and autoclaved freshwater sapropel (gyttja) derived in the eastern Latvia K. Stankevica, Z. Vincevica-Gaile, O. Muter and M. Klavins

111

PLFAs as bioindicators of the structural biodiversity of microorganisms in soil under aided phytostabilization D. Wasilkowski, J. Chojniak, G. Płaza and A. Mrozik

116

Quantification of viable Enterococcus spp., and Pseudomonas aeruginosa through propidium monoazide dye assisted qPCR array in surface water and sediments G. Singh, T.A. Stenstrom and R. Shanker

121

Redox mediator evaluation in the azo dye biodegradation G. C. Santos, J. Forss, U. Welander and C. R. Corso

126

Removal of free cyanide present on gold mine wastewater using a rotating biological contactor Pablo Xavier Jumbo Pacheco and Diego Alejandro Nieto Monteros

130

Studies on biodegradation of phenolic compounds by Aspergillus glaucus strain M. Gerginova, K. Litova, J. Manasiev, N. Peneva, R. Bibova, A. Krastanov and Z. Alexieva

135

Towards the selection of the best discriminating parameters of microbiological water quality: a case study of an urban recreational water resource involving a dam complex in Córdoba, Argentina J.V. Pavan, G. Masachessi, C.A. Mateos, P.A. Barril, V.E. Prez, L.C. Martínez, M.O. Giordano, L.J. Ferreyra, M.B Isa, A. Welter, M. Martinez Wassaf, V. Ré and S.V. Nates

140

Treatment of simulated textile effluent with acid red 151 dye by electrolytic process and evaluation of its toxicity with Artemia salina ecotoxicological test E. D. Bidoia and J. R. Moraes Júnior

145

Food microbiology

A novel culture medium for Lactobacillus plantarum based on defatted rice bran L.M. Nunes Pinto, J. Garavaglia, A.P. Cavedon Spohr, A. Neuhaus Ferronatto, C. Nunes, R. Cassanta Rossi, J. de Castilhos, D. de Souza, I.C. Kasper Machado, R.C. de Souza Ramos and D.D. Righetto Ziegler

150

Antimicrobial activity of olive (Olea europaea) leaf extract to be used during the elaboration process of green table olives T. Schaide, D. Martín-Vertedor, A. Hernández, J. Delgado-Adámez, M.G. Córdoba and F. Pérez-Nevado

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Bacterial identification by LC-ESI-IT-MS/MS P. Calo-Mata, K. Böhme, Monica Carrera, S. Caamaño-Antelo, J.M. Gallardo, J Barros-Velázquez and B. Cañas

160

Bacteriocin: Antimycobacteria Activity in Milk and its Molecular Characterization K.E.N. Nwanekwu, R.N. Nwabueze, J.C. Orji and S.U. Oranusi

165

Biochemical and microbiological description of spontaneous wheat sourdough from two Spanish bakeries E. Gordún, R. Carbó and M. Ginovart

170

Biodiversity of the characteristic microbiota in refrigerated, frozen and heat-treated donkey milk S. Ozturkoglu-Budak

175

Biomarker discovery for foodborne pathogen detection by LC-MS/MS K. Böhme, S. Caamaño-Antelo, M. Carrera, J. Gallardo, J. Barros-Velázquez, B. Cañas and P. Calo-Mata

180

Detection of Salmonella and Campylobacter in chicken rinse water using a surface plasmon resonance sensor Fur-Chi Chen, Suping Zhou, Samuel N. Nahashon and Roger C. Bridgman

186

Effect of drying conditions of probiotic microorganism impregnated in an extruded commercial feed of fry of tilapia on viability and stability during storage Jorge Iván Quintero-Saavedra and Ayda Rodriguez de Stouvenel

191

Effect of postharvest application of Metschnikowia pulcherrima L672 as biocontrol agent on `Ambrunés´ sweet cherries (Prunus avium L.). E. de Paiva, S. Ruiz-Moyano, M.C. Villalobos, M.J. Serradilla, M.G. Córdoba, F. Pérez-Nevado and A. Hernández

196

Horizontal gene transfer and the evolving definition of coliforms Felipe Molina, Alfredo Simancas, Rafael Tabla, Isidro Roa and José Emilio Rebollo

200

Influence of starter culture and ripening temperature on survival of L. monocytogenes in traditional Portuguese dry-fermented sausages V. Cadavez, A. P. Pereira, U. Gonzales-Barron and T. Dias

205

Inhibition of Rhizopus stolonifer spot-inoculated in tomato fruit by a mixture of crustacean chitosan and lemongrass essential oil A. J. A. A. Athayde, L. R. R. Berger, I. C. D. Guerra, P. D. L. de Oliveira, K. A. R., Oliveira, T. C. Montenegro Stamford and E. L. de Souza

209

Occurrence of mycotoxin-producing fungi in conventional and organic corn in Spain E.M. Mateo, J.V. Gimeno–Adelantado, M.A. García-Esparza, D. Romera, R. Mateo-Castro and M. Jiménez

214

Potentiation of functional and antimicrobial activities through synergistic growth of probiotic Pediococcus acidilactici with natural prebiotics (garlic, basil) Sharmistha Samanta Koruri, Ranjana Chowdhury and Pinaki Bhattacharya

219

Ribotyping the Bacterial Community in Fresh Fruit Juices M. Albokari, M. Alshehri, I. Mashhour and A. Alsalman

225

Strategies for the control of Fusarium langsethiae, an emerging risk as a source of T-2 and HT-2 toxins in cereals E.M. Mateo, J.V. Gimeno-Adelantado, M.A. García-Esparza, D. Romera, R. Mateo-Castro and M. Jiménez

229

yaiO, a new target for highly specific detection of Escherichia coli by PCR amplification Alfredo Simancas, Felipe Molina, Rafael Tabla, Isidro Roa and José Emilio Rebollo

234

Medical microbiology. Antimicrobial agents and chemotherapy. Antimicrobial resistance

Antibiofilm activity of major compounds of essential oils against Salmonella enteritidis J.A.A. Nascimento Junior, B.S. Santos, C.B. Malafaia, T.D. Silva, R.A de Paula, M.S Melo, N.G.P Maciel , I.B.S Santos, P.A. Moura, L.C.A. Araújo, M.B.M. Oliveira, T.H. Napoleão,

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P.M.G. Paiva, M.V. Silva, M.T.S. Correia

Antiphage activity of sera from patients receiving staphylococcal phage preparations Marzanna Łusiak-Szelachowska, Maciej Żaczek, Beata Weber-Dąbrowska, Marlena Kłak, Ryszard Międzybrodzki, Wojciech Fortuna, Paweł Rogóż, Krzysztof Szufnarowski, Ewa Jończyk-Matysiak, Andrzej Górski

245

Bacteria as resistant microorganisms in decomposition of mummified human remains H. Vojtková

250

Biodiversity and antimicrobial activity from endophytic fungi isolated from Morinda citrifolia I.A.T.A. Ribeiro, S.T.S. Veras, A.P.S. Silva, B.S. Santos, C.M.A.S. Bessa, P.F.F. Mercês, A.D.A. Uchôa, N.M. Santos, M.V. Silva, M.T.S. Correia, V.L.M. Lima and M.S. Cavalcanti

255

Biological activities of methanolic extract of Buchenavia tetraphylla J.R.N. Cavalcanti Filho, K.P.M. Silva, L.M. Cruz, T.F. Silva, B.S. Santos, F.V.F. Arruda, M.C.V. Vicalvi-Costa, N.B. Gusmão, M.V. Silva, L.C. N. Silva and M.T. S. Correia

260

Endophytic Nocardiopsis dassonvillei and Amycolatopsis orientalis isolated from Brazilian tropical savannah presented antibiosis against pathogens A.C.M.T. Piza, L.C.P.S. Lima, C.O. Hokka, and C.P. de Sousa

264

Genetic characterization of antibiotic resistance in Enterococcus spp. from ready-to-eat salads W. Chajęcka-Wierzchowska, A. Zadernowska, Ł. Łaniewska-Trokenheim

267

In vitro antibacterial activity of ethanolic extracts of Mentha spicata L. and Artemisia campestris from Algeria A. Houicher, E. Kuley, B. Bendeddouche and F. Özogul

272

In vitro study of Antimicrobial and Antioxidant Activities of Essential oils from the leaf of three Khaya species B.O. Opawale, A.K. Onifade and A.O. Ogundare

277

Introducing Clinical Microbiology to secondary school science teachers in Catalonia Javier Méndez and Josep M. Fernández-Novell

282

Investigation of Trichomonas vaginalis infection among women of child bearing age in Owerri, Imo State, Nigeria Egbuobi, R.C.; Dike-Ndudim, J.N.; Uduji, H.I.; Ukogo, I.; Okorie, H.I.; Ogamaka, I.A.; Nwakwe, M.E. and Opara, A.U.

287

Microbial and crustacean Chitosan: Isolation, characterization and antimicrobial activity J.C. Vilar Junior, P.C.V. Souza Maia, V. Pimentel Santos, C.F.B. Costa Filho, G.M. Souza Lima, A. Elesbão do Nascimento and G.M. de Campos-Takaki

290

Nanostructural Silver Particles in Aqueous and Organic Dispersions and their Microbiological Activity K.Z. Gumargalieva, I.G. Kalinina, V.P. Gerasimenya, S.V. Zakharov, M.A. Klykov and A.L. Schwartz

295

Pore forming drugs: antimicrobial mechanism and clinical applications D. A. Aliverdieva, M.H. Efendieva and D. V. Mamaev

302

Postoperative infections associated with semiautomatic retractors O'Sullivan O' Connors, Balfour and Soriano Ángel R. Soriano-Sánchez, Armando Ahued-Ortega and Eleuterio Ortiz-Cruz

307

Industrial microbiology. Microbial production of high-value products

A low-cost solid fermentation medium for potential prodigiosin production by Serratia marcescens UCP/WFCC 1549 D. Montero-Rodríguez, R. F. S. Andrade, D. Rubio-Ribeaux, R. A. Lima, D. L. R. Ribeiro, G. K. B. Silva, H. W. C. Araújo and G. M. Campos-Takaki

312

Antimicrobial activity of silver nanoparticles synthesized by Pseudomonas rhodesiae SA-1 N. Sivakumar and A. Al Zadzali

316

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Biotechnological production of biosurfactant in economic medium by Candida sp. and evaluation of biodegradation potential of petro derivates D. Rubio-Ribeaux, R. F. S. Andrade, D. Montero-Rodríguez, M. A. C. Luna, M. A. Pele, A. Antunes, L. O. Franco, M. A. B. Lima, G. M. Campos-Takaki

321

Chitin, chitosan and biosurfactant production by Cunninghamella phaeospora using agroindustrial wastes T.C. Silva, D.G. Souza, A.P.M. Bione, A.B. Lins, N.S.A.A. Marques, N.S. Rosa-Leão, D.L. Cavalcanti, D.S.P. Silva and G.M. Campos-Takaki

326

High value nutraceutical products from Microalgae Archana Tiwari and Thomas Kiran M

331

Influence of nutritional supplementation on ethanol production by Scheffersomyces shehatae UFMG HM52.2 using sugarcane bagasse hydrolysate K. Dussán, D.D.V. Silva, A.D. Ferreira and S.S. da Silva

334

Low-cost production of biosurfactant by Cunninghamella phaeospora using agro-industrial wastes A. Barbosa Lins, A.P. Melo Bione, T. Cavalcanti Silva, D.G. De Souza and G.M. Campos-Takaki

339

Optimization of the medium components in biosurfactant production by Mucor circinelloides UCP 001 with response surface methodology T.A.L. Silva, N.S.A. Amaral Marques, M.C. Freitas Silva, P.M. Souza, L.O. Truan, V.P. Santos, D.G. Souza, M.C.L.B. Silva, A.B. Souza Júnior, P.C.V.S. Maia, C.F.B. Costa Filho, E.R. Santos, K. Okada and G.M. Campos Takaki

344

Pigment production by Penicillium spp isolated from Caatinga-Brazil soil using different carbon and nitrogen sources A. de Araújo Alencar, C.A. Alves da Silva, J.C. Vilar Junior, A. Elesbão do Nascimento, G.M. de Campos-Takaki

349

Production and optimization of extracellular invertase from a novel fungal strain via solid state fermentation Dina Helmy El-Ghonemy

353

Production of biosurfactant by Cunninghamella phaeospora in submerged fermentation using water soluble substrates A.P. Melo Bione, A. Barbosa Lins, T. Cavalcanti Silva, D. Montero-Rodríguez, D.G. de Souza, P. Rego Barros Filizola, P.C. de Véras Souza Maia, H. Félix Nobrega and G.M. Campos-Takaki

361

Protease production by Cunninghamella echinulata (SIS 40) through submerged fermentation by using factorial design T. C. Souza, T. A. Ostendorf, F. A. P. Cunha Amaral, P. S.C. Sales, G.M. Campos- Takaki and C. A. Alves da Silva

366

Tannase production by Penicillium chrysogenum (SIS 25) through submerged fermentation using alternative media containing agro-industrial residues C.M. R. Moura, B.F. Lima, J.F.Silva, G.K.B. Silva, M.C. Sá Muniz, N.R. Andrade Silva, M.A.B. Correia, D.K.S.T. Maciel, E.C. Vasconcelos, G.M. Campos- Takaki and C. A. Alves da Silva

371

The preparation of constructs for secretome pathway study in yeast Candida utilis V. Lišková, J. Krahulec, H. Boňková, A. Lichvariková and J. Turňa

376

Biotechnologically relevant enzymes and proteins

An Input-Output Linearizing controller for producing Angiogenin from E. coli P. Kathiresan, Jashwant Kumar, Viki Chopda, Sanket Jain, Manidipa Banerjee, Anurag S. Rathore, and James Gomes

379

Application of response surface methodology to optimize purification of recombinant oxidoreductases S. Moein,R. Mahdizadeh, H. Shahbazmohammadi

385

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Biochemical and structural studies of laccase isozymes from Ganoderma lucidum MDU-7 Krishna Kant Sharma, Deepti Singh and Amit Kumar

390

Immobilization of the human enteropeptidase light chain on bead cellulose particles Ján Krahulec, Martin Šafránek, Veronika Palušová, Kristína Jiríčková and Ján Turňa

397

LAGLIDADG homing endonucleases encoded within the chloroplast psbB gene from lichen phycobionts C. Suárez-Lledó, E.M. del Campo, A. del Hoyo, F. Gasulla, L.M. Casano

399

Medium optimization of a dihydrolipohyl dehydrogenase with diaphorase activity from Bacillus spharicus F. Zahedi, H. Shahbazmohammadi

406

Partial characterization of the cellulolytic enzyme produced by filamentous fungi L. Toscano Palomar, G. Montero Alpirez, L. Cervantes Díaz, R. M. Félix Navarro, M.G. Amado Moreno, J. L. Vázquez Méndez

411

Methods and technology development

A comparative study of Staphylococcus aureus adhesion to the fluorine-containing surfaces obtained by various methods of ion-plasma technology L.V. Didenko, V.M. Elinson, N.V. Shevlyagina, G.A. Avtandilov, A.N. Lyamin, L.I. Kravets, G.Dinesku and M.Y. Yablokov

417

Be-CoDiS and Be-FAST: Mathematical models to predict the spread of human and livestock diseases with real data. Application to the 2014-15 Ebola Virus Disease epidemic and livestock diseases A.M. Ramos, B. Ivorra, E. Fernández Carrión, B. Martinez-Lopez, D. Ngom and J.M. Sanchez Vizcaino

422

Bioreactor dynamic modelling and control in a decentralised wastewater treatment plant with spectral online monitoring L. Fernandes, C. Leitão, T. Arriaga, R. Ribeiro, M.C. Almeida, C.I.C. Pinheiro, and H.M. Pinheiro

427

Comparative studies of ion exchange chromatography and yeast application for desalting fibersol-2 Aboubacar Oumar Bangoura, Doussou Lanciné Traoré, Fodé Mohamed Sylla, Qian He, Yao Weirong and Aïssata N’Diaye

432

Comparison of various DNA extraction methods for Fusarium solani detection and enumeration with real-time PCR B. Dalecka and L. Mezule

439

Mass degradation to reduce cytotoxic products as an individual behavior-rule embedded in a microbial model for the study of the denitrification process P. Araujo, A. Gras and M. Ginovart

444

Palm Oil Mill Effluent as Yeast Growth Medium A. Hamzah and H.N. Mokhtardin

449

Production of a new Agar medium for non-fastidious bacteria Sara Shahabi, Zarrintaj Bidar, Nima Hosseini Jazani

454

Microbial physiology

Alterations of ubiquinone system and fatty acids compositions of Rhizopus arrhizus UCP402 and R. arrhizus UCP402x (mutant) mediated by pyrene R.K. Shiosaki, P.H. Silva, K. Morant, E.R. Santos, C.D.C. Albuquerque, K. Okada and G.M. Campos-Takaki

458

Classical rsbU- strains of S. aureus exhibit better growth in synthetic nasal medium Aya J Takemura, Yuri Ushijima, Le Thuy Nguyen Thi and Kazuya Morikawa

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Competence development and horizontal plasmid transfer in natural Escherichia coli strains Akiko Matsumoto, Ayuka Sekoguchi, Yukako Murakami, Junko Imai, Kumiko Kondo, Yuka Shibata, and Sumio Maeda

468

E. coli Dps without its DNA binding activity (Dps18) can compensate for S. aureus MrgA in hydrogen peroxide resistance Yuri Ushijima and Kazuya Morikawa

474

Effect of ionizing radiation on the motility of Escherichia coli Mikio Kato

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IX

Introduction This book contains a selection of papers presented at the VI International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2015), which was held at the beautiful Historic Building of the University of Barcelona (UB), in Barcelona, Spain, during October 28th – 30th 2015. BioMicroWorld (which evokes the concept/image “biological microscopic world”) is a well-established forum that has brought together researchers in the applied microbiology field every two years since 2005. This forum is aimed at communicating current progress in the fields of environmental, industrial, food, or medical microbiology, as well as in microbial biotechnology or technology development. This sixth edition of the BioMicroWorld conference series gathered 511 participants, coming from 66 countries. Some of the research works presented at that conference are discussed in this book within the following topics:

Agricultural and environmental microbiology. Biodeterioration, biodegratation, bioremediation.

Food microbiology.

Medical microbiology. Antimicrobial agents and chemotherapy. Antimicrobial resistance.

Industrial microbiology. Microbial production of high-value products.

Biotechnologically relevant enzymes and proteins.

Methods and technology development.

Microbial physiology. We thank all authors for their contributions to this proceedings book. And we gratefully acknowledge the essential work of the reviewers and the members of the International Advisory Committee for their effort and assistance. We hope readers will find the content of this book interesting, inspiring and useful. A. Méndez-Vilas Editor BioMicroWorld2015 General Coordinator Formatex Research Center C/Zurbarán 1, Planta 2, Oficina 1 06002 Badajoz Spain

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Agricultural and environmental microbiology.

Biodeterioration, biodegratation, bioremediation

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A comparative microbiological assessment of the Isipingo River and Palmiet River in Kwa-Zulu Natal province to elucidate health risks

A. Sithebe*,1 G. Singh*,1 I. D. Amoah*,1 and T. A. Stenstrom1

1Institute for Water and Wastewater Technology, Durban University of Technology, ML Sultan Campus 2PO Box 1334, Durban, 4000 South Africa. *Corresponding author: e-mail: [email protected], Phone: +27 313736704

The microbiological quality of the Palmiet and Isipingo Rivers in KwaZulu-Natal (South Africa) was investigated. The study involved an assessment of historical data collected over the past 66 months on each river based on Escherichia coli (E.coli) values obtained from the Municipality (eThekwini Water and Sanitation Dept). This was followed by microbiological quality assessments in 2015 for surface water and sediments with IDEXX Colilert 18 for coliforms and E.coli quantifications and Enterolert for the enumeration of Enterococci. Physiochemical properties included temperature, pH, conductivity, TDS, salinity and dissolved oxygen. The selected sampling points in both rivers expressed a water quality classified as unsuitable for crop irrigation and recreational use. The observed microbial concentrations in both rivers exceeded the World Health Organization guideline values and standard limits defined by Department of Water Affairs and Forestry (SA). It was further concluded, Isipingo River had an inferior quality than the Palmiet River.

Keywords Water quality; risk assessment; Escherichia coli; Enterococci

1. Introduction

The deteriorating quality of river water is a major threat to South Africa’s ability to provide adequate water of appropriate quality to meet its population’s needs and to ensure a sustainable environment. Thus, a further understanding of the quantitative variability is important for accurate and comprehensive assessment of microbial river water quality which accounts for seasonal and spatial variations. Diarrheal diseases ranked 2nd

after HIV in the global burden of diseases in South Africa with an impact of 4.9% (1 138 000) life years lost per year solely due to diarrhea [1]. Children who are malnourished or have impaired immunity as well as people living with HIV are at risk of life-threatening diarrhoea. Approximately 2.2 million people die each year worldwide due to diarrheal diseases [2]. This is partly due to the lack of access and use of potable water, which has forced many inhabitants of the informal settlements to rely on surface water sources for their daily needs, thus exposing them to waterborne diseases [3]. According to the World Health Organization report, the global disease burden could be decreased by 10% simply by improving people’s access and use of safe drinking water [4]. The Isipingo catchment is located in the South Africa, KwaZulu-Natal province, roughly 20 kilometers south of the Durban Central Business District (CBD). The length is approximately 27 km, originating approximately 16 km in a north-westerly direction from the mouth of the Isipingo Estuary [5]. It is a comparatively small catchment that is characterized by informal settlements, formal developments as well as intensive industrial activities especially in the lower catchment, thus it is greatly influenced by anthropogenic sources. According to a study conducted by the [6] on water pollution, this river and interlinked canals had the highest E.coli and other bacterial concentrations as well as ammonia levels on the Natal coast. Phenolic contaminants, pesticides and metals like lead, zinc, and lithium were also found from industrial and agricultural pollution [5]. The second catchment is a 25 km long catchment that is located in the northerly peripheral of the city of Durban. The Palmiet river was named after the Prionium Serratum known as the palmiet plant. It is also a relatively small catchment area of 500 km2, with 5 irrigation and hydropower dams along its length, and provides water for agricultural and industrial uses [3]. It is also highly influenced by industrial areas as well as informal settlements, as it flows directly through the Quarry road informal settlement (which is a major pollution source and therefore our main focus for the purpose of this study). This catchment joins the Umngeni river which provides water to over 3,5 million people [3].

Microbes in the spotlight: recent progress in the understanding of beneficial and harmful microorganisms

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2. Materials and methods

2.1 Historical data analysis:

The analysis of the historical data was conducted using the E.coli CFU/100 mL values obtained from the Municipality (eThekwini Water and Sanitation Department) and expressed as the mean arithmetic values of the data collected at various sampling points over 66 months (Jan 2009 – June 2014) for each river.

2.2 Microbiological Assessment (Data collected in 2015):

The microbiological quality of surface water and sediments from the Isipingo and Palmiet rivers at 8 sampling points (4 points at each river) were analysed using the Colilert -18 and Enterolert (IDEXX), and Quanti-Tray Test to determine E.coli and Enterococci respectively.

2.3 Statistical analysis:

All microbiological data were log10 transformed. Statistical analyses were performed using Microsoft Excel and GraphPad prism: Log transformation and graphs were conducted in Microsoft Excel. Pearson correlation analysis, and unpaired t-tests were performed using Graphpad prism.

3. Results

3.1 Historical data analysis

Figure 1: Comparison of the mean E.coli (Log10 CFU/ 100 ml) concentration along the Isipingo River and Palmiet River for 5 years and 6 months (Jan 2009-June 2014) (bars represent ±SD) The concentration of the E.coli values ranged from 3.31 (2010) to 3.62 (2012) and 3.45 (2011) to 4.53 (2013) E.coli log10 CFU/ 100 mL for the Palmiet and the Isipingo rivers respectively (n=36: 2009 to 2014). Based on an unpaired t-test, there was statistical difference in the concentration of E. coli between the two rivers (p= 0.0076). Isipingo reported higher concentrations than the Palmiet river as shown in Figure 1.


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3.2 Microbial Assessment (Data collected in 2015)

Figure 3: Comparison of the mean concentrations (Log10) of E. coli and Enterococci in the surface water and sediments of the Palmiet River and Isipingo River (n=12 ±SD) The mean E.coli and Enterococci values in surface water and sediment samples in the Palmiet and Isipingo rivers are shown in Fig 3. In the Palmiet river the indicator microorganisms ranged from 3.44 to 4.15 Log10 MPN/100 mL and at the Isipingo river it ranged from 4.17 to 4.80 Log10 MPN/100 mL. The red line indicates the eThekwini standard limits <3.33 shows that the values above this line are in a poor state. Table 1: Comparison of the mean concentrations (Log10) MPN/100 mL of E. coli and Enterococci in the surface water of the two rivers

Surface Water

E. coli Enterococci P-value Pearson correlation

Palmiet 4.0 ± 0.1 3.4 ± 0.2 0.009 r= 0.55

Isipingo 4.6 ± 0.6 4.2 ± 1.1 0.900 r= 0.90

2,5

3

3,5

4

4,5

5

s/w sediments s/w sediments

E.coli ENT

Log

10 E

.col

i MP

N/ 1

00 m

L

Indicator microorganisms

Palmiet Isipingo

Figure 2: Represents E.coli log10 CFU/100 mL verses rainfall (mm) E.coli log10 CFU/ 100 mL concentrations were correlated with rainfall events (on the day of sampling) (r= 0.2646 and p-value = 0.2724) using linear regression for values of rainfall >5 mm (n=19).

0 35 700

2

4

6

Rainfall (mm)

E.co

liLo

g 10

MPN

/100

mL

---- Standard limits

S/W: Surface water; ENT: Enterococci


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4. Discussion

Historical data analysis helps researchers to establish existing conditions of a particular catchment. A comparison of the two rivers of interest is illustrated by figure1 above. High levels of E.coli were found at the Isipingo river from the year 2009 to 2014 except for the year 2011, where Palmiet river (3.59 Log10 CFU/100 mL) and Isipingo river (3.45 Log10 CFU/100 mL) had values in a similar range. The generally high levels of E.coli at the Isipingo river over the years is due to its surroundings, represented by the sampling points (1) Upstream of the Isipingo wastewater treatment plant (WWTP), (2) next to the WWTP, (3) Downstream of the WWTP (this sampling point is surrounded by informal settlements) and (4) at the river mouth. Therefore, pipe outbursts and overflows from the WWTP contribute to river’s pollution in addition to the contaminants washed off during heavy rains particularly from the informal settlements as this area is highly polluted. This situation is typical for many African catchments and thereby represents a general trend. According to a study conducted by Cha et al [7], variability in fecal coliform concentration tends to be positively related to precipitation. Therefore an increase in total rainfall will elevate contamination levels. In order to determine this relationship, E.coli log10 CFU/ 100 mL concentrations were correlated with rainfall events (on the day of sampling) from the Palmiet river using linear regression for values of rainfall >5 mm (n=19). A positive correlation between E.coli concentrations and rainfall was found (r= 0.2646 and p-value = 0.2724), this indicated that the amount of precipitation had a direct relation with E.coli concentration, although it was a weak association as shown by the r-value and figure 2. Elevated levels of Coliforms/E.coli during rainfall are due to contamination that is washed off into to the rivers or streams. When surface water and sediments samples were compared (figure 3), the bacterial concentration of both E.coli and Enterococci was higher in sediment samples, in both the Isipingo and Palmiet rivers. The high bacterial concentration in sediments is probably due to the accumulation of the pathogens in the river bed/sediments over time which comes from sewage discharge during an event of a pipe outburst or contamination that is washed off into the river during heavy rains. Another interesting factor that contributes to this is the attachment of bacterial indicators to soil particles. Ana et al [8] showed that sediment samples yield higher levels of indicator bacteria than surface water. The study, tried to correlate E.coli densities in water and in sediment, but was not successful and further concluded that E.coli in water is not much of a good indicator for densities in sediment. Another study conducted in Congo found that microbial sediment analysis provides complementary and important information for assessing sanitary quality of surface water under tropical conditions [9]. Our study was in concurrence with previous reports, and we conclude that, the addition of sediment samples in river water quality analysis would give a better picture of the level of contamination. In relation to the eThekwini Water and Sanitation standard limits (>3.3 log10) both rivers were in a poor state in terms of water quality, hence water from both the Isipingo and Palmiet rivers is not suitable for crop irrigation purposes (≤1000 Fecal coliforms/100 mL) [10] and (≤1 E,coli /100 mL) [11]. The surface water from the Isipingo river is used to irrigate small scale market gardens (the produce is sold in the local community) and subsistence gardens by the community members in the surrounding area. Irrigation is conducted manually using plastic containers or buckets directly onto the produce. This potentially puts the members of the community at risk of being exposed to water-borne diseases. Both subsistence and commercial farmers potentially reduce the microbiological quality and safety of fresh produce by using feacally contaminated water for irrigation, this is thus not just restricted to direct wastewater reuse. Although it is widely known that using fecal contaminated water can be beneficial as it provides nutrients for crops. According to Beuchat et al [12] sufficient evidence of the presence of defecated pathogens found on the surface of vegetables irrigated with water containing fecal matter contamination. The relationship that microorganisms (bacteria, viruses, protozoa and helminths) can survive for days, weeks or even months on crops irrigated with microbially contaminated wastewater was established by Shuval et al [13] several decades ago. The presence of pathogens in waste water and receiving water bodies such as rivers is therefore still a serious concern especially if used for irrigating minimally processed crops such as lettuce, spinach and parsley [12]. The municipality rely upon the assumed predictive relationship between E.coli as an indicator organisms and pathogen survival/ transport in surface water, in order to ensure that the surface water does not put the public’s health at risk. Although it is impossible to test surface water for all possible pathogens it is important to use more than one indicator, and to also ensure that the indicator used can be correlated with a broad variety of waterborne pathogens [14, 15]. Statistical analyses were conducted in order to determine whether or not E.coli can be used to simulate the presence of other microorganism such as Enterococci. Statistical difference between the concentrations of E. coli and Enterococci was determined using an unpaired t-test, a significant difference was found in the Palmiet river (p-value = 0.009). However in the Isipingo river, no significant difference (p-value = 0.900) was observed in the concentration of these two indicators in surface water. A Pearson correlation analysis was conducted in order to determine the correlation between E.coli and Enterococci, the values in table 1 indicated that there was a stronger relationship between these indicators in the Isipingo river than the Palmiet river. The strong correlation between the E. coli and Enterococci figures in the


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Isipingo river could be attributed to the high concentrations of the indicator organisms (figure 3, table 1). Therefore, E.coli can be used as an indicator organism alone in extremely polluted areas, but using E.coli and Enterococci might be necessary in moderately polluted rivers such as the Palmiet river. Overall, a comparison of the two rivers indicates a higher contamination level in the Isipingo than the Palmiet. In conclusion, both the rivers were found to be over the statuary local guidelines, basically giving a representation of diluted feaces (especially the Isipingo river). The risks associated with irrigation and human health is still a major concern as the community members continue to use these rivers even though they do not comply with international and local guidelines. These two case studies represent a general trend for many of the African rivers. There is a correlation between E.coli concentration and rainfall. Lastly, it was found that sediment samples give an enhanced picture with regards to pollution level as appose to surface water only. Also, the correlation between E.coli and enterococci showed that these two indicators should be used in parallel in rivers that exhibits low contamination levels. Overall, Palmiet river was in a far better river health condition than the Isipingo river according to local guidelines.

Acknowledgements The support from Durban University of Technology and South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa as well as the EThekwini Water and Sanitation is sincerely acknowledged.

5. References

[1] World Health Organization, Water Sanitation and Health. Child hood and maternal undernutrition [Internet]. Geneva; c2015 [Cited 18/10/2015]. Available from: http://www.who.int/whr/2002/chapter4/en/index3.html

[2] World Health Organization, Water Sanitation and Health. Water related diseases- Diarrhoea [Internet]. Geneva; c2015 [Cited 18/10/2015]. Available from http://www.who.int/water_sanitation_health/diseases/diarrhoea/en/

[3] Olaniran AO, Naicker K, Pillay. Antibiotic resistance profiles of Escherichia coli isolates from river sources in Durban, South Africa. World Journal of Microbiology and Biotechnology. 2009; 25(10): 1743-1749.

[4] World Health Organization, Water Sanitation and Health. Costs and benefits of water and sanitation improvements at the global level [Internet]. Geneva; c2015 [Cited 18/10/2015]. Available from: http://www.who.int/water_sanitation_ health/wsh0404summary/en/.

[5] Pillay RK. An integrated study of the Isipingo River and Estuary: water and sediment quality, estuary-nearshore material fluxes, anthropogenic impacts and management [dissertation]. University of KwaZulu-Natal; 2013.

[6] Council for Scientific Industrial Research. A CSIR perspective on water in South Africa–2010. Council for Scientific and Industrial Research, CSIR Report No. CSIR/NRE/PW/IR/2011/0012/A [ISBN: 978-0-7988-5595-2]; 2010.

[7] Cha SM, Lee SW, Park YE, Cho KH, Lee S, Kim JH. Spatial and temporal variability of fecal indicator bacteria in an urban stream under different meteorological regimes. Water Science and Technology. 2010; 61(12): 3102-3108.

[8] Ana Y, Kampbellb DH, Breidenbach GP. Escherichia coli and total coliforms in water and sediments at lake marinas. Environmental Pollution. 2002; 120(3): 771-778.

[9] Mwanamoki PM, Devarajan N, Thevenon F, Atibu EK, Tshibanda JB, Ngelinkoto P, et al. Assessment of pathogenic bacteria in water and sediment from a water reservoir under tropical conditions (Lake Ma Vallée), Kinshasa Democratic Republic of Congo. Environmental monitoring and assessment. 2014; 186(10): 6821-6830.

[10] World Health Organization. Guidelines for Drinking water quality: Health Criteria and other supporting information. World Health organization, Geneva; 2006.

[11] Department of Water Affairs and forestry and Department of Health. A Guide for Health Related Assessment of Quality of Water Supplies. Interdepartmental Coordinating and Liaison Committee for water Supply and sanitation, Department of Water Affairs and Forestry and Department of Health; Institute for water Quality Studies, Pretoria; 1996.

[12] Beuchat LR. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection. 2002; (4): 413–423

[13] Shuval HI, Adin A, Fattal B, Rawitz E, Yekutiel P. Wastewater irrigation in developing countries: Health effects and technical solutions. Washington, DC; 1986.

[14] Rose JB, Farrah SR, Harwood VJ, Levine AD, Lukasik J, Menendez P, et al. Reduction of pathogens, indicator bacteria, and alternative indicators by wastewater treatment and reclamation processes, Water Environment Research Foundation; 2004.

[15] Noble RT, Moore DF, Leecaster MK, McGee CD, Weisberg SB. Comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality testing. Water Research. 2003; 37(7): 1637-1643


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Aerobic degradation of lindane in the presence of bacteria and zero valent iron nanoparticles

A.V Menéndez1,*, L.R Osuna1, V. Mesa1, J.L.R Gallego1, H. Sastre1 and A.I Peláez1 1Environmental Technology, Biotechnology and Geochemistry Group, Biotechnology Institute of Asturias,

University of Oviedo, C/ Gonzalo Gutiérrez Quirós s/n, 33600, Mieres-Asturias, Spain *Corresponding author: e-mail: [email protected], Phone: +34 985103000 ext.5836

Lindane is the common name for the gamma isomer of the hexaclorocyclohexane (HCH) a chemical compound widely use throughout the world due to its insecticidal properties. It is included in the Persistent Organic Pollutants (POP) list of the Stockholm Convention, and banned in the EU since 2002. Currently one of the main problems is the management and treatment of contaminated soils from past manufacturing of the compound. Nanoscale zero valent iron nanoparticles (nZVI) have shown to be effective as environmental decontamination tool, especially in the treatment of halogenated hydrocarbons. Also, bioremediation by bacteria and fungi constitutes an alternative treatment for lindane contaminated environments. The aim of this work was to explore the possibility of improving the removal of lindane with a combined treatment of bioaugmentation and nanoremediation with nZVI particles.

Keywords: Lindane; Iron nanoparticles, nZVI; Bacteria; Bioremediation.

1. Introduction

Lindane (ɣ-HCH) is a halogenated organic compound derived from benzene. Due to its insecticidal properties it has been widely used throughout the world in agriculture, forestry, wood manufacturing and as human and animal treatment against ectoparasites. Because of its durability in the environment, toxicity and easy bioaccumulation along the trophic chain lindane was included in the POP list of the Stockholm Convention, and its use is banned in the EU since 2002. One of the main problems nowadays is the manufacturing and treatment of contaminated soils from the past manufacturing of the compound. In Spain, one of the principal sources of pollution is located in Sabiñánigo (Aragón), a region near to The Pyrenees where around 20,000 tons of lindane residues coming from an old abandoned factory are stored in dumps. Bacterial lindane biodegradation has been observed in both anaerobic and aerobic ecosystems and thus bioremediation has therefore been considered a strategy of choice for cleaning soils contaminated with lindane [1]. Both bioremediation approaches, biostimulation and bioaugmentation have been applied to HCH-contaminated sites. Most of the lindane aerobic degrading bacteria known to date are members of the family Sphingomonadaceae (genera Sphingobium and Sphingomonas [1]). On the other hand the treatment with nanoscale zero valent iron nanoparticles has shown to be an effective tool in environmental decontamination process, especially in the treatment of halogenated hydrocarbons, including lindane degradation in aqueous solution [1] [2] [3] or soil [4]. The effectiveness of nanoparticles lies in its nanometric scale, which increases the specific surface area making them more reactive. In presence of oxygen the nanoparticles oxidized creating an environment which promotes the reduction of surrounding compounds. The main purpose of this work was the study of a combined treatment of bioaugmentation and nanoremediation with nZVI particles for the total or partial degradation of lindane. The approach was made by isolation of potential degrading bacteria from lindane contaminated soils. The isolated bacteria were submitted to a screening process to select the most appropriate for the combined decontamination treatments.

2. Experimental

2.1 Isolation and identification of bacteria

Enrichment cultures and microcosm assays were carried out with polluted soil from Sabiñánigo to isolate potentially lindane degrading bacteria and/or nZVI tolerant bacteria. Enrichment cultures were performed by adding 1 g of soil in 100 ml of minimal liquid medium (Bushnell Haas broth, Fluka Analytical) with lindane at final concentration of 50 µg/ml as sole carbon source. The lindane was previously diluted in dimethyl sulfoxide due to its low solubility. In the microcosm assay 20 g of contaminated soil were mixed with 40 ml of distilled sterile water in which nZVI particles were diluted at final concentrations of 1, 5 and 20 g/l. The nanoparticles were NANOFER STAR, supplied by Nano Iron s.r.o., Czech Republic. In both cases aliquots from different sampling times were taken and cultured in rich solid media. In the enrichment culture the sample was taken after


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three passes; in the microcosm were taken after 24 hours, 10 days and 30 days. The isolated bacteria were identified by amplification and sequencing of 16S rRNA gene, using OptiTaq PCR Master Mix (2X) (Eurx) for amplification and Big Dye terminator kit (Applied Biosystems) for sequencing. Nucleotide sequences were analysed by ABI PRISM ® 3130 Genetic Analyzer (Thermofisher). The sequence homology search was performed using BLAST program available al National Centre for Biotechnology Information website.

2.2 Screening tests

Several screening tests related with their capability to degrade lindane were carried out to select the most suitable bacteria among the previously isolated. The bacteria selection was made combining the result obtained in each test.

2.2.1 Growth and degradation of lindane as carbon source

The purpose of these experiments was to evaluate the capability of the bacteria to growth in a minimum liquid medium with lindane as sole carbon source, assuming that a visible growth implies the degradation of the pollutant. Bacteria were preincubated in 10 ml of TSB medium diluted 1:10 (Tryptone Soy Broth, Merck) during 72 hours at 30ºC. A saturated solution of lindane was prepared with 500 ml of liquid minimum media and 100 mg of lindane (Sigma-Aldrich), stirred at room temperature during 48 hours and filtered using membrane disc filters with pore size of 0.45 µm (Pall Corporation) [5]. Preincubated bacteria were centrifuged and washed twice with sterile water and resuspended in 3 ml. 1 ml of this suspension was added to Erlenmeyer flask with 20 ml of minimum lindane saturate medium and incubated at 30ºC and 150 rpm. Growth was assessed comparing the observed turbidity of the medium over time at 600 nm λ in a spectrophotometer, with respect to the same bacterial suspension in the minimum medium without lindane. The capability of selected bacteria to degrade lindane was tested by quantification of the total amount of lindane in the medium, comparing with the control (lindane medium without bacteria), after 7 days of incubation. The contaminant was extracted with a mixture of hexane and acetone (1:1) in a Soxtherm equipment and measured by Gas Chromatography-Mass Spectrometry (GC-MS) (Method 8270D, EPA).

2.2.2 Presence of lin Genes

A proposed lindane degradation pathway has been described in Sphingobium japonicum UT26 [6], where a cascade of enzymes is involved in the transformation of lindane into succinyl-CoA and acetyl-CoA, which are finally metabolized in the citrate/tricarboxylic acid cycle. We analysed the presence of the gene linA which codes for the first enzyme (dehydrochlorinase) of the metabolic pathway by PCR, which was performed as described previously [7] using OptiTaq PCR Master Mix (2X) (Eurx) and primers LinAF (5’-ATGAGTGATCTAGACAGACTTGC-3’) and LinAR1 (5’-TTATGCGCCGGACGGTGCG-3’) [8].

2.3 Effects of nZVI on selected bacteria

After the screening tests a few bacteria were selected and assayed again on the presence of nZVI. Each bacterium was preincubated as described above and 1 ml of the final suspension inoculated in Erlenmeyer flask with 18 ml of lindane saturated minimum medium and 1 ml of nZVI solution previously sonicated during 15 minutes to disperse nanoparticles and avoid agglomeration, at a final concentration of 1 g/L. The same bacteria inoculated in lindane-minimum medium without nZVI served as control. Cultures were incubated as described above. Bacterial growth was measured in both cases by dilution and plate-count methods in solid TSA. These experiments were complemented with a viability analysis by confocal laser scanning microscopy observations of the bacterial samples, after staining with vital dyes (SYTO 9 and IP, LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes) [9].

2.4 Combined decontamination experiments

These microcosm experiments were performed in 50 ml conical tubes with 13 g of soil, 15 ml of lindane saturated solution prepared as described above, 1 ml of nZVI solution (final concentration 1g/L) and 1 ml of bacterial suspension prepared as described previously; the tubes without nanoparticles were completed with sterile distilled water to reach the same volume in all. Soil was sterilised with two autoclave cycles (1 atm, 121 ºC, 20 minutes) separated by 24 hours. The soil suspensions were incubated at room temperature with manual agitation twice daily. After 72 hours and 15 days the soil was dried for further lindane quantification (see above).


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3. Results

3.1. Selection and screening of bacteria

As a result of the initial screening 75 bacteria were isolated from the first microcosm assay, named B51 to B73 and two bacteria, ES1 and ES2 were isolated from enrichment culture. The isolated bacteria were submitted to several consecutive growth and degradation tests (Section 2.2). At the end of the process ten bacteria were selected and identified (Table 1). The results obtained with these bacteria in the presence of lindane are also shown in Table 1. Streptomyces, Arthrobacter, Cupriavidus and Sinorhizobium show higher reduction of lindane in the medium, being chosen for further assays. Table 1. Identification and lindane utilization of the bacteria isolated and selected in the present work.

Lab Code

Highly similar sequences (Identification percentage) Accesion number

Lindane ppm ∗

B51 Streptomyces sp. MN40 (99) KF95308.1 7.91 B59 NI - 14.34 B61 B62 B63 B64 B65 B68 ES1 ES2

Promicromonospora sukumoe HBUM174119 (98) Bacterium JP15 (98) Arthrobacter sp. E7 (99) Nocardioides albus YJ-R21 (99) Sinorhizobium sp. MR-I13 (99) Pseudomonas sp DOC21 (98) Cupriavidus alkaliphilus odb12 (98) Sinorhizobium medicae MP-7H1 (98)

FJ486347.1 KC602251.1 FJ535482.1 KF876862.1 KJ811554.1 JN695041.1 KR812323.1 KF468788.1

8.48 8.28 6.76 9.14

16.24 9.09 8.03 6.73

*Data obtained after 7 days of incubation. Control value (lindane medium without bacteria; see 2.2.1) was 14.7 ppm. NI: Not identified

Presence of linA genes was tested in the above bacteria. As shown in Fig. 1 clear positive result were obtained only with Cupriavidus alkaliphilus, Sinorhizobium medicae and Arthrobacter, although an additional band is observed in Arthrobacter.

Fig. 1. Gel electrophoresis analysis of the PCR products of the linA gene amplification in C. alkaliphilus (lane 1) S. medicae (lane 2) and Arthrobacter (lane 3).

3.2. Effects of nZVI on selected bacteria

The effects of nZVI particles were tested on the four bacteria which gave best results in the lindane experiments: Streptomyces, Arthrobacter, Cupriavidus and Sinorhizobium. Streptomyces was included also due to former reports showing its capability to degrade organochlorines compounds, including lindane [1] [10]. Growth of the bacteria in the presence and absence of lindane was analysed at 18 and 72 hours (Fig. 2). The bacterial counts showed a similar growth dynamic of the bacteria in presence and absence of nanoparticles, although there is an initial transitory decrease when exposed to nanoparticles. Viability analysis was directly observed by confocal

1 2 3


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laser scanning microscopy. The microscopy observations (Fig. 3) show a predominance of green stained bacteria (live) than dead (red), which is consistent with the above growth results.

Fig. 2. Colony forming units (cfu) analysis of the selected bacteria in presence and absence of nanoparticles. a) Streptomyces sp. b) Arthrobacter sp. c) C. alkaliphilus. d) S. medicae.

Fig. 3. Images from the confocal microscopy observations of the bacteria samples in the presence of nZVI particles. a) Streptomyces sp. b) Arthrobacter sp. c) C. alkaliphilus. d) S. medicae.

3.3. Combined decontamination experiments in soil.

The ability to degrade lindane of two of the selected bacteria (Arthrobacter sp. and Cupriavidus alkaliphilus) alone or in combination (consortium) was tested in a microcosm assay with sterilized soil, in the absence or presence of nZVI particles (Table 2). Best results were obtained in the presence of nZVI alone or in the presence of Arthrobacter sp. at 3 days or the consortium plus nZVI after 15 days. Remarkably, the presence of bacteria apparently seems to impair the effectivity of the nanoparticles to the extent that when C. alkaliphilus was added, the levels of lindane were even higher that in the control. Possible explanations of this phenomenon are discussed below.

1,00E+07

1,00E+08

1,00E+09

1,00E+10

0 18 72Hours

B51

Without Np's

With NP's

1,00E+07

1,00E+08

1,00E+09

1,00E+10

0 18 72Hours

B63

Without Np's

With NP's

1,00E+08

1,00E+09

1,00E+10

0 18 72Hours

ES2

Without Np's

With NP's

1,00E+06

1,00E+07

1,00E+08

1,00E+09

0 18 72Hours

ES1

Without Np's

With NP's

a) b)

c) d)


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Table 2. Lindane degradation experiments in soil microcosms.

Treatment Sample time (days)

Lindane (μg/g soil)

Lindane (%)

Control 3 1.846 100 Control 15 1.579 85.5 nZVI 3 0.273 14.8 nZVI 15 0.096 5.2 Arthrobacter sp. 3 1.235 66.9 Arthrobacter sp. 15 1.501 81.3 C. alkaliphilus 3 2.542 137.7 C. alkaliphilus 15 1.829 99.0 Arthrobacter + nZVI 3 0.395 21,4 Arthrobacter + nZVI 15 0.824 44.6 C. alkaliphilus + nZVI 3 1.677 90.8 C. alkaliphilus + nZVI 15 1.75 94.8 Arthrobacter + C. alkaliphilus + nZVI 3 1.782 96.5 Arthrobacter + C. alkaliphilus + nZVI 15 1.104 59.8 Control: sterilized soil with lindane saturated minimum medium.

4. Discussion

We have isolated several soil bacteria capable to use lindane as the sole carbon source. One of them (Cupriavidus) had not been described previously as lindane degrader. An additional aim of the work was the design of a combined treatment of bioaugmentation and nanoremediation with nZVI particles for the total or partial degradation of lindane, exploiting the advantages that each technique offers. Nanoscale zero valent iron (nZVI) particles have specifically shown to be very effective for environmental remediation, especially for the treatment of chlorinated hydrocarbons or organochlorine pesticides [3] [4] [6]. As in our work, effectivity of nZVI towards lindane has been demonstrated previously by other authors in batch tests [1] [2] or soil [4]. However, as shown by our results, the presence of bacteria previously selected for their capability to use lindane as carbon source, does not seem to clearly improve the degradation of this compound. This was unexpected, as Arthrobacter has also been described by other authors as capable to degrade lindane [1] [10] [11]. One possible explanation is that the bacteria change the mobilization characteristics of the compound, making it less accessible to the nanoparticles. Lindane must first sorb onto the iron surface, where the highly electronegative chlorine substituents act as the electron acceptors and Fe0 serves as the electron donor [2] and these processes could be impaired by the bacteria. Other possibility is that the bacteria affect the concentration of lindane in the samples by enhancing the desorption of this compound from soil. Further investigations will necessary to clarify that bacterial effect. In this regard, sequential, instead of simultaneous treatment with nZVI particles and bacteria could provide useful approaches for the decontamination of lindane and/or other organochlorine compounds. In fact, accompanying the chemical transformation of lindane by nZVI particles, other by-products, as chlorobenzene, tetrachloroethylene, benzene and additional smaller by-products, not detected by the current methodology used, are formed [2] [3]. The subsequent treatment with the selected bacteria could decisively contribute to the elimination of these potential harmful derivatives.

Acknowledgements. Work was funded by the State Secretary of Investigation, Development and Innovation (Ministry of Economy and Competitiveness, Government of Spain), Project MINECO-13-CTM2012-38522-C02-01. Our special thanks to Kateřina Jašková from NANO IRON s.r.o for her advice and technical support.

References

[1] Lal R, Pandey G, Sharma P, Kumari K, Malhotra S, Pandey R, et al. Biochemistry of microbial degradation of Hexachlorocyclohexane and prospects for bioremediation. Microbiology and molecular biology reviews. 2010; 74 (1); 58-80.

[2] Wlliott D.W, Lien H-L, Zhang W-X. Degradation of lindane by zero-valent iron nanoparticles. Journal of environmental engineering. 2009; 135 (5); 317-324.

[3] San Román I, Alonso ML, Bartolomé L, Galmades A, Goiti E, Ocejo M, et al. Relevance study of bare and coated zero valent iron nanoparticles for lindane degradation from its by-product monitorization. Chemosphere. 2013; 93 (7); 1324-1332.


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[4] Singh R, Singh A, Misra V, Singh RP. DEgradation of lindane contaminated soil using zero-valent iron nanoparticles. J

Biomed Nanotechnol. 2011; 7 (1); 175-176. [5] Elango V. Biodegradation and bioremediation of hexachlorocyclohexane isomers, chlorinated ethenes, chlorinated

benzenes and benzene. All Dissertations. 2010; Paper 524. [6] Nagata Y, Endo R, Ito M, Ohtsubo Y, Tsuda M. Aerobic degradation of lindane (ɣ-hexachlorocyclohexane) in bacteria

and its biochemical and molecular basis. Appl Microbiol Biotechnol. 2007; 76; 741-752. [7] Cérémonie H, Boubakri H, Mavingui P, Simonet P, Vogel T.M. Plasmid-encoded ɣ-hexachlorocyclohexane degradation

genes and insertion sequences in Sphingobium francense (ex-Sphingomonas paucimobilis Sp+). Federation of European Microbiological Societies. 2006; 257; 243-252.

[8] Tabata M, Endo R, Ito M, Ohtsubo Y, Kumar A, Tsuda M, et al. The lin genes for γ-Hexachlorocyclohexane degradation in Sphingomonas sp. MM-1 proved to be dispersed across multiple plasmids. Biosci. Biotechnol. Biochem. 2011; 75 (3); 466-472.

[9] Menendez-Vega D, Gallego J.L.R, Pelaez A.I, Fernandez de Cordoba G, Moerno J, Muñoz D, et al. Engineered in situ bioremediation of soil and groundwater polluted with weathered hydrocarbons. European Journal of Soil Microbiology. 2007. 43 (5-6); 310-321.

[10] Alvarez A, Benimeli C.S, Saez J.M, Fuentes M.S, Cuozzo S.A, Polti M.A, et al. Bacterial Bio-resources for remediation of hexachlorocyclohexane. International journal of molecular sciencies. 2012; 13; 15086-15106.

[11] De Paolis M.R, Lipp D, Guerriero E, Polcaro C.M and Donati E. Biodegradation of α-, β-, and γ-Hexachlorocyclohexane by Arthrobacter fluorescens and Arthrobacter giacomelloi. Appl Biochem Biotechnol. 2013; 170; 514-524.


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Altruistic model of N2-fixing microbe-plant symbiosis: evolutionary and agronomic aspects

N. Provorov*,1, O. Onishchuk1, S. Yurgel2, O. Kurchak1, E. Chizhevskaya1 and N. Vorobyov1 1All-Russia Research Institute for Agricultural Microbiology, Podbelsky sh., 3, Saint-Petersburg, 196608, Russia 2Department of Environmental Sciences, Dalhousie University, Truro, NS, B2N5E3 Canada *Corresponding author: e-mail: [email protected], Phone: +7 812 470 51 00

Operation of N2-fixing microbe-plant (rhizobia-legume) symbiosis is described using the altruistic model which suggests that increase of the mutualism efficiency (ability to improve the host fitness) in microsymbionts results from their evolution towards differentiation into non-reproducible cellular forms (bacteroids). They express the enormous nitrogenase activity due to repressing the majority of free-living functions including the key stages of C/N metabolism and of cell envelope biogenesis. This altruistic evolutionary strategy is implemented via the kin selection pressures operating within the endosymbiotic rhizobia populations which are reorganized due to metabolic and signaling feedbacks with the hosts. The altruistic strategy represents a conduit for constructing the rhizobia strains for the legume crop inoculation: symbiotic efficiency in these strains may be improved due to inactivation of negative regulators of symbiosis (eff genes) responsible for adaptations to the soil environments (e.g., accumulation of nutritional/energy resources and synthesis of stress-protective surface components).

Keywords N2-fixing bacteria; leguminous plants; root nodule bacteria (rhizobia); plant-microbe symbioses; interspecies altruism; kin selection; symbiotic efficiency; genetic construction; microbial inoculants; poly-β-hydroxybutyrate; capsular and exo-polysaccharides

1. Introduction

Plant-microbe N2-fixing symbioses are broadly distributed in natural ecosystems and are used widely in sustainable agriculture to substitute the environmentally hazardous and expensive nitrogen fertilizers. Many of these symbioses are studied comprehensively at the molecular, cellular and ecological levels providing the excellent models for developing different areas of genetics and evolutionary biology [1]. A special interest to these models is related to the irreversible differentiation of N2-fixing bacteria into non-reproducible forms which develop very high N2-fixing activity and completely donate its products to the plants thus expressing an altruistic behavior towards their hosts (Table 1). Table 1 N2-fixing bacteria forming non-reproducible cellular forms in symbiosis with plants

Bacteria Taxonomy Hosts Non-reproducible N2-fixing forms

Location of N2-fixing forms

Rhizobia (Rhizobium, Sinorhizobium) [2]

α-proteo-bacteria

Galegoid legumes Bacteroids (enlarged, polyploid)

Intracellular (symbiosomes within the nodular cells)

Nostoc [3] Cyano-bacteria

All types of vascular plants (from mosses to angiosperms)

Multiple (over 30% of total cell number) heterocysts*

Intracellular (Gunnera), in tissue cavities (Anthoceros, Azolla**), in intercellular spaces (Cycas)

Azoarcus [4] β-proteo-bacteria

Cereals (Leptochloa fusca, Oryza sativa)

Non-cultivable cellular forms

In intercellular spaces

*In free-living cyanobacteria, 10% of cells are differentiated into heterocysts while mutants with multiple heterocysts have a severely reduced viability [5].

**N. azollae (microsymbiont of Azolla filiculoides) completely lost the ex planta viability due to a deep genome reduction supported by vertical transmission (via megaspores) of microsymbionts in the host generations [6].

In legume-rhizobia associations, these symbiotic forms are represented by intracellular bacteroids residing

within symbiosomes, which are most deeply differentiated in the “galegoid” legumes (including Vicieae, Trifolieae and Galegae tribes [2]). Bacteroids provide the analogs of cellular organelles (surrounded with double membranes with a highly reduced peribacteroid space, ensuring an intensive metabolic exchange with plant cytosol) therefore suggesting a useful model for reconstructing the evolution of eukaryotic cells.


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